13th international double-stranded rna · 14 abstract book | 13th international double-stranded rna...

13th International Double-Stranded RNA

Virus Symposium

24-28 September 2018Houffalize, Belgium

ABSTRACT BOOK

ISBN 978 94 6165 258 4D/2018/1869/41

Table of contents

1. Welcome ....................................................................................................................52. Organisers .................................................................................................................93. Travel grant awardees ..............................................................................................114. Meeting venue .........................................................................................................175. Sponsors ..................................................................................................................196. Program .................................................................................................................. 237. Abstracts oral and shotgun presentations .............................................................. 53

Workshop 1: Viral Diversity ................................................................................. 55Workshop 2: Evolution and Epidemiology ...........................................................71Workshop 3: Molecular and Cellular Virology ..................................................... 89Workshop 4: Immunity and Pathogenesis .........................................................105Workshop 5: Structure........................................................................................121Workshop 6: Using and Abusing Host Pathways ..............................................133Workshop 7: Applied dsRNA Virology ...............................................................143Rotavirus Satellite symposium ...........................................................................149

8. Abstracts poster presentations ..............................................................................159Workshop 1: Viral Diversity ................................................................................161Workshop 2: Evolution and Epidemiology .........................................................185Workshop 3: Molecular and Cellular Virology ....................................................215Workshop 4: Immunity and Pathogenesis .........................................................241Workshop 5: Structure....................................................................................... 263Workshop 6: Using and Abusing Host Pathways ............................................. 267Workshop 7: Applied dsRNA Virology .............................................................. 277

9. Student classes..................................................................................................... 29310. Author Index .......................................................................................................... 30311. Attendees .............................................................................................................. 325

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1. WELCOME

WELCOME TO THE 13TH INTERNATIONAL dsRNA VIRUS SYMPOSIUM

We have the pleasure of welcoming you to the 13th International dsRNA virus symposium, which is held in Houffalize, Belgium, from September 24 to 28, 2018.

This meeting started in 1982 in St. Thomas, US Virgin Islands, followed by Oxford, UK (1986), Kona Island, Hawaii, USA (1990), Scottsdale, Arizona, USA (1992), Djerba, Tunisia (1995), Cocoyoc, Morelos, Mexico (1997), Palm Beach, Aruba (2000), Il Ciocco, Lucca, Italy (2002), Cape Town, South Africa (2006), Hamilton Island, Australia (2009), San Juan, Puerto Rico (2012) and Goa, India (2015).

Historically, these meeting have always had an informal character allowing attendees from every stage in their career to interact in a friendly and open atmosphere, and discuss a wide range of scientific topics related to dsRNA virology. A major goal of the meeting is to promote the careers of international young scientists and trainees, including those from the developing world. We have been very fortunate to receive generous support from several sponsors allowing us to award a large number of travel grants. A big THANK YOU goes to the Bill & Melinda Gates foundation, GSK, the Burroughs Wellcome Fund, MSD, Bharat Biotech, The Serum Institute of India, the FWO, the NIH and the Dep. Microbiology and Immunology of the KU Leuven.

As you may have noticed, we have introduced a few new items to the meetings. In additional to the classical keynote lecture, workshops, poster session and the Jean Cohen lecture, we have added a student class, a live TWiV, shotgun presentations and two round table discussion to the program.

The student class will allow (early career) researchers to refresh and increase their background knowledge on the various dsRNA viruses, which will be discussed this week, in order to maximize their scientific experience during the symposium. A big THANK YOU goes to John Parker, Pranav Danthi, Sarah McDonald, Mark Boyce and Michelle Arnold for taking on this educational challenge.

We are also very happy to host a life session of the podcast TWiV (This Week in Virology; http://www.microbe.tv/twiv/), produced by Vincent Racaniello. We thank Vincent to travel all the way to Belgium and be with us here this week.

The shotgun presentations (4 minutes of presentation without questions) are an experiment that will allow a larger group of people to place their research in the spotlight with a short presentation AND a poster. The presented scientific topics can be further discussed during the poster sessions, coffee breaks, lunches, dinners or social events. We hope it will be a successful format.

6 Abstract book | 13th International Double-Stranded RNA Virus Symposium

Two round table discussion about “Rotavirus reverse genetics” and “Reoviridae and the ICTV” have been scheduled upon request of the scientific community. Feel free to contribute and learn from the discussions.

With all of this being said we welcome you again in Belgium, and hope you will enjoy the meeting and the company of your fellow colleagues.

Jelle MatthijnssensJohn ParkerPranav DanthiPierre Van Damme

71. Welcome

2. ORGANISERS

1. Scientific Committee

ARNOLD, Michelle Louisiana State University, USABOYCE, Mark University of Oxford, UKCOULSON, Barbara University of Melbourne, AustraliaCRAWFORD, Sue Baylor College of Medicine, USAITURIZZA-GOMARA, Miren University of Liverpool, UKKOBAYASHI, Takeshi Osaka University, JapanMAINOU, Bernardo Emory University, USAMCDONALD, Sarah Wake Forest University, USAPARRENO, Viviana Instituto Nacional de Tecnología Agropecuaria,

ArgentinaSHMULEVITZ, Maya University of Alberta, CanadaSTEYER, Andrej University of Ljubljana, SloveniaSUZUKI, Nobuhiro Okayama University, JapanTAO, Yizhi (Jane) Rice University, USAVAN STADEN, Vida University of Pretoria, South Africa

2. Local Organising Committee

MATTHIJNSSENS, Jelle KU Leuven, Belgium [email protected] PARKER, John Cornell University, USA [email protected] DANTHI, Pranav Indiana University, USA [email protected] VAN DAMME, Pierre Antwerp University, Belgium [email protected]

3. Conference Coordinator

DE BRABANTER, Dominique KU Leuven, Belgium [email protected]


3. TRAVEL GRANT AWARDEES

1. Full grant

Agbemabiese, Chantal Ama Noguchi Memorial Institute for Medical Research, Ghana

Ahmed, Mohamed Lemine Cb. National Institute of Research on Public Health, Mauritania

Amadu, Dele Ohinoyi University of Ilorin Teaching Hospital, Nigeria

Apondi, Ernest Wandera Nagasaki University, Kenya

Bonifacio, Joseph Manzano Research Institute for Tropical Medicine, Philippines

Boula, Angeline yvette Centre Mère et Enfant, Cameroon

Bucardo, Filemón National Autonomous University of León, Nicaragua

Castells, Matias University of the Republic, Uruguay

Chagas, Elaine Evandro Chagas Institute, Brazil

Chilakalapudi, Durga Rao Indian Institute of Science, India

Damanka, Susan Afua Noguchi Memorial Institute for Medical Research, Ghana

Degiuseppe, Juan Ignacio INEI - ANLIS “Dr. Carlos G. Malbrán”, Argentina

Dey, Shuvra Jahangirnagar University, Bangladesh

Donato, Celeste Michelle Monash University, Australia

Garcés Suárez, Yasel Autonomous National University of Mexico, Mexico

Gómez, FabiánInstituto de Diagnóstico y Referencia Epidemiológicos, Mexico

Joshi, Madhuri Shantanu National Institute of Virology, India

Kadoya, Syun-suke Tohoku University, Japan

Kawagishi, Takahiro Research Institute for Microbial Diseases, Osaka university, Japan

Mandile, Marcelo Gastón Universidad Nacional de Quilmes, Argentina

Martinez, Iara Magaly Health Sciences Research Institute, National University of Asuncion, Paraguay

Masachessi, Gisela Universidad Nacional de Córdoba, Argentina

Miño, Samuel INTA, Argentina

Mogotsi, Milton Tshidiso University of the Free State, South Africa


Mokoena, Fortunate Sefako Makgatho Health Science University, South Africa

Mukhopadhyay, Urbi National Institute of Cholera and Enteric Diseases, India

Ngomane, Thenjiwe Grace Sefako Makgatho health Sciences university, South Africa

Ngoy Kiluba, Médard Pediatric Kalembelembe hospital, Democratic Republic of the Congo

Ngoya, RogerCentre Mère et Enfant de la Fondation Chantal BIYA, Cameroon

N’guessan-Kouame, Amani Rebecca Teaching Hospital of Yopougon, Côte d’Ivoire

Nkolo Mviena, Gaston Eric Mother and Child Center Chantal BIYA Foundation, Cameroon

Oni, Oluwole Oyetunde Federal University Of Agriculture Abeokuta, Nigeria

Patra, Upayan National Institute of Cholera and Enteric Diseases, India

Rakau, Kebareng Sefako Makgatho Health Sciences University, South Africa

Ranshing, Sujata Sudhir National Institute of Virology, India

Rezende da Silva, Julia Evandro Chagas Institute, Brazil

Sander, Wico University of the Free State, South Africa

Sharma, Kuldeep National Institute for Research in Tribal Health, India

ShipraMolecular Virology and Vaccinology Lab SCLS, India

Strydom, Amy University of the Free State, South Africa

Tatte, Vaishali ICMR-National Institute of Virology, India

Varanasi, Gopalkrishna National Institute of Virology, India

Vega, Celina Guadalupe INTA, Argentina

Wall, Gayle Victoria University of Pretoria, South Africa

2. Partial grant

Ahmad, Irfan SK.University of Agricultural Sciences & Technology of Kashmir, India

Alshaikhahmed, Kinda London School of Hygiene and Tropical Medicine, UK

133. Travel grant awardees

Attoui, Houssam UMR1161 Virologie INRA-ANSES-ENVA, France

Bekker, Shani May University of Pretoria, South Africa

Ben Hadj Fredj, Mouna University Hospital Sahloul, Tunisia

Bennour, Haifa University Hospital Sahloul, Tunisia

Borodavka, Alexander University of Leeds, UK

Boughan, Shareen University of Pretoria, South Africa

Chen, Meng-Jung Chi-Mei Medical Center, Taiwan

Crawford, Sue Ellen Baylor College of Medicine, USA

Dennis, Francis Ekow Noguchi Memorial Institute for Medical Research, Ghana

Ferreira-Venter, Linda University of Pretoria, South Africa, South Africa

Fodha Bouzgarrou, Imene University Hospital Sahloul, Tunisia

Ghosh, Ananta Kumar Indian Institute of Technology Kharagpur, India

Hardy, Alexandra MRC - University of Glasgow Centre for Virus Research, UK

Hsieh, Chia-Jung Chung-Hwa University of Medical Technology, Taiwan

Hyser, Joseph Baylor College of Medicine, USA

Kanai, Yuta Osaka University, Japan

Karatas, Fidan University of Pretoria, South Africa

Kerviel, Adeline London School of Hygiene and Tropical Medicine, UK

Lartey, Belinda NaaNoguchi Memorial Institute for Medical Research, Ghana

Lazaro, C. Eures Iyar Garcia Research Institute for Tropical Medicine, Philippines

Llauger, Gabriela National Institute of Agricultural Technology (INTA), Argentina

Luchs, Adriana Adolfo Lutz Institute, Brazil

Malik, Yashpal Indian Veterinary Research Institute, India

Matsuo, Eiko Kobe Univesity, Japan

Miller, Cathy Iowa State University, USA


Mukherjee, Anupam ICMR-National Institute of Cholera and Enteric Diseases, India

Munlela, Benilde António Instituto Nacional de Saúde, Mozambique

Nyaga, Martin University of the Free State, South Africa

Oberholster, Larise University of the Free State, South Africa

Pieters, Ané University of Pretoria, South Africa

Prez, Verónica Emilse Instituto de Virologia Dr JM Vanella, Argentina

Qiao, Rui Peking university, ChinaTribhuvan University Institute of Medicine, Nepal

Sherchand, Jeevan Bahadur Tribhuvan University Institute of Medicine, Nepal

Soares, Luana da Silva Evandro Chagas Institute, Brazil

Suzuki, Nobuhiro Okayama University, Japan

van Rijn, Piet A. Wageningen BioVeterinary Research, Netherlands

Yssel, Litia University of Pretoria, South Africa

3. NIH grant

Aravamudhan, PavithraUPMC Children’s Hospital of Pittsburgh, USA

Criglar, Jeanette MarieBaylor College of Medicine, USA

Ding, SiyuanStanford University, USA

Hu, LiyaBaylor College of Medicine, USA

Kumar, DilipBaylor College of Medicine, USA

Mainou, Bernardo Emory University, USA

Ogden, Kristen MarieVanderbilt University Medical Center, USA

Philip, Asha AnnIndiana University, USA

Ramani, SasirekhaBaylor College of Medicine, USA

Shepherd, Frances KatenUniversity of Minnesota, USA

Velasquez Portocarrero, Daniel EduardoCenters for Disease Control and Prevention, USA

153. Travel grant awardees

4. MEETING VENUE

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Vayamundo HouffalizeOl Fosse d’Outh 16660 HouffalizeBELGIUM


5. SPONSORS

Platinum Sponsors

Silver Sponsors

Bronze Sponsors

Others

http://www.gatesfoundation.org/

http://www.seruminstitute.com/

https://www.bwfund.org/

https://gbiomed.kuleuven.be/english/research/50000698

http://www.msd.com

https://www.gsk.com/The Organization does not in any way promote, stimulate or suggest off-label use of any GSK or third party product.

http://www.fwo.be/en/

https://www.nih.gov/ Research reported in this publication was supported by the National Institute Of Allergy And Infectious Diseases of the National Instiitutes of Health under Award Number R13AI140632. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

https://www.bharatbiotech.com/

Department of Microbiology and Immunology

KU Leuven This symposium is approved by Mdeon.Visa number: 18/V2/10177/006268.


6. PROGRAM

DAY 1: 24 SEPTEMBER 201812:00 – 19:00 Arrival and check-in15:30 – 17:30 Student classes, with the following presentations:

John Stuart Lesie Parker: Overview of different dsRNA viruses Pranav Danthi: Structure and entry Sarah McDonald: RNA synthesis mechanisms of dsRNA viruses Mark Boyce: Packaging of segmented genomes Michelle Arnold: Immunology

17.30 – 19:00 Welcome reception19:00 – 19:15 Welcome by the conference organizers19:15 – 20:15 Keynote Lecture by Julie Pfeiffer:

How transkingdom interactions influence viral infection20:15 – 22:15 Dinner

DAY 2: 25 SEPTEMBER 201809.00 – 09:15 Introduction and welcome by the conference organizersWorkshop 1: Viral DiversityConveners: Nobuhiro Suzuki and Andrej Steyer09:15 – 09:40 Plenary presentation by Nobuhiro Suzuki:

Another nude virus: a capsidless ssRNA virus hosted by an unrelated dsRNA virus

09:40 – 09:55 Oral presentation by Souvik Ghosh, Yashpal S Malik, Nobumichi Kobayashi:Molecular characterization of complete gene segment-2 of picobirnaviruses from different animal host species: Evidence for high genetic diversity, and identification of conserved features in putative RdRps

09:55 – 10:10 Oral presentation by Luan Wang, Jingze Zhang, Hailong Zhang, Dewen Qiu, Lihua Guo: Two Novel Relative Double-stranded RNA Mycoviruses Infecting Fusarium Poae Strain SX63

10:10 – 10:15 Shotgun presentation by Kinda Alshaikhahmed, Po-Yu Sung, Polly Roy:RNA-RNA interactions hold the key to assortment in the multipartite dsRNA virus

10:15 – 10:20 Shotgun presentation by Juan Ignacio Degiuseppe, Juan Andrés Stupka: Genetic diversity of Group A Rotavirus in Latin America before and after universal vaccination, 2001-2017


10:20 – 10:25 Shotgun presentation by Frances Katen Shepherd, Fangzhou Chen, Michael Murtaugh, Douglas Marthaler: In silico analysis of the VP7 for porcine rotavirus B and C illustrates genotype-specific antigenic epitopes

10:25 – 11:00 Coffee break11:00 – 11:25 Plenary presentation by Marilyn Roossinck:

Evolution in the Family Partitiviridae and Novel Aspects of the Polymerases

11:25 – 11:40 Oral presentation by Joseph Hughes, Anna Janowicz, Massimo Palmarini:Bluetongue virus replication in culicoides cells increases the number of genetic variants in the viral population

11:40 – 11:55 Oral presentation by Andrea Kaup Erickson, Julie Pfeiffer:Impact of host microbiota on enteric RNA virus evolution.

11:55 – 12:10 Oral presentation by Houssam Attoui, Fauziah Mohd Jaafar, Peter Mertens:The Reovirales a new taxonomic order: families Sedoreoviridae and Spinareoviridae

12:10 – 12:15 Shotgun presentation by Josh Singer, Rob Gifford, Kyriaki Nomikou, Alexander MacLeod, Peter Mertens, Massimo Palmarini:BTV-glue: a web-based bioinformatics resource for Bluetongue Virus sequence data

12:15 – 12:20 Shotgun presentation by Samuel Miño, Matías Adúriz, Viviana Parreño:Rotavirus species tropism: new insights on P-type classification

12:20 – 12:25 Shotgun presentation by Leen Beller, Ward Deboutte, Chenyan Shi, Daan Jansen, Claude Kwe Yinda, Mark Zeller, Marc Van Ranst, Jelle Matthijnssens: Exploring the diversity and dynamics of the gut virome of infants during the first year of life

12:25 – 12:30 Shotgun presentation by Øystein Wessel, Turhan Markussen, Torstein Tengs, Dhamotharan K, Salman Malik, Maria Dahle, Espen Rimstad:Piscine orthoreovirus (PRV); in vivo propagation in erythrocytes enables virus purification, causality to important disease in farmed Atlantic salmon and protection by inactivated vaccine

12:30 – 14:30 Lunch breakwith live postcast ‘This Week in Virology (TWiV) by Vincent Racaniello and special guest Harry Greenberg (between 13:00 and 14:00)

256. Program

Workshop 2: Evolution and EpidemiologyConveners: Kristen Marie Ogden and Sarah McDonald14:30 – 14:55 Plenary presentation by Kristen Marie Ogden, Yi Tan, Asmik

Akopov, Laura S Stewart, Rendie McHenry, Christopher J Fonnesbeck, Bhinnata Piya, Maximilian H Carter, Nadia B Fedorova, Rebecca A Halpin, Meghan H Shilts, Kathryn M Edwards, Daniel C Payne, James D Chappell, John T Patton, Natasha B Halasa, Suman R Das:Multiple introductions and antigenic mismatch contribute to increased predominance of G12P[8] rotaviruses in the United States

14:55 – 15:10 Oral presentation by Yashpal S Malik, Sharad Saurabh, Jobin Jose Kattoor, S. Shanmuganathan, Shubhankar Sircar, Kuldeep Dhama, Souvik Ghosh, Nadia Touil, Raj Kumar Singh:Epidemiology and evolution of Rotavirus C (RVC) in India

15:10 – 15:25 Oral presentation by Thenjiwe Grace Ngomane, Ina Peenze I, Mapaseka Seheri:Genetic diversity of group a rotavirus strains circulating in porcine from five provinces in south africa during 2007, 2008 and 2015.

15:25 – 15:30 Shotgun presentation by Sebastiaan Theuns, Quinten Bernaert, Philip Vyt, Hans J Nauwynck:Intriguing successive (sub)clinical shedding patterns of different rotavirus genotypes in suckling and weaned pigs

15:30 – 15:35 Shotgun presentation by Mohamed Lemine Cb.Ahmed, Abdellahi Weddih, Mohemed Abdellahi Boullah, Meriam Sidatt, Abdelkarim Filali-Maltouf, Mohammed Benhafid:Rotavirus among children aged 0-5 years in Nouakchott Mauritania

15:35 – 15:40 Shotgun presentation by Tohru Suzuki: Genetic characteristics of porcine rotaviruses H detected recently in Japan

15:40 – 15:45 Shotgun presentation by Frances Katen Shepherd, Michael Murtaugh, Douglas Marthaler:Genetic variability and implications for identifying neutralizing epitopes in swine rotavirus A strains

15:45 – 15:50 Shotgun presentation by Samuel Miño, María Barrandeguy, Viviana Parreño:Worldwide evolution of equine RVA, four voices, one story

15:50 – 16:20 Coffee break16:20 – 16:45 Plenary presentation by Eeva Vainio, Anna Poimala, Tuula Piri,

Muhammad Kashif, Jarkko Hantula:Partitiviruses and related unclassified viruses in plant pathogenic fungi and oomycetes


16:45 – 17:00 Oral presentation by Susie Roczo-Farkas, Celeste Michelle Donato, Carl Kirkwood, Daniel Cowley, Graeme Barnes, Ruth Bisop, Nada Bogdanovic-Sakran, Karen Boniface, Julie Bines:Analysis of the Impact of Rotavirus Vaccines on Genotype Diversity in Australia

17:00 – 17:15 Oral presentation by Filemón Bucardo, Yaoska Reyes, Ylva Rönnelid, Fredman González, Lennart Svensson, Johan Nordgren:Lewis and Secretor phenotypes affect rotavirus vaccine shedding in Nicaraguan children

17:15 – 17:20 Shotgun presentation by Ward Deboutte, Leen Beller, Claude Kwe Yinda, Nádia da Conceição Neto, Piet Maes, Dirk C de Graaf, Jelle Matthijnssens:A viral metagenomics view on the honeybee virome reveals a plethora of undescribed dsRNA viruses

17:20 – 17:25 Shotgun presentation by Hiroaki Shirafuji, Natsumi Kobayashi, Katsunori Murota, Shogo Tanaka, Tohru Yanase:Epizootic hemorrhagic disease virus identified in affected cattle in Japan during 2015−2017

17:25 - 17:30 Shotgun presentation by Chantal Ama Agbemabiese, Toyoko Nakagomi, Susan Afua Damanka, Francis Ekow Dennis, Belinda Larteley Lartey, Frederick Karikari Asamoah, Michael Fokuo Ofori, George Enyimah Armah, Osamu Nakagomi:A proposal for the lineage framework for sub-genotype phylogeny of the backbone genes of DS-1-like Rotavirus A strains.

17:30 - 17:35 Shotgun presentation by Isidore Juste O. Bonkoungou, Ylva Rönnelid, Nafissatou Ouedraogo, Nicolas Barro, Lennart Svensson, Johan Nordgren:Predominance of norovirus in children hospitalized with gastroenteritis after rotavirus vaccine introduction in Burkina Faso

17:35 - 17:40 Shotgun presentation by Syun-suke Kadoya, Syun-ichi Urayama, Takuro Nunoura, Masaaki Kitajima, Satoshi Okabe, Toyoko Nakagomi, Osamu Nakagomi, Daisuke Sano:Neutral Evolutionary Rate of Rhesus Rotavirus

17:40 - 17:45 Shotgun presentation by Daniel E Velasquez Portocarrero, Baoming Jiang:Comparative analysis of trivalent P2-VP8 subunit rotavirus vaccine strains and P[8], P[4], and P[6] human rotaviruses reported globally during 1974 and 2017

Poster Session 1-2-318:00 – 20:00 Poster presentations from Workshop 1-2-3. All shotgun

presentations from Workshop 1, 2 and 3 will also be presented as poster. A list of all poster presentations in this session can be found on pages 36 through 45.

276. Program

20:00 – 22:15 DinnerRound Table discussion: Reoviridae and the ICTVChairs: Houssam Attoui and Jelle Matthijnssens21:00 – … Round table discussion led by Houssam Attoui and Jelle

Matthijnssens

DAY 3: 26 SEPTEMBER 2018Workshop 3: Molecular and Cellular VirologyConveners: Takeshi Kobayashi and Mark Boyce08:45 – 09:10 Plenary presentation by Takeshi Kobayashi:

Reverse genetics systems for orthoreoviruses and rotaviruses 09:10 – 09:35 Plenary presentation by Angela K Berger, Jameson TL Berry,

Roxana M Rodriguez Stewart, Bernardo Mainou:From Bench to Bedside: Screening of Small Molecules to Better Understand Virus Biology and Develop Improved Therapeutics

09:35 – 09:50 Oral presentation by Asha Ann Philip, Siyuan Ding, Heather E Heaton, Chantal A Agbemabiese, Maya Shmulevitz, Harry G Greenberg, John T Patton:Generation of Recombinant Rotaviruses Expressing UnaG Fluorescent Protein Using the RNA Capping Enzyme of African Swine Fever Virus

09:50 – 10:05 Oral presentation Marta Fratini, Tina Wiegan, Joachim Spatz, Ada Calvacanti-Adams, Steeve Boulant:Measuring forces during the cellular uptake of mammalian reovirus: Physico-chemical regulation of early events of clathrin-mediated endocytosis of viral particles

10:05 – 10:10 Shotgun presentation by Shani May Bekker, Christiaan Potgieter, Vida van Staden, Jacques Theron:The use of a soluble African horse sickness virus VP7 protein to study virus replication and vaccine development

10:10 – 10:15 Shotgun presentation by Durga Rao Chilakalapudi, Poonam Dhillon:Viroplasms and remodelled stress granules and P-bodies: Union of the triad to promote progeny rotavirus production

10:15 – 10:20 Shotgun presentation by Po-yu Sung, Cheng Kao, Polly Roy:Mapping the RNA binding and packaging motifs of Bluetongue virus capsid protein VP6 by biophysical and molecular analysis

10:20 – 10:50 Coffee break10:50 – 11:15 Plenary presentation by Piet van Rijn:

Orbivirus replication and reverse genetics


11:15 – 11:30 Oral presentation by Damien Vitour, Grégory Caignard, Cindy Kundlacz, Aurore Fablet, Marie Pourcelot, Rayane Amaral, Corinne Sailleau, Emmanuel Bréard, Cyril Viarouge, Stephan Zientara:Study of molecular interactions between BTV and its hosts

11:30 – 11:45 Oral presentation by Catherine C Eichwald, Antonino Buttafuoco, Kevin MIchaelsen, Kurt Tobler, Stuart Sims, Ohad Medalia, Cornel Fraefel, Mathias Ackermann:Dissection of rotavirus replication intermediates NSP5 and VP2: A step towards an antiviral

11:45 – 11:50 Shotgun presentation by Eric N. Salgado, Brian Garcia Rodriguez, Nagarjun Narayanaswamy, Yamuna Krishnan, Stephen C. Harrison:Visualization of Ca2+ loss from rotavirus during cell entry

11:50 – 11:55 Shotgun presentation by Marc Guimera Busquets, Gillian Pullinger, Massimo Palmarini, Karin E. Darpel, Rennos Fragkoudis, Peter P. C. Mertens:Molecular determinants of bluetongue virus that drive replication in the insect vector cell

11:55 – 12:00 Shotgun presentation by Marno GJ Huyzers, Abraham Christiaan Potgieter, Louis Jeremia Cornelius Theart, Alberdina Aike van Dijk:Local implementation and optimisation of entirely plasmid-based SA11 rotavirus reverse genetics

12:00 – 12:05 Shotgun presentation by Joerg Bormann, Cornelia Heinze, Christine Blum, Michael Mentges, Anke Brockmann, Arne Alder, Svenja Kim Landt, Brian Josephson, Daniela Indenbirken, Michael Spohn, Birte Plitzko, Sandra Loesgen, Michael Freitag, Wilhelm Schäfer:Expression of a structural protein of the mycovirus FgV-ch9 negatively affects the transcript level of a novel symptom alleviation factor and causes virus-infection like symptoms in Fusarium graminearum

12:05 – 12:10 Shotgun presentation by Anirban Kundu, Amit Kumar Das, Ananta Kumar Ghosh:Molecular mechanism of polymerase activity and RNA-binding properties of E. coli expressed different domains of Antheraea mylitta cytoplasmic polyhedrosis virus RNA-dependent RNA polymerase

12:10 – 13:15 LunchJean Cohen Lecture13:15 – 13:25 Introduction by Ulrich Desselberger13:25 – 14:15 Jean Cohen Lecture by Harry Greenberg:

The generation and function of innate and acquired immunity to rotavirus infection in vitro and in vivo

296. Program

Poster Session II14:15 – 16:15 Poster presentations from Workshop 4-5-6-7. All shotgun

presentations from Workshop 4, 5, 6 and 7 will also be presented as poster. A list of all poster presentations in this session can be found on pages 45 through 51.

Social Program17:00 - 20:15 Social activities

Visit to the Bastogne War museum Mountain biking Visit to the Achouffe brewery

20:15 – 22:15 DinnerRound Table discussion: Rotavirus Reverse GeneticsChairs: Takeshi Kobayashi, Ulrich Desselberger and John Patton21:15 – … Round table discussion led by Takeshi Kobayashi, Ulrich

Desselberger and John Patton

DAY 4: 27 SEPTEMBER 2018Workshop 4: Immunity and PathogenesisConveners: Michelle Arnold and Sue Crawford08:45 – 09:10 Plenary presentation by Cathy Miller, Luke Bussiere, Choudhury

Promisree:Factories, Granules, and Hypoxia: Does stress granule disruption by mammalian orthoreovirus factory proteins contribute to hypoxia downregulation in tumor cells?

09:10 – 09:35 Plenary presentation by Karl Boehme: Reovirus non-structural protein σ1s enhances viral resistance to type-1 interferons

09:35 – 09:50 Oral presentation by Marie Hagbom:Neurotropic factors from enteric glial cells contribute to maintain the intestinal epithelial barrier during rotavirus infection

09:50 – 10:05 Oral presentation by Alexandra Hardy, Meredith Stewart, Mariana Varela, Andrew Shaw, Sam Wilson, Richard Randall, Massimo Palmarini:Species-specific restriction of Bluetongue virus replication correlates to host resilience

10:05 – 10:10 Shotgun presentation by Siyuan Ding, Peter K. Jackson, John T. Patton, Jia Liu, Harry B.Greenberg: Interferon-stimulated gene SAMD9 restricts rotavirus replication and is counteracted by non-structural protein 1


10:10 – 10:15 Shotgun presentation by Megan Stanifer, Stephanie Muenchau, Dorothee Albrecht, Sina Bartfeld, Franck van Kuppeveld, Takashi Kanaya, Steeve Boulant:Polarised interferon-mediated immune response against mammalian reovirus reveal novel mechanisms of immune tolerance in the human gut

10:15 – 10:20 Shotgun presentation by Lyndsay Cooke, Katy Moffat, Laura Tugwell, Nicolas Locker, Geraldine Taylor, Karin Darpel:Bacterial products within Culicoides midge saliva enhance Bluetongue virus infection in bovine monocytes

10:20 – 10:50 Coffee break10:50 – 11:15 Plenary presentation by Barbara Coulson:

Evidence of a distinct mechanism for rotavirus acceleration of type 1 diabetes development

11:15 – 11:30 Oral presentation by Gwen Taylor, Andrea J. Pruijssers, Terence S. Dermody:Neuron-specific NF-κB-dependent genes mediate reovirus neuropathogenesis

11:30 – 11:45 Oral presentation by Scott Handley, George Armah, Lindsay Droit, Barry Hykes, Chandni Desai, Herbert Virgin, Vanessa Harris:Transkingdom viral, bacterial and fungal microbiome correlation with rotavirus vaccine immunogenicity in rural Ghana

11:45 – 11:50 Shotgun presentation by Yuta Kanai, Takahiro Kawagishi, Misa Onishi, Pannacha Pimfhun, Ryotaro Nouda, Jeffery Nurdin, Keiichiro Nomura, Hiroshi Ushijima, Takeshi Kobayashi:Antigenicity of simian and human reassortant rotaviruses generated by reverse genetics

11:50 – 11:55 Shotgun presentation by Yi Jin, Maxime Ratinier, Siddharth Bakshi, Marco Caporale, Aislynn Taggart, Meredith Stewart, Massimo Palmarini:The African horse sickness virus NS4 counteracts the antiviral response and is a determinant of viral virulence

11:55 – 12:00 Shotgun presentation by Katherine Dulwich, Efstathios S. Giotis, Michael A. Skinner, Andrew J. Broadbent:The VP4 protein from a very virulent IBDV strain antagonises type I IFN responses, in contrast to the VP4 from a classical strain

316. Program

12:00 – 12:05 Shotgun presentation by Miguel Avia, Lisa Miorin, José Manual Rojas, Elena Pascual, Verónica Martín, Adolfo García-Sastre, Noemí Sevilla:Bluetongue virus uses the lysosome-autophagy pathway to degrade STAT2 and block IFN signaling

12:05 – 12:10 Shotgun presentation by Takahiro Kawagishi, Yuta Kanai, Yusuke Sakai, Ryotaro Nouda, Hideki Tani, Masayuki Shimojima, Masayuki Saijo, Yoshiharu Matsuura, Takeshi Kobayashi:Nelson Bay Orthoreovirus cell attachment protein σC determines strain-specific differences in viral replication and pathogenesis

12:10 – 13:15 LunchWorkshop 5: StructureConveners: Yizhi Tao and Vida Van Staden13:15 – 13:40 Plenary presentation by Aaron M. Collier, Outi L. Lyytinen, Jason

Kaelber, Yusong R. Guo, Yukimatsu Toh, Xiaoying Lei, Wah Chiu, Minna M. Poranen, Yizhi Tao: Structure and Function of Human Picobirnavirus

13:40 – 13:55 Oral presentation by Yasel Garcés Suárez, José Luis Martínez Guevara, David Torres Hernández, Haydee Olinca Hernandez Avina, Juan Manuel Rendón Mancha, Adán Oswaldo Guerrero Cárdenas, Susana Lopez Charreton, Carlos Federico Arias OrtizNanoscopic organization of rotavirus replication machineries by super resolution microscopy

13:55 – 14:10 Oral presentation by Alexander Borodavka, Eric C Dykeman, Waldemar Schrimpf, Don C Lamb:These can go up to eleven: shedding light on the mechanisms of multi-segmented genome assembly

14:10 – 14:15 Shotgun presentation by Liya Hu, Wilhelm Salmen, Banumathi Sankaran, Sasirekha Ramani, Mary Estes, B. V. Venkataram Prasad:Structural basis of glycan specificity in human rotaviruses

14:15 – 14:20 Shotgun presentation by Dilip Kumar, Xinzhe Yu, Sue E Crawford, Rodolfo Moreno, Anish Ramakrishnan, Liya Hu, Mary K Estes, Zhao Wang, B.V.Venkataram Prasad:Near-atomic resolution cryo-EM structure of Rotavirus capping enzyme VP3

14:20 – 14:25 Shotgun presentation by Adeline Kerviel, Mason Lai, Jonathan Jih, Peng Ge, Hong Zhou, Polly Roy:Atomic structure of translation regulatory protein NS1 of bluetongue virus in tubule form

14:25 – 14:55 Coffee break


14:55 – 15:20 Plenary presentation by Hong Zhou:Imaging viral RNA genomes and RNA transcription in action by cryoEM

15:20 – 15:35 Oral presentation by Courtney L. Steger, Crystal E. Boudreaux, Courtney A. Cohen, Sarah M. McDonald:Importance of Residues Surrounding +RNA Exit Tunnel Interface of the Rotavirus Polymerase for Genome Replication

15:35 – 15:50 Oral presentation by David Stuart, Mark Boyce, Corey Hecksel, Abhay Kotecha, Xiaofeng Fu, Geoff Sutton, Peijun Zhang:Dissection of the assembly pathway of Mammalian orthoreovirus using focused ion beam milling and in situ tomographic analysis of native assembly intermediates in infected cells

15:50 – 15:55 Shotgun presentation by José R. Castón, Carlos P. Mata, Daniel Luque, Nobuhiro Suzuki, Said A. Ghabrial:Structure of replicative cores of dsRNA fungal viruses

15:55 – 16:00 Shotgun presentation by Serban Ilca, Xiaoyu Sun, Kamel El Omari, Jonathan Grimes, David Stuart, Minna Poranen, Juha Huiskonen:Multiple Liquid Crystalline Geometries of Highly Compacted Nucleic Acid in a dsRNA Virus

16:00 – 16:30 Coffee breakWorkshop 6: Using and Abusing Host PathwaysConveners: Bernardo Mainou and Barbara Coulson16:30 – 16:55 Plenary presentation by Roy Duncan:

Structure, Function and Evolution of Reovirus Membrane Fusion Proteins

16:55 – 17:20 Plenary presentation by Michelle Arnold:Inhibition of IRF and NF-κB nuclear translocation by rotavirus NSP1

17:20 – 17:25 Shotgun presentation by Gayle Wall, Christiaan Potgieter, Fourie Joubert, Vida van Staden:African horse sickness virus non-structural protein NS4 suppresses host innate immunity

17:25 – 17:30 Shotgun presentation by Jonathan Knowlton, Isabel Fernández De Castro, Daniel Gestaut, Paula Zamora, Judith Frydman, Cristina Risco, Terence Dermody:The TRiC chaperonin is required for reovirus assembly

17:30 – 17:35 Shotgun presentation by Sarah Katen, John Patton:Comparative Activities of the Phosphodegrons for beta-TrCP in Rotavirus NSP1 and IkappaB

18:15 – 00:00 Dinner and dance

336. Program

DAY 5: 28 SEPTEMBER 2018Workshop 6: Using and Abusing Host PathwaysConveners: Bernardo Mainou and Barbara Coulson08:45 – 09:10 Plenary presentation by Alexandra L Chang-Graham., Jacob L.

Perry, Melinda A Engevik., Heather A. Danhof, Alicia C Strtak., Jeanette M. Criglar, Mary K. Estes, Joseph Hyser:Rotavirus Induces Intercellular Calcium Waves through Paracrine Purinergic Signaling from Infected Cells

09:10 – 09:25 Oral presentation by Pavithra Aravamudhan, Jennifer Konopka-Anstadt, Krishnan Raghunathan, Amrita Pathak, Bruce D Carter, Terence S Dermody:Reovirus uses macropinocytosis-mediated entry and fast axonal transport to infect neurons

09:25 – 09:40 Oral presentation by Urbi Mukhopadhyay, Mamta Chawla-Sarkar, Anupam Mukherjee:MicroRNAs, Autophagy, and Rotavirus Infection: New Insights in Pathogenesis and Therapy

09:40 – 09:55 Oral presentation by Jeanette Marie Criglar, Ramakrishnan Anish, Liya Hu, Sue Ellen Crawford, Banumathi Sankaran, B.V. Venkataram Prasad, Mary K Estes:Phosphorylation cascade regulates the formation and maturation of rotavirus viroplasms

09:55 – 10:30 Coffee breakWorkshop 7: Applied dsRNA VirologyConveners: Viviana Parreno and Maya Shmulevitz10:30 – 10:55 Plenary presentation by Don Morris:

Reovirus: Bench to Bedside and back again (this time with re-purposed therapeutics)

10:55 – 11:20 Plenary presentation by Viviana Parreño:IgY from chicken egg yolk and Camelid derived nanobodies: Two strategies to control rotavirus diarrhea in animals and humans

11:20 – 11:45 Plenary presentation by Minna Porannen: dsRNA bacteriophage as a tool for production of RNA-based treatments and crop protectants

11:45 – 12:00 Oral presentation by Celina Guadalupe Vega, Claudio Bernardo Santos, Lorena Laura Garaicoechea, Marina Bok, Lucía Rocha, Andres Wigdorovitz, Marcelo Criscuolo, Mauricio Berro, Viviana Parreño:Puna: the first transgenic cow expressing RVA-specific VHH antibodies in milk


12:00 – 12:15 Oral presentation by Jana Van Dycke, Francesca Arnoldi, Guido Papa, Justine Vandepoele, Oscar R. Burrone, Eloise Mastrangelo, Delia Tarantino, Elisabeth Heylen, Johan Neyts, Joana Rocha-Pereira:One nucleoside viral polymerase inhibitor that blocks the replication of norovirus, rotavirus and sapoviruses.

12:15 – 13:00 Executive Meeting13:00 – 14:00 LunchRotavirus Satellite Symposium14:00 – 14:20 Presentation by Umesh Parashar (Centers for Disease Control

and Prevention):Global Rotavirus Vaccine Introduction and Impact

14:20 – 14:40 Presentation by Bernd Benninghof (GlaxoSmithKline):Lessons learned from long term rotavirus vaccine impact: Belgium data from 2007 to 2017

14:40 – 15:00 Presentation by Esmée de Wolde (MSD): Update on the Pentavalent Human-Bovine (WC3) Rotavirus Vaccine

15:00 – 15:20 Presentation by Krishna Mohan, Ella Raches, Sai Prasad (Bharat Biotech International Ltd.):ROTAVAC® (nHRV) – Product Development and Programmatic Implementation

15:20 – 15:40 Coffee break15:40 – 16:00 Presentation by Carl Kirkwood (Bill and Melinda Gates

Foundation):The pipeline of live, attenuated and non replicating rotavirus vaccines

16:00 – 16:20 Presentation by Baoming Jiang (Centers for Disease Control and Prevention):Progress towards the development of an inactivated rotavirus vaccine

16:20 – 16:40 Presentation by Alan Fix, Michelle J. Groome, Lee Fairlie, Julie Morrison, Anthonet Koen, Maysseb Masenya, Nicola Page, Lisa Jose, Shabir A. Madhi, Monica McNeal, Len Dally, Iksung Cho, Maureen Power, Jorge Flores, Stanley Cryz (PATH):Safety and immunogenicity of a parenteral trivalent P2-VP8 subunit rotavirus vaccine

18:00 - 20:00 Dinner

356. Program

List of all poster presentations (including posters from shotgun presentations)Poster session 1: Viral DiversityP1: D. O Amadu, M.B Abdulkadir, S.A Saka, A Fadeyi, A.S Aderibigbe, D.B Tijani,

C. E. Nwabuisi, A.A Akanbi Ii, J.M. Nwenda, A. Isiaka., Armah George: Prevalence and circulating genotypes of rotavirus induced diarrhoea amongst under fives at the University of Ilorin Teaching Hospital (UITH), Ilorin, Nigeria

P2: Joseph Bonifacio, Mary Ann Igoy, Amado III Tandoc, Socorro Lupisan: Emergence of rare G2P[8] rotavirus genotype among children <5 years of age in the Philippines

P3: Michael D. Bowen, Mathew D. Esona, Charity Perkins, Slavica Mijatovic-Rustempasic, Geoffrey A. Weinberg, Amy Kinzler, John Williams, Gail A. Bloom, Mary Allen Staat, Monica M. McNeal, Mary E. Wikswo, Margaret M. Cortese, Daniel C. Payne, Umesh D. Parashar: Emergence of G8P[8] rotavirus strains of apparent Asian origin in four US cities, 2016-17

P4: Matias Castells, Dario Caffarena, Laura Casaux, Carlos Schild, Federico Giannitti, Daniel Castells, Franklin Riet-Correa, Viviana Parreño, Rodney Colina: Genetic diversity of bovine rotavirus in Uruguay: detection of common and uncommon genotypes

P5: Luis Eduardo Cottet, Christiaan Potgieter, Antonio Castillo: Molecular characterization of a new botybirnavirus that infects the phytopathogenic fungus Botrytis cinerea CCg427

P6: Susan Afua Damanka, Francis Ekow Dennis, Belinda Larteley Lartey, Chantal Ama Agbemabiese, Frederick Asamoah, George Enyimah Armah: VP4 Gene Polymorphism of human rotavirus and its impact on P-genotyping by RT-PCR

P7: Francis Ekow Dennis, Yen Hai Doan, Chantal Ama Agbemabiese, Belinda Larteley Lartey, Susan Afua Damanka, Frederick Karikari Asamoah, George Enyimah Armah, Kazuhiko Katayama: Detection and full genome characterization of human Group C rotavirus in Ghana

P8: Mathew D Esona, M. Leanne Ward, Mary E Wikswo, Rashi Gautam, Naga S Betrapally, Slavica Mijatovic-Rustempasic, Charity Perkins, Eric Katz, Jose Jaimes, Rangaraj Selvarangan, Christopher J Harrison, Julie A Boom, Janet Englund, Eileen J Klein, Mary Allen Staat, Monica M McNeal, Natasha Halasa, James Chappell, Geoffrey A Weinberg, Daniel C Payne, Parashar Umesh, Michael D Bowen: Comparative genomic analysis of genogroup 1 (Wa-like) and genogroup 2 (DS-1-like) rotaviruses circulating in seven US cities, 2013-2016.


P9: Shipra Gupta, Vasundhara Razdan Tiku, Satender Aneja, Praveen Kumar, Mohammad Islamuddin, Pratima Ray: Epidemiology and Genetic Diversity of Group A Rotavirus in Acute Diarrhea Patients (<5 Years) in Pre-vaccination Era in Delhi.

P10: Belinda Naa Larteley Lartey, Susan Afua Damanka, Francis Ekow Dennis, Chantal Ama Agbemabiese, Frederick Asamoah, George Enyimah Armah: Changes in Amino Acid Profiles in Antigenic Epitopes of the VP7 Protein of circulating Rotavirus Strains in Ghana

P11: Antonio Charlys da Costa, Elcio Leal, Flavio Augusto de Padua Milagres, Shirley Vasconcelos Komninakis, Rafael Brustulin, Maria da Aparecida Rodrigues Teles, Márcia Cristina Alves Brito Sayão Lobato, Rogério Togisaki das Chagas, Maria de Fátima Neves dos Santos Abrao, Cassia Vitória de Deus Alves Soares, Xutao Deng, Eric Delwart, Ester Cerdeira Sabino, Adriana Luchs: Complete genome sequence of two cucumis melo endornavirus strains discovery during deep sequencing investigation in human stool samples in Brazil

P12: Monica Malone McNeal, Amy Singh, Victoria Fabry, Nicole Meyer, Rebecca Grais, Celine Langendorf, Shelia Isanaka, Ousmane Guindo: Epidemiology of Rotavirus Strains Circulating in Niger, Africa from 2014 to 2016

P13: Tina Mikuletič, Danijela Rihtarič, Peter Hostnik, Nataša Toplak, Simon Koren, Urška Kuhar, Urška Jamnikar-Ciglenečki, Denis Kutnjak, Andrej Steyer: Identification of novel reassortant mammalian orthoreovirus serotypes 1, 2, 3 from bats in Slovenia

P14: Milton Tshidiso Mogotsi, Peter Nthiga Mwangi, Hester Trudi O’Neill, Martin Munene Nyaga: Genomic investigation of the faecal RNA virome in children from Oukasie clinic, North West province, South Africa

P15: Eva Dora João, Amy Strydom, Benilde Munlela, Martin Nyaga, Assucênio Chissaque, Jerónimo Langa, Ezequias Sitoe Sitoe, Jorfélia Chilaúle, Elda Anapakala, Júlia Sambo, Esperança Guimarães, Diocreciano Bero Bero, Marta Cassocera, Lena Manhique, José Paulo Langa, Idalécia Cossa-Moiane, Isabel Maurício, Hester G. O’Neill, Nilsa de Deus: Whole genome characterization of rotavirus strains from National Rotavirus Surveillance of acute diarrhea in Mozambique

P16: Martin Nyaga, Mapaseka Seheri, Duncan Steele, Jason Mwenda, Jeffery Mphahlele: Whole-genome sequencing and analyses identifies high genetic heterogeneity and diversity of rotavirus genotype P[6] strains circulating in Africa

P17: Shipra Gupta, Sanjeev Chaudhary, Pratima Ray: G and P Types of Circulating Rotavirus Strains in Himachal Pradesh, India During 2013-2016: Three Years of Prevaccine Data

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P18: Chenyan Shi, Raul Yhossef Tito Tadeo, Leen Beller, Ward Deboutte, Claire Belkhou, Jeroen Raes, Jelle Matthijnssens: Characterization of human gut virome among populations in four regions of Peru with different urbanization level

P19: Julia Rezende Silva, Elaine Hellen Nunes Chagas, Renato da Silva Bandeira, Bruno de Cássio Veloso Barros, Ceyla Maria Oeiras Castro, Delana Andreza Melo Bezerra, Joana D’Arc Pereira Mascarenhas: Picobirnavirus in humans and livestock animals from areas of environmental change in Brazil

P20: Ana Carolina Silva Serra, Delana Andreza Melo Bezerra, Jonas França Cruz, Patrícia Santos Lobo, Sylvia de Fátima dos Santos Guerra, Joana d’Arc Pereira Mascarenhas, Luana da Silva Soares: Molecular analysis of G3P[6] rotavirus collected from children with acute gastroenteritis in the Brazilian Amazon region

P21: Amy Strydom, Eva D Joao, Martin M Nyaga, Christiaan A Potgieter, Assa Cuamba, Inacia Mandomando, Marta Cassocera, Nilsa de Deus, Hester G O’Neill: Whole genome characterisation of Rotavirus A circulating in southern Mozambique during 2012-2013 reveals diverse strains and several reassortment events

P22: Gopalkrishna Varanasi: Rotavirus: Genotype distribution and changing trends in children with acute gastroenteritis: A study from western India

P23: Kinda Alshaikhahmed, Po-Yu Sung, Polly Roy: RNA-RNA interactions hold the key to assortment in the multipartite dsRNA virus

P24: Juan Ignacio Degiuseppe, Juan Andrés Stupka: Genetic diversity of Group A Rotavirus in Latin America before and after universal vaccination, 2001-2017

P25: Frances Katen Shepherd, Fangzhou Chen, Michael Murtaugh, Douglas Marthaler: In silico analysis of the VP7 for porcine rotavirus B and C illustrates genotype-specific antigenic epitopes

P26: Josh Singer, Rob Gifford, Kyriaki Nomikou, Alexander MacLeod, Peter Mertens, Massimo Palmarini: BTV-glue: a web-based bioinformatics resource for Bluetongue Virus sequence data

P27: Samuel Miño, Matías Adúriz, Viviana Parreño: Rotavirus species tropism: new insights on P-type classification

P28: Leen Beller, Ward Deboutte, Chenyan Shi, Daan Jansen, Claude Kwe Yinda, Mark Zeller, Marc Van Ranst, Jelle Matthijnssens: Exploring the diversity and dynamics of the gut virome of infants during the first year of life


P29: Øystein Wessel, Turhan Markussen, Torstein Tengs, Dhamotharan K, Salman Malik, Maria Dahle, Espen Rimstad: Piscine orthoreovirus (PRV); in vivo propagation in erythrocytes enables virus purification, causality to important disease in farmed Atlantic salmon and protection by inactivated vaccine

Poster session 2: Evolution and EpidemiologyP30: Irfan Ahmad, Anjum Afshan, Aasifa Jan, Taniya Kirmani:

Molecular epidemiology of rotavirus gastroenteritis in Kashmir Himalaya. Tourism and climatic disturbance - A potent platform for transmission.

P31: Ernest Wandera Apondi, Satoshi Komoto, Shah Mohammad, Martin Bundi, James Nyangao, Cyrus Kathiiko, Amina Galata, Sora Guyo, Gabriel Miring’u, Tomihiko Ide, Koki Taniguchi, Yoshio Ichinose: Increased Detection of G3P[6] Human Rotaviruses after Introduction of Rotavirus Vaccination in Kenya

P32: Mouna Ben Hadj Fredj, Haifa Bennour, Asma Bouazizi, Amira Jerbi, Meriam Ben Hamida-Rebai, Besma Abdi, Samia Lakhal, Saoussen Kacem, Zouhour Fekih, Imene Fodha, Jelle Matthijnssens, Abdelhalim Trabelsi: Distribution of NSP4 genotypes of group a rotavirus strains circulating in Tunisian children over a 10 years period (2006 to 2016)

P33: Haifa Bennour, Imene Fodha, Asma Bouazizi, Amira Jerbi, Samia Lakhal, Mouna Ben Hadj Fredj, Meriam Ben Hamida-Rebai, Ouafa Kallala, Saoussen Kacem, Zouhour Fekih, Akila Mili, Noureddine Boujaafar, Abdelhalim Trabelsi: Characterization of group A rotavirus strains identified in Tunisian adults and children more than 5 years old (2015 to 2017)

P34: Angeline Yvette Boula, Rose Ngoh, Mina Njiki Kinkela, Grace Kemajou, Roger Ngoya, Eric Nkolo, Franky Baonga Ba Pouth, Marcellin Nimpa, Jason Mwenda, Paul Koki Ndombo: Rotavirus genotype distribution after Rotavirus vaccine introduction in Cameroon

P35: Elaine Hellen Nunes Chagas, Júlia Rezende Silva, Bruno de Cássio Veloso Barros, José Wandilson Barboza Duarte Junior, Delana Andreza Melo Bezerra, Edivaldo Costa Sousa Júnior, Renato da Silva Bandeira, Sylvia de Fátima dos Santos Guerra, Ceyla Maria Oeiras de Castro, Joana D’Árc Pereira Mascarenhas: Picobirnavirus and Reovirus in faecal samples of animals, mammals and birds, from areas of environmental change of Brazil

P36: Juan Ignacio Degiuseppe, Vanina Sol Eibar, Sandra Fabiana Grucci, Juan Andrés Stupka: Unexpected emergence of novel Group A Rotavirus G9P[4] in Mendoza, Argentina

P37: Shuvra Kanti Dey, Sadia Farzana Sifat, Md. Almamun, Nadim Sharif, Nasir Uddin Nobel, Sadia Nowshin Sany, Md. Baki Billah: High prevalence and changing genotyping pattern of rotavirus infection among Bangladeshi pediatric patients

396. Program

P38: Imene Fodha Bouzgarrou, Anissa Chouikha, Mouna Ben Hadj Fredj, Amal Moussa, Haifa Bennour, Meriam Ben Hamida, Asma Bouazizi, Abdelhalim Trabelsi, Andrew Duncan Steele: Tunisian Rotavirus Surveillance (1995-2017): A Brief Overview.

P39: Fabián Gómez-Santiago, Atenea Estela Andrés, Sergio Issac de la Cruz, Heidi Terán, Héctor Méndez, Herlinda García: Molecular characterization of a group A rotaviruses in Mexico during 2018, emergence of G3P[8] strain variants.

P40: Madhuri Shantanu Joshi, Shalu Akash Arya, Atul Madhukar Walimbe, Varanasi Gopalkrishna: Rotavirus C: Genetic evolutionary dynamics based on VP3 and VP7 Genes, and Detection in Fecal Specimens of Pigs from Western India

P41: C. Eures Iyar Garcia Lazaro, Joseph Bonifacio, Mary Ann Igoy, Amado Tandoc III: Etiology of non-rotavirus-associated gastroenteritis among hospitalized children under five years of age in the Philippines, 2015-2016

P42: Adriana Luchs, Antonio Charlys da Costa, Audrey Cilli, Shirley Cavalcante Vasconcelos Komninakis, Lais Boen, Rita de Cassia Compagnoli Carmona, Simone Guadagnucci Morillo, Ester Cerdeira Sabino, Maria do Carmo Sampaio Tavares Timenetsky: Spread of the emerging equine-like G3P[8] ds-1-like genetic backbone rotavirus strain in Brazil

P43: Marcelo Gastón Mandile, Estefanía Peri Ibañez, Marcelo Argüelles, Facundo Temprana, Dalila Silvestre, Alicia Mistchenko, Graciela Glikmann, Alejandro Castello: Surveillance of Group A Rotavirus in Buenos Aires, Argentina after the introduction of the monovalent vaccine and the study of a rare strain in an unusual immune environment.

P44: Iara Magaly Martinez, Maria Eugenia Galeano, Graciela Russomando, Gabriel Ignacio Parra: Differences in VP6 Predict Restriction on the Reassortment of Co-circulating AU-1-like and Wa-like Rotaviruses Strains

P45: Fabiolla da Silva dos Santos, Edivaldo Costa Sousa Junior, Sylvia de Fátima dos Santos Guerra, Patrícia dos Santos Lobo, Edvaldo Tavares Penha Junior, Ana Beatriz Figueiredo Lima, Caio Breno Gomes Vinente, Maria Cleonice Aguiar Justino, Alexandre da Costa Linhares, Luana da Silva Soares, Joana D’Arc Pereira Mascarenhas: G1P[8] Rotavirus in childrens with acute diarrhea after vaccine introduction in Brazil: evidence of reassortments and structural modifications in VP7 and VP4 genes

P46: Sarita Yadav, Kyriaki Nomikou, Sushila Maan, Narender Maan, Massimo Palmarini, Peter Paul Clement Mertens: Isolation and Molecular Characterization of Bluetongue Virus Isolates from Southern India


P47: Sushila Maan, Narender Singh Maan, Sunil Kumar Mor, Nitish Bansal, Deepika Chaudhary, Panduranga P. Rao, Peter Paul Clement Mertens: Whole genome sequence-based characterisation of bluetongue virus serotype 5 from India

P48: Amani Rebecca N’Guessan-Kouame, Marie-Helene Ake-Assi-Konan, Catherine Boni, Flore Zaba, Hamidou Kone, Nicaise Aka, Laurence Adonis-Koffy: Epidemiological and clinical aspects of gastroenteritis in the pediatrics departement of teaching hospital of Yopougon one year after introduction rotateq vaccine (abidjan rci)

P49: Gaston Eric Nkolo Mviena, Angeline Boula, Rose Ngoh, E. Mugyia Akongnwi, Mina Njiki Kinkela, Grace Kemajou, Roger Ngoya Ebiguide, Ba Pouth Baonga, Marcellin Nimpa, Jason Mwenda, Paul Koki Ndombo: Norovirus Co-infection with Rotavirus in Yaoundé,Cameroon

P50: Kyriaki Nomikou, Ana Da Silva Filipe, Roman Biek, Joseph Hughes, Sreenu Vattipally, Stephan Zientara, Emmanuel Breard, Damien Vitour, Bernd Hoffmann, Giovani Savini, Piet van Rijn, Peter Mertens, Massimo Palmarini: BTV-8 in Europe: direct sequencing of the full viral genome from clinical samples

P51: Oluwole Oyetunde Oni, Ademola Amubieya Owoade, Christopher Adeyinka Adeyefa: Association of rotavirus gastroenteritis with bacterial infection in poultry birds.

P52: Verónica Emilse Prez, Laura Cecilia Martínez, Gisela Masachessi, Miguel Oscar Giordano, Patricia Angélica Barril, Silvia Viviana Nates: Rotavirus and enterovirus: through irrigation waters to green vegetables.

P53: Ngoya Roger, Boula Angeline, Kemajou Grace, Nkolo Eric, Ngoh Rose, Koki Paul: Epidemiological, clinical and virological aspects of Rotavirus diarrhea in Yaounde-Cameroon (2014-2016)

P54: Kuldeep Sharma, Nitish S Parihar, Arti Shrivas, Vaibhav K Tamrakar, Jyothi Bhat: Development of a novel, visual Polymerase Spiral Reaction for detection of Rotavirus C

P55: Vaishali Tatte, Deepthy Maran, Gopalkrishna Varanasi: Genetic characterization of structural and non-structural genes of unusual rotavirus strains associated with acute gastroenteritis

P56: Celina Guadalupe Vega, Marina Bok, Juan Ignacio Degiuseppe, Alejandra Antonella Rivolta, Ana Paula Piantanida, Matías Adúriz Guerrero, Gustavo Asenzo, Andrés Wigdorovitz, Juan Stupka, Lorena Laura Garaicoechea, Viviana Parreño: Rotadial: the first RVA diagnostic kit based on patented VHH antibodies

P57: Zoe Yandle, Suzie Coughlan, Cillian De Gascun: Detection of rotavirus and distribution of genotypes before and after the introduction of Rotarix in Ireland

416. Program

P58: Sebastiaan Theuns, Quinten Bernaert, Philip Vyt, Hans J Nauwynck: Intriguing successive (sub)clinical shedding patterns of different rotavirus genotypes in suckling and weaned pigs

P59: Mohamed Lemine Cb.Ahmed, Abdellahi Weddih, Mohemed Abdellahi Boullah, Meriam Sidatt, Abdelkarim Filali-Maltouf, Mohammed Benhafid: Rotavirus among children aged 0-5 years in Nouakchott Mauritania

P60: Tohru Suzuki: Genetic characteristics of porcine rotaviruses H detected recently in Japan

P61: Frances Katen Shepherd, Michael Murtaugh, Douglas Marthaler: Genetic variability and implications for identifying neutralizing epitopes in swine rotavirus A strains

P62: Samuel Miño, María Barrandeguy, Viviana Parreño: Worldwide evolution of equine RVA, four voices, one story

P63: Ward Deboutte, Leen Beller, Claude Kwe Yinda, Nádia da Conceição Neto, Piet Maes, Dirk C de Graaf, Jelle Matthijnssens: A viral metagenomics view on the honeybee virome reveals a plethora of undescribed dsRNA viruses

P64: Hiroaki Shirafuji, Natsumi Kobayashi, Katsunori Murota, Shogo Tanaka, Tohru Yanase: Epizootic hemorrhagic disease virus identified in affected cattle in Japan during 2015−2017

P65: Chantal Ama Agbemabiese, Toyoko Nakagomi, Susan Afua Damanka, Francis Ekow Dennis, Belinda Larteley Lartey, Frederick Karikari Asamoah, Michael Fokuo Ofori, George Enyimah Armah, Osamu Nakagomi: A proposal for the lineage framework for sub-genotype phylogeny of the backbone genes of DS-1-like Rotavirus A strains.

P66: Isidore Juste O. Bonkoungou, Ylva Rönnelid, Nafissatou Ouedraogo, Nicolas Barro, Lennart Svensson, Johan Nordgren: Predominance of norovirus in children hospitalized with gastroenteritis after rotavirus vaccine introduction in Burkina Faso

P67: Syun-suke Kadoya, Syun-ichi Urayama, Takuro Nunoura, Masaaki Kitajima, Satoshi Okabe, Toyoko Nakagomi, Osamu Nakagomi, Daisuke Sano: Neutral Evolutionary Rate of Rhesus Rotavirus

P68: Daniel E Velasquez Portocarrero, Baoming Jiang: Comparative analysis of trivalent P2-VP8 subunit rotavirus vaccine strains and P[8], P[4], and P[6] human rotaviruses reported globally during 1974 and 2017

Poster session 3: Molecular and Cellular VirologyP69: Carolina Allende-Ballestero, Carlos P. Mata, José R. Castón:

Analysis of ribonucleoprotein complexes of IBDV


P70: Alice Gray, Andrew James Broadbent: Determining the dynamics of birnavirus replication organelle movement in vitro using a split-GFP tagged infectious bursal disease virus (IBDV).

P71: Oscar R. Burrone, Catherine Eichwald, Giuditta De Lorenzo, Elisabeth Schraner, Guido Papa, Michela Bollati, Paolo Swec, Matteo de Rosa, Mario Milani, Eloise Mastrangelo, Mathias Ackermann, Francesca Arnoldi: A novel rotavirus inhibitor impairs the structural integrity of rotavirus particles and viroplasms

P72: Durga Rao Chilakalapudi, Varsha Tandra, Poonam Dhillon: Cytoplasmic re-localization and colocalization with viroplasms of host nuclear and cytoplasmic proteins, and their role in rotavirus infection

P73: Sue Ellen Crawford, Sarah E. Blutt, Sasirekha Ramani, Daniel R. Laucirica, Winnie Zou, Xi-Lei Zeng, Umesh Karandikar, James Broughman, Mary K. Estes: A New Form of Human Rotavirus Produced from Human Intestinal Enteroids

P74: Sue Ellen Crawford, Zheng Liu, Jeanette Criglar, Mary K. Estes: The Role of DGAT1 in Rotavirus Viroplasm/Lipid Droplet Formation

P75: Selene Glück, Antonino Buttafuoco, Anita F Meier, Francesca Arnoldi, Vogt Bernd, Elisabeth M Schraner, Mathias Ackermann, Catherine C Eichwald: Rotavirus replication is correlated with S/G2 interphase arrest of the host cell cycle.

P76: Catherine C Eichwald, Mathias Ackermann, Max L Nibert: The dynamics of both filamentous and globular mammalian reovirus viral factories rely on the microtubule network.

P77: Michael Burwinkel, Alexander Falkenhagen, Eva Trojnar, Reimar Johne: Adaptive mutations occurring during serial passages of an avian/mammalian rotavirus reassortant in cell culture

P78: Linda Ferreira-Venter, Eudri Venter, Vida van Staden: Unravelling the complexity of AHSV NS3: a targeted investigation of its functional domains.

P79: Thomas Labadie, Sophie Jegouic, Polly Roy: Polybasic motif within non-structural protein 3 of Bluetongue virus is important for cell release

P80: Delphine Lanoie, Emmanuelle Degeorges, Stéphanie Côté, Véronique Sandekian, Guy Lemay: Combining classical and reverse genetics to study the multiple proteins of mammalian orthoreoviruses affecting induction of and sensitivity to the interferon response

P81: Simon Boudreault, Martin Bisaillon, Guy Lemay: Modifications to the cellular transcriptome and alternative RNA splicing after mammalian orthoreovirus infection

436. Program

P82: Gabriela Llauger, Demián Esteban Monti, Carlos María Figueroa, Alberto Álvaro Iglesias, Mariana del Vas: Mal de Río Cuarto virus minor viroplasm component P6 interacts with an atypical wheat thioredoxin

P83: Outi L Lyytinen, Daria A Starkova, Minna M Poranen: Pseudomonas Phage Phi6 Envelope Formation and Phi6-Specific Vesicle Production in Escherichia coli

P84: Eiko Matsuo, Marina Hamaji, Hiroko Omori, Hideyuki Tsuji, Akari Saito, Polly Roy, Keiichi Saeki, Takeshi Kobayashi, Junichi Kawano: Further analysis of Ibaraki virus VP6 to produce fluorescence-labeled orbiviruses

P85: Fortunate Mokoena, Mapaseka Seheri, Martin Nyaga, Nonkululeko Magagula, Arnold Mukaratirwa, Augustine Mulindwa, Almaz Abebe, Angelina Boula, Tsolenyanu Enyonam, Kebareng Rakau, Ina Peenze, Jason Mwenda, Jefferey Mphahlele: Whole genome analysis of African G12P[6] and G12P[8] rotavirus strains with evidence of porcine-human reassortment at NSP3 and NSP4 genes

P86: Guido Papa, Francesca Arnoldi, Giuditta De Lorenzo, Gianluca Petris, Marco Bestagno, Oscar R. Burrone: Development of a novel reverse genetics system for Rotavirus

P87: Jin Dai, Asha Ann Philip, Chantal A Agbemabiese, Guido Papa, John Patton: Impaired growth phenotype of recombinant rotaviruses expressing VP3 with a defective 2’,5’-phosphodiesterase (PDE)

P88: Asha A Philip, Jin Dai, Chantal A Agbemabiese, Guido Papa, Promisree Choudhury, Sarah Katen, John Patton: Modifications of the Rotavirus Plasmid-Based Reverse Genetics System

P89: Mariela Duarte, Patrice Vende, Annie Charpilienne, Cécile Laroche, Didier Poncet: Rotavirus infection induces alternative splicing of the stress-regulated transcription factor XBP1.

P90: Kebareng Rakau, Maemu Gededzha, Mapaseka Seheri: Molecular characterization of rotavirus infections in children attending Dr George Mukhari Academic Hospital and Oukasie Primary Healthcare and their association with histo-blood group antigen profiles

P91: Sujata Ranshing, Ashish Bavdekar, Umesh Vaidya, Manas Behera, Gopalkrishna Varanasi: Molecular Characterization of Unusual Rotaviruses G12P[11] Strain Identified in Neonates Admitted at Neonatal Intensive Care Units (NICUs) : A study from Pune, Western India

P92: Carlos Sandoval-Jaime, Carlos Arias, Susana López: Detection of Rotavirus NSP3 interactions during infection

P93: Litia Yssel, Eudri Venter, Vida van Staden: Investigating the interaction of the viral inclusion bodies formed during African horse sickness virus infection with the host protein synthesis apparatus


P94: Shani May Bekker, Christiaan Potgieter, Vida van Staden, Jacques Theron: The use of a soluble African horse sickness virus VP7 protein to study virus replication and vaccine development

P95: Durga Rao Chilakalapudi, Poonam Dhillon: Viroplasms and remodelled stress granules and P-bodies: Union of the triad to promote progeny rotavirus production

P96: Po-yu Sung, Cheng Kao, Polly Roy: Mapping the RNA binding and packaging motifs of Bluetongue virus capsid protein VP6 by biophysical and molecular analysis

P97: Eric N. Salgado, Brian Garcia Rodriguez, Nagarjun Narayanaswamy, Yamuna Krishnan, Stephen C. Harrison: Visualization of Ca2+ loss from rotavirus during cell entry

P98: Marc Guimera Busquets, Gillian Pullinger, Massimo Palmarini, Karin E. Darpel, Rennos Fragkoudis, Peter P. C. Mertens: Molecular determinants of bluetongue virus that drive replication in the insect vector cell

P99: Marno GJ Huyzers, Abraham Christiaan Potgieter, Louis Jeremia Cornelius Theart, Alberdina Aike van Dijk: Local implementation and optimisation of entirely plasmid-based SA11 rotavirus reverse genetics

P100: Joerg Bormann, Cornelia Heinze, Christine Blum, Michael Mentges, Anke Brockmann, Arne Alder, Svenja Kim Landt, Brian Josephson, Daniela Indenbirken, Michael Spohn, Birte Plitzko, Sandra Loesgen, Michael Freitag, Wilhelm Schäfer:Expression of a structural protein of the mycovirus FgV-ch9 negatively affects the transcript level of a novel symptom alleviation factor and causes virus-infection like symptoms in Fusarium graminearum

P101: Anirban Kundu, Amit Kumar Das, Ananta Kumar Ghosh: Molecular mechanism of polymerase activity and RNA-binding properties of E. coli expressed different domains of Antheraea mylitta cytoplasmic polyhedrosis virus RNA-dependent RNA polymerase

Poster session 4: Immunity and PathogenesisP102: Davide Agnello, Damien Denimal, Amandine Lavaux, Cecile Pitoiset,

Pierre Pothier, Alexis de Rougemont: Asymmetric recirculation of rotavirus-specific memory B cells induced by intra-rectal immunization with 2/6-virus-like particles (2/6-VLPs) throughout the gut-associated lymphoid tissues.

P103: Sarah Caddy, Leo James: Intracellular neutralisation of rotavirus by antibodies

P104: Tanner Gill, Geoffrey Holm: Identification of Interferon Stimulated Genes That Restrict Reovirus Infection

456. Program

P105: Gennaro Iaconis, Miren Iturriza-Gomara, Mark Boyce, Steve Goodbourn, Neil Blake, Ben Jackson, Julian Seago: Characterization of molecular targets for differential regulation of the type I and III interferon induction and signalling pathways by rotavirus NSP1

P106: Fidan Karatas, Wilma Fick: Investigating the role of akirin, a conserved transcription factor of arthropods, in the pathogenesis of C. sonorensis derived KC cells caused by African Horse Sickness Virus infection

P107: Tina Mikuletič, Tanja Švara, Mitja Gombač, Marko Kolenc, Andrej Steyer: Bat MRV isolate SI-MRV01 dissemination after oral inoculation of neonatal BALB/c mice

P108: Médard Ngoy Kiluba: Choice of vaccine and study of the careful use of pharmacy during the introduction of rotavirus vaccine in DRC

P109: Larise Oberholster, Jasmin Aschenbrenner, Christiaan Potgieter, Hester Gertruida O’Neill: Evaluation of recombinant Newcastle Disease Viruses (NDV) as candidate vaccine delivery vectors for rotavirus VP7 and NSP4 in mice

P110: Alejandro Marin-Lopez, Eva Calvo-Pinilla, Diego Barriales, Gema Lorenzo, Sergio Utrilla-Trigo, Ruyman Alonso-Ravelo, Alejandro Brun, Juan Anguita, Javier Ortego: CD8 T cell responses to an immunodominant epitope within the non-structural protein NS1 provides wide immunoprotection against bluetongue virus in ifnar(-/-) mice

P111: Eva Calvo-Pinilla, Alejandro Marín-López, Natalia Barreiro-Piñeiro, Diego Barriales, Sergio Utrilla-Trigo, Ruyman Alonso-Ravelo, Javier Benavente, Alejandro Brun, José Manuel Martínez-Costas, Javier Ortego: Cross-protective immune responses against ashv after vaccination with avian reovirus muns microspheres and modified vaccinia virus ankara

P112: Rui Qiao, Yu Wang: A complement factor NcTEP3 transcriptional regulated by Toll pathway inhibits the RDV infection of leafhopper by inducing antimicrobial peptides

P113: Sasirekha Ramani, Christopher Stewart, Daniel Laucirica, Nadim Ajami, Liya Hu, B. V. Venkataram Prasad, Lars Bode, Gagandeep Kang, Mary Estes: Modulation of Neonatal Rotavirus Infection by Interplay between Human Milk Oligosaccharides, Milk Microbiome and Infant Gut Microbiome

P114: Jesús Rodríguez-Díaz, Antonio Rubio-del-Campo, Javier Buesa, Roberto Gozalbo-Rovira, Susana Vila-Vicent, Cristina Santiso-Bellón, Delgado Susana, Natalia Molinero, Abelardo Margolles, Vicente Monedero, Maria Carmen Collado: Determination of rotavirus binding bacteria by fluorescence-activated cell sorting and next generation sequencing.


P115: Linda J. Saif, Ayako Miyazaki, Sukumar Kandasaamy, Husheem Michael, Stephanie Langel, Francine Paim, Juliet Chepngeno, Moyasar Alhamo, David Fischer, Huang-Chi Huang, Vishal Srivastava, Dipak Kathayat, Loic DeBlais, Gireesh Rajashekara, Anastasia Vlasova: Protein deficiency reduces the efficacy of an oral attenuated human rotavirus vaccine in a human infant fecal microbiota transplanted gnotobiotic pig model

P116: Wico Sander, Trudi G O’Neill, Carlien H Pohl: The effect of supplementation of fatty acids, with varying degrees of saturation, on rotavirus yield and replication in MA104 cells

P117: Adrish Sen, Harry Greenberg: Rotavirus regulates the STAT1 antiviral response in infected and uninfected bystander cells by two discrete mechanisms.

P118: Piet A. van Rijn, Femke Feenstra, Rene G.P. van Gennip: A novel vaccine approach for insect borne diseases; Disabled Infectious Single Animal (DISA) vaccine for Bluetongue

P119: Celina Guadalupe Vega, Marina Bok, Maren Ebinger, Lucia Rocha, Verónica Pinto, Christian Knapp, Alejandra Antonella Rivolta, Viviana Parreño, Andrés Wigdorovitz Bioinnovo IgY DNT: towards the end of newborn calf diarrhea

P120: Daniel E Velasquez Portocarrero, Paulina Rebolledo, Umesh Parashar, Baoming Jiang, Juan Leon: Pre-vaccination rotavirus serum IgA and IgG, and stunting are associated with low rates of seroconversion to Rotarix® at 1 year of age among Bolivian infants

P121: Lijuan Yuan: Using gnotobiotic (Gn) pig model for pre-clinical evaluation of candidate rotavirus vaccines

P122: Piet A. van Rijn, Sandra G.P. van de Water, Christiaan A. Potgieter, Rene G.P. van Gennip: Disabled Infectious Single Animal (DISA) vaccine for African Horse Sickness

P123: Siyuan Ding, Peter K. Jackson, John T. Patton, Jia Liu, Harry B.Greenberg: Interferon-stimulated gene SAMD9 restricts rotavirus replication and is counteracted by non-structural protein 1

P124: Megan Stanifer, Stephanie Muenchau, Dorothee Albrecht, Sina Bartfeld, Franck van Kuppeveld, Takashi Kanaya, Steeve Boulant: Polarised interferon-mediated immune response against mammalian reovirus reveal novel mechanisms of immune tolerance in the human gut

P125: Lyndsay Cooke, Katy Moffat, Laura Tugwell, Nicolas Locker, Geraldine Taylor, Karin Darpel: Bacterial products within Culicoides midge saliva enhance Bluetongue virus infection in bovine monocytes

476. Program

P126: Yuta Kanai, Takahiro Kawagishi, Misa Onishi, Pannacha Pimfhun, Ryotaro Nouda, Jeffery Nurdin, Keiichiro Nomura, Hiroshi Ushijima, Takeshi Kobayashi: Antigenicity of simian and human reassortant rotaviruses generated by reverse genetics

P127: Yi Jin, Maxime Ratinier, Siddharth Bakshi, Marco Caporale, Aislynn Taggart, Meredith Stewart, Massimo Palmarini: The African horse sickness virus NS4 counteracts the antiviral response and is a determinant of viral virulence

P128: Katherine Dulwich, Efstathios S. Giotis, Michael A. Skinner, Andrew J. Broadbent: The VP4 protein from a very virulent IBDV strain antagonises type I IFN responses, in contrast to the VP4 from a classical strain

P129: Miguel Avia, Lisa Miorin, José Manual Rojas, Elena Pascual, Verónica Martín, Adolfo García-Sastre, Noemí Sevilla: Bluetongue virus uses the lysosome-autophagy pathway to degrade STAT2 and block IFN signaling

P130: Takahiro Kawagishi, Yuta Kanai, Yusuke Sakai, Ryotaro Nouda, Hideki Tani, Masayuki Shimojima, Masayuki Saijo, Yoshiharu Matsuura, Takeshi Kobayashi: Nelson Bay Orthoreovirus cell attachment protein σC determines strain-specific differences in viral replication and pathogenesis

Poster session 5: StructureP131: Deepti Thete, Anthony Snyder, Pranav Danthi:

Mu1 protein dependent aggregation of reovirus particles promotes reassortment

P132: Alvaro Ortega-Esteban, Carlos P. P. Mata, M. Jesús R. Espinosa, Pedro J. de Pablo, José R. Castón: Structural and biophysical analysis of human picobirnavirus capsid

P133: Liya Hu, Wilhelm Salmen, Banumathi Sankaran, Sasirekha Ramani, Mary Estes, B. V. Venkataram Prasad: Structural basis of glycan specificity in human rotaviruses

P134: Dilip Kumar, Xinzhe Yu, Sue E Crawford, Rodolfo Moreno, Anish Ramakrishnan, Liya Hu, Mary K Estes, Zhao Wang, B.V.Venkataram Prasad: Near-atomic resolution cryo-EM structure of Rotavirus capping enzyme VP3

P135: Adeline Kerviel, Mason Lai, Jonathan Jih, Peng Ge, Hong Zhou, Polly Roy: Atomic structure of translation regulatory protein NS1 of bluetongue virus in tubule form

P136: José R. Castón, Carlos P. Mata, Daniel Luque, Nobuhiro Suzuki, Said A. Ghabrial: Structure of replicative cores of dsRNA fungal viruses


P137: Serban Ilca, Xiaoyu Sun, Kamel El Omari, Jonathan Grimes, David Stuart, Minna Poranen, Juha Huiskonen: Multiple Liquid Crystalline Geometries of Highly Compacted Nucleic Acid in a dsRNA Virus

Poster session 6: Using and Abusing Host PathwaysP138: Fauziah Mohd Jaafar, Pierre Olivier Vidalain, Damien Vitour, Stephan

Zientara, Peter P.C. Mertens, Houssam Attoui: NS4 and NS5 of orbiviruses: modulation of pathways in insect and mammalian cells

P139: Shareen Boughan, Christiaan Potgieter, Vida van Staden: Nuclear localisation of African horse sickness virus non-structural protein NS4 differs across strains

P140: Cindy Kundlacz, Aurore Fablet, Rayane Amaral Da Silva Moraes, Marie Pourcelot, Cyril Viarouge, Corinne Sailleau, Axel Gorlier, Emmanuel Breard, Stephan Zientara, Damien Vitour, Grégory Caignard: Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway

P141: Andrew McNamara, Pranav Danthi: Reovirus inhibits NF-kappaB signaling in infected cells by at least two different mechanisms

P142: Luis Alejandro de Haro, Sofía Maité Arellano, Ondrej Novák, Regina Feil, Analía Delina Dumón, María Fernanda Mattio, Danuše Tarkowská, Gabriela Llauger, Miroslav Strnad, John Lunn, Stephen Pearce, Carlos María Figueroa, Mariana del Vas: Integrative hormone, metabolomic and transcriptomic profiling reveal hormonal imbalance and strong sugar partitioning alteration in Mal de Rio Cuarto virus-infected wheat

P143: Angela K Berger, Alison W Ashbrook, Maximilian H Carter, Karla D Passalacqua, Kristen M Ogden, Charles M Rice, Christiane E Wobus, Bernardo A Mainou: Loperamide, a mu-opioid receptor agonist, inhibits reovirus infection

P144: Urbi Mukhopadhyay, Shampa Chanda, Mamta Chawla-Sarkar: Rotavirus hinders antiviral RNA interference by degradation of Argonaute-2

P145: Upayan Patra, Urbi Mukhopadhyay, Mamta Chawla-Sarkar: Rotavirus infection down-regulates redox-sensitive transcription factor Nrf2 and Nrf2-driven transcription units

P146: Ané Pieters, Vida van Staden: Investigating cytosolic conveyance of newly synthesised mature African horse sickness virus particles

P147: Gayle Wall, Christiaan Potgieter, Fourie Joubert, Vida van Staden: African horse sickness virus non-structural protein NS4 suppresses host innate immunity

496. Program

P148: Jonathan Knowlton, Isabel Fernández De Castro, Daniel Gestaut, Paula Zamora, Judith Frydman, Cristina Risco, Terence Dermody: The TRiC chaperonin is required for reovirus assembly

P149: Sarah Katen, John Patton: Comparative Activities of the Phosphodegrons for beta-TrCP in Rotavirus NSP1 and IkappaB

Poster session 7: Applied dsRNA VirologyP150: Akhtar Ali, Samira Mokhtari:

Isolation and identification of dsRNA viruses from Fusarium speciesP151: Meng-Jung Chen:

Non- vaccine prevention of dsRNA virus infectionP152: Carolina Cornejo, Helena Braganca, Daniel Rigling:

Identification and initial characterization of dsRNA mycoviruses infecting the chestnut pathogen Cryphonectria naterciae

P153: Shuvra Dey, Sadia Farzan Sifat, Md Almamun, Nadim Sharif, Nasir Uddin Nobel, Shilpi Sarkar, Anowar Khasru Parvez, Ali Azam Talukder: Comparative evaluation of sensitivity and specificity of Immunochromatography kit (IC Kit) for the rapid detection of rotavirus in Bangladesh

P154: Chia-Jung Hsieh: Epidemiology and burden of dsRNA virus in Taiwan

P155: Soizick F. Le Guyader, My Phan, Sofia Strubbia, Julien Schaeffer, Marion Koopmans, Matthew Cotton: Metagenomic approach to detect dsRNA viruses and other human enteric viruses in wastewater samples.

P156: Gisela Masachessi, Verónica Emilse Prez, Laura Cecilia Martínez, Miguel Oscar Giordano, Patricia Angélica Barril, Jorge Vitorio Pavan, Silvia Viviana Nates: Assessing the use of picobirnavirus and infective enterovirus as indicators for faecal contamination tracking in freshwater and its potential to report the presence of rotavirus.

P157: Patricia Angélica Barril, Hugo Ramiro Poma, Georgina Gisela Giordano, Verónica Emilse Prez, Silvia Viviana Nates, Verónica Beatriz Rajal: Diffuse contamination of surface waters from the north region of Argentina: human rotavirus detection and characterization.

P158: Jeevan Bahadur Sherchand, Sony Shrestha, Ocean Thakali, Laxman Shrestha, Ajit Raymajhi: Rotavirus-gastroenteritis and molecular characterization of hospitalized children under 5 years of age with acute diarrhea in Nepal

P159: Francisca Cristi-Munoz, Yip Wan Kong, Hitt Mary, Maya Shmulevitz: Combination of Mutations to Produce Reovirus Variants with Improved Oncolytic Potency


P160: Meredith Stewart, Alejandro Marin-Lopez, Vanessa Herder, Javier Ortego, Massimo Palmarini: A synthetic biology approach for African horse sickness vaccine platforms

P161: Sebastiaan Theuns, Bert Vanmechelen, Quinten Bernaert, Nádia Conceição Neto, Ward Deboutte, Marilou Vandenhole, Leen Beller, Piet Maes, Jelle Matthijnssens, Hans J Nauwynck: Nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus and picobirnavirus as important enteric viruses

P162: Matias Aduriz Guerrero, Cecile Vincke, Ema Romao, Marina Bok, Celina Vega, Andres Wigdorovitz, Serge Muyldermans, Viviana Parreño: First immune nanobody library against Rotavirus derived from a guanaco

P163: Houping Wang, Jackelyn Murray, Les Jones, Ralph Tripp, Baoming Jiang: Assessment of improved rotavirus vaccine production in gene-edited Vero cells

P164: Yuhuan Wang, Theresa Resch, Mathew D Esona, Sung-Sil Moon, Baoming Jiang: A DS-1 like human rotavirus G9P[6] vaccine strain for improving efficacy in target populations

516. Program

7. ABSTRACTSOral and shotgun presentations

(in chronological order)

WORKSHOP 1: VIRAL DIVERSITY

Another nude virus: a capsidless ssRNA virus hosted by an unrelated dsRNA virus

Nobuhiro Suzuki

Okayama University, Japan

Recent studies illustrate that fungi as virus hosts provides a unique platform for hunting viruses and exploring virus/virus and virus/host interactions. Such studies have revealed a number of as-yet-unreported viruses and virus/virus interactions. Among them is a unique intimate relationship between a (+)ssRNA virus, yado-kari virus (YkV1) and an unrelated dsRNA virus, yado-nushi virus (YnV1). YkV1 dsRNA, a replicated form of YkV1, and RNA-dependent RNA polymerase (RdRp), are trans-encapsidated by the capsid protein of YnV1. While YnV1 can complete its replication cycle, YkV1 relies on YnV1 for its viability. We previously proposed a model in which YkV1 diverts YnV1 capsids as the replication sites. YkV1 is neither satellite virus nor satellite RNA, because YkV1 appears to encode functional RdRp and enhance YnV1 accumulation. This represents a unique mutualistic virus/virus interplay and similar relations in other virus/host fungus systems are detectable. We propose to establish the family Yadokariviridae that accommodates YkV1 and recently discovered viruses phylogenetically related to YkV1. In this presentation I overview what is known and unknown about the YkV1/YnV1 interactions. Also discussed are the YnV1 Phytoreo_S7 and YkV1 2A-like domains that may have been captured via horizontal transfer during the course of evolution and are conserved across extant diverse RNA viruses. Lastly, evolutionary scenarios are envisioned for YkV1 and YnV1.


Molecular characterization of complete gene segment-2 of picobirnaviruses from different animal host species: Evidence for high genetic diversity, and identification of conserved features in putative RdRps

Souvik Ghosh (1), Yashpal S Malik (2), Nobumichi Kobayashi (3)

1: Ross University School Veterinary Medicine, Saint Kitts and Nevis; 2: Indian Veterinary Research Institute, India;3: Sapporo Medical University, Japan

Picobirnaviruses (PBVs), family Picobirnaviridae, are opportunistic enteric pathogens that infect a wide variety of mammals and birds. PBVs possess a bi-segmented double-stranded RNA genome. The PBV gene segment-1 (~2.2–2.7 kb) encodes the viral capsid protein and a putative polypeptide of unknown function, whilst gene segment-2 (~1.2–1.9 kb) codes for the viral RNA-dependent RNA polymerase (RdRp). During the last decade, we detected PBVs in different animal species, expanding the host range of these viruses. Using a non-specific primer-based amplification method, we determined the full-length nucleotide (nt) sequences of gene segment-2/RdRp-gene of PBV strains detected in a cow, a cat, a dog, vervet monkeys, and a rat. The gene segment-2 of the bovine, canine, feline, simian, and rattus PBV strain was 1758 bp, 1689 bp, 1784 bp, 1707 bp, and 1730 bp long, respectively, and encoded a putative RdRp of 554 amino acid (aa), 531 aa, 534 aa, 538 aa, and 535 aa, respectively. By nt and deduced aa sequence identities and phylogenetic analyses, the complete gene segment-2 of the bovine, canine, feline, simian and rattus PBV strains exhibited a high degree of genetic diversity with those of PBVs from other host species. On the other hand, these PBV strains retained the 5′- and 3′- end nt sequences and the three domains of putative RdRp that appear to be conserved in PBVs. Taken together, our findings provided for the first time important insights into the features of putative RdRps, and diversity of gene segment-2 of PBVs in these animal species.

57Oral and shotgun presentations Workshop 1: Viral Diversity

Two Novel Relative Double-stranded RNA Mycoviruses Infecting Fusarium Poae Strain SX63

Luan Wang, Jingze Zhang, Hailong Zhang, Dewen Qiu, Lihua Guo

Institute of Plant Protection,Chinese Academy of Agricultural Sciences, Beijing, China

Two novel double-stranded RNA (dsRNA) mycoviruses, termed Fusarium poae dsRNA virus 2 (FpV2) and Fusarium poae dsRNA virus 3 (FpV3), were isolated from the plant pathogenic fungus, Fusarium poae strain SX63, and molecularly characterized. FpV2 and FpV3, with respective genome sequences of 9518 and 9419 base pairs (bps), are both predicted to contain two discontinuous open reading frames (ORFs), ORF1 and ORF2. A hypothetical polypeptide (P1) and a RNA-dependent RNA polymerase (RdRp) are encoded by ORF1 and ORF2, respectively. Phytoreo_S7 domain (pfam07236) homologs were detected downstream of the RdRp domain (RdRp_4; pfam02123) of the ORF2-encoded proteins of both FpV2 and FpV3. The same shifty heptamers (GGAAAAC) were both found immediately before the stop codon UAG of ORF1 in FpV2 and FpV3, which could mediate programmed -1 ribosomal frameshifting (-1 PRF). Phylogenetic analysis based on RdRp sequences clearly place FpV2 and FpV3 in a taxonomically unassigned dsRNA mycovirus group. Together with comparison of genome organization, a new taxonomic family termed Fusagraviridae is proposed to be created to include FpV2- and FpV3-related dsRNA mycoviruses, within which FpV2 and FpV3 would represent two distinct virus species.


RNA-RNA interactions hold the key to assortment in the multipartite dsRNA virus

Kinda Alshaikhahmed, Po-Yu Sung, Polly Roy

London School of Hygiene and Tropical Medicine, UK

Bluetongue virus (BTV), a member of the Orbivirus genus within the Reoviridae family, has a genome of 10 double-stranded RNA segments (S1-S10), which it has been suggested to follow a sequential packaging pathway during capsid assembly. In order to elucidate the mechanism of RNA segment assortment and packaging, we undertook an interdisciplinary approach, combining bespoke bioinformatics and network models of RNA-RNA interactions together with previously reported experimental data. A set of sequences, amongst the five smallest segments were identified, which appear to be vital for the formation of RNA complexes in stages. Predicted interaction sites for complex formation were validated by in vitro RNA-RNA interactions, targeted mutagenesis and virus recovery. The results obtained, revealed that segment interactions occur at multiple specific sites, which are dispersed across each segment. Further, these multiple RNA-RNA interacting sites are crucial for an assortment of different segments and pre-packaging complex formation. The correct RNA network is an essential criterion for genome packaging and virus infectivity. Thus, such an integrated interdisciplinary approach provides unique methods and insights into the formation of RNA complexes and can be applied to other segmented RNA viruses.


Genetic diversity of Group A Rotavirus in Latin America before and after universal vaccination, 2001-2017

Juan Ignacio Degiuseppe, Juan Andrés Stupka

INEI - ANLIS “Dr. Carlos G. Malbrán”, Argentine Republic

Introduction. During the last decade, most Latin American countries have implemented oral live rotavirus (RVA) vaccines in their national vaccination programs with remarkable results. It has been suggested that massive vaccination could produce the replacement of circulating genotypes or the emergence of new G/P type associations. The objective was to analyze the genetic diversity of RVA before and after the introduction of universal vaccination in Latin America.Methods. Literature search in PubMed and Scielo was conducted. There were considered only Latin American countries with rotavirus massive vaccination strategy which had described circulating genotypes surveillance data from 2001-2017, spanning for pre and post implementation periods.Results. Out of 18 countries that incorporated any rotavirus vaccine, 7 presented studies of RVA genetic diversity before and after implementation. Regarding to circulating genotypes, an increase in the proportion of G2P[4] association, G3P[8], G9P[8] and G12P[8] has been observed in the residual cases of rotavirus diarrhea. More specifically, Argentina, Brazil, Colombia and Nicaragua experienced a rapid switch from Wa-like to DS-1 like strains. G1P[8] association, considered the most predominant worldwide in the pre-vaccination era, decreased significantly and was only frequently detected in Venezuela and Nicaragua. In general, no defined pattern of emergence of unusual associations was observed, except for some evidence of G9P[4] in Argentina and Colombia.Conclusions. The diverse information provided by the studies conducted in Latin America is not still sufficient to assess the effect of vaccines on viral ecology or whether genetic diversity is influenced by natural fluctuation mechanisms.


In silico analysis of the VP7 for porcine rotavirus B and C illustrates genotype-specific antigenic epitopes

Frances Katen Shepherd (1), Fangzhou Chen (2), Michael Murtaugh (1), Douglas Marthaler (3)

1: University of Minnesota College of Veterinary Medicine, Department of Veterinary Biomedical Sciences USA; 2: Huazhong Agricultural University College of Veterinary Medicine, State Key Laboratory of Agricultural Microbiology, China; 3: Kansas State University College of Veterinary Medicine, Veterinary Diagnostic Laboratory, USA

Rotaviruses are responsible for the majority of acute diarrhea in swine, causing morbidity and mortality in piglets. Modified live vaccines for rotavirus B and C (RVB and RVC) are lacking due to difficulty in cell culture adaptation, but recombinant protein (RP) vaccines can be engineered to induce immunity to the major antigenic proteins, viral protein (VP)7 and VP4. However, antigenic targets for RVB and RVC are unknown to inform RP vaccine design. The VP7 genes of 174 RVB and 369 RVC strains from North American swine were sequenced to describe G genotypic diversity, genetic variability, and antigenic sites. The RVB strains belonged to 11 G genotypes, with the discovery of three new genotypes while 5 RVC G genotypes were identified. Eight variable regions (VRs) were identified in both RVB and RVC. The discrepancies between the VRs of swine RVC and previously identified VRs in other hosts suggest swine RVCs have unique variability patterns. In silico analysis utilized EPCES (Epitope Prediction using ConsEnsus Scoring) algorithm to predict the B cell epitopes for the predominant genotypes of RVB (G12, n=21; G14, n=15 ; G16, n=21; G18, n=19; G20 n=16) and RVC (G1, n=15; G5, n=25; G6, n=25; G9, n=23). The predicted epitopes of RVB and RVC were genotype-specific and several sites were highly conserved, which may be effective RP vaccine targets. These results provide valuable information on RVB and RVC antigenicity and illustrate the potential for incorporating in silico analysis into RP vaccine development.


Evolution in the Family Partitiviridae and Novel Aspects of the Polymerases

Marilyn J Roossinck

Penn State University, USA

Plants are frequently infected with persistent viruses that are maintained via seed transmission for many generations, and in some cases probably thousands of years. Most of these viruses have double-stranded RNA genomes, and families for these viruses are shared among plants and fungi. The Partitiviridae family includes about 80 virus isolates from a diverse array of plants, fungi and oomycetes that have complete genome sequences available in GenBank. Most isolates contain only two RNAs, encoding an RNA dependent RNA polymerase (RdRp) and a coat protein, although some contain additional RNAs. The RdRp is clearly monophyletic, and reliable phylogenies can be constructed that show both plant and fungal partitiviruses in some of the same clades, implying cross-kingdom transmission. However, the coat protein genes are very diverse, and are almost certainly polyphyletic, even within some of the proposed genera for the family. Protein secondary structures support the hypothesis that these proteins have been coopted from diverse sources and have converged to have a common function. This scenario for the deep evolution of a virus family that seems to have originated from an RdRp that diverged and then acquired coat proteins from various sources has important implications for the origins and evolutionary history of modern viruses. In addition, the RdRps of these and related dsRNA viruses have conserved domains related to other types of polymerases, and have novel activities in vitro that cannot easily be explained in the virus life cycle.


Bluetongue virus replication in culicoides cells increases the number of genetic variants in the viral population

Joseph Hughes, Anna Janowicz, Massimo Palmarini

MRC-University of Glasgow Centre for Virus Research, UK

RNA viruses exist as a diverse population possessing closely related genomes but distinct from their consensus sequence.Bluetongue virus (BTV) is the causative agent of bluetongue, one of the major infectious diseases of ruminants. BTV is an arbovirus transmitted by Culicoides biting midges. Like any arbovirus, BTV must adapt rapidly to replicate in host cells as diverse as those of a warm-blooded mammal and those of an insect. Understanding how the viral population evolves in different host cells can provide important insights into BTV evolution. We investigated the microevolution of the BTV population passaged either in culicoides cells (KC, grown at either 28 or 37C), or in sheep cells (CPT-Tert) or passaged alternatively in sheep and culicoides cells. We show that the BTV viral population becomes more diverse in insect cells than sheep cells. This increasing diversity is not the result of differences in bottleneck size or differences in survival of variants in the different cell types. The differences in diversity observed are thus the result of either an increase in errors during replication in the KC cell line or a result of host RNA editing. Interestingly, we noticed a bias in the type of mutations observed in insect cells, which were not evident in sheep cells.Our data suggest that Culicoides cells might function as a natural source of new BTV variants. The genetic diversity acquired in culicoides cells may provide the opportunity for BTV to adapt more quickly to selective pressures.


Impact of host microbiota on enteric RNA virus evolution.

Andrea Kaup Erickson, Julie K. Pfeiffer

University of Texas Southwestern Medical Center at Dallas, USA

RNA viruses have high genetic diversity, which is thought to enhance viral fitness. Genetic diversity occurs through error-prone replication as well as through exchange of genetic material during recombination and reassortment. To generate novel viral progeny, these genetic exchange processes require the presence of multiple viral variants within a single cell (co-infection). Co-infection is presumably a rare event during initial infection, where the ratio of virions to cells is low. Previously we found that multiple poliovirus virions bound to a single bacterium, and bacteria increased the frequency of poliovirus co-infection under low multiplicity of infection (MOI) conditions in vitro. Additionally, bacteria strains that increased viral co-infection facilitated viral recombination, resulting in viral progeny with increased fitness. Here we use reovirus, a segmented enteric virus, to investigate the impact of bacteria on viral co-infection and reassortment in vitro and in vivo. Using a flow cytometry assay, we found that bacteria facilitated co-infection of reovirus serotype 1 Lang (T1L) and serotype 3 strain Dearing (T3D) under low MOI conditions in vitro. To examine reassortment in vivo, germ-free and conventional mice were orally infected with T1L and T3D strains and the relative frequency of viral reassortment in intestinal tissues was quantified using shRNA-expressing cell lines that prevent growth of the parental viruses. Our preliminary results suggest that germ-free mice had relatively high viral titers, but reduced reassortant viruses compared to mice colonized with bacteria. Our findings provide further understanding on the impact of the host microbiota on the evolution of enteric viruses.


The Reovirales a new taxonomic order: families Sedoreoviridae and Spinareoviridae

Houssam Attoui (1), Fauziah Mohd Jaafar (1), Peter P.C. Mertens (2)

1: UMR1161 Virology, INRA-ANSES-ENVA, France, 94700;2: The School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Leicestershire UK, LE12 5RD

Family Reoviridae contains 15 distinct genera, classified within two subfamilies Spinareovirinae (turreted) and Sedoreovirinae (non-turreted) that are recognised by the International Committee on Taxonomy of Viruses (ICTV). Analysis of RdRp sequences suggests that the turreted and non-turreted reoviruses evolved from a common ancestor.It has been recently suggested that RdRps of turreted reoviruses are more closely related to those of birnaviruses than to RdRps of non-turreted reoviruses. However, several findings argue against this suggestion: 1-Birnavirus RdRp is a Vpg and thus distinct from the RdRps of reoviruses; 2-Reovirus RdRp uses a fully conservative mechanism, while birnavirus RdRp is semi-conservative and in this respect is more like a DNA polymerase; 3-Birnavirus RdRp possesses a reverse-transcriptase activity, bringing it closer to DNA polymerases; 4-Infectious bursal disease virus (a birnavirus) RdRp reveals a characteristic rearrangement of motifs, from A–B–C to C–A–B, in the RNA polymerase catalytic palm domain, which is not found in RdRps from other dsRNA viruses. In addition birnaviruses have only two genome-segments, one of which encodes a polyprotein, again distinguishing them from reoviruses. Although members of family Birnaviriade possess a T13 capsid, like reoviruses, the evolutionary origin of these capsids is distinct.Based on conserved structural and functional properties and despite high genetic diversity between genera of the family Reoviridae, we propose the creation of a new taxonomic level - the order ‘Reovirales’. Collectively these data suggest that this order should only encompass families Sedoreoviridae and Spinareoviridae, which correspond to the current subfamilies of the Reoviridae.


BTV-GLUE: A WEB-BASED BIOINFORMATICS RESOURCE FOR BLUETONGUE VIRUS SEQUENCE DATA

Josh Singer (1), Rob Gifford (1), Kyriaki Nomikou (1,2), Alexander MacLeod (1), Peter Mertens (2,3), Massimo Palmarini (1)

1: MRC-University of Glasgow, Glasgow, UK; 2: The School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, Leicestershire, UK; 3: The Pirbright Institute, Pirbright, Woking, UK

Bluetongue virus (BTV) is an arbovirus transmitted by biting midges (Culicoides pp.). BTV causes a severe disease (bluetongue) in domestic and wild ruminant species with high levels of morbidity and mortality. Bluetongue has emerged as an important disease in sheep and cattle worldwide. The BTV genome is composed by ten linear dsRNA segments, packaged within a triple-layered icosahedral protein-capsid, and encode 7 structural and 4/5 non-structural proteins. To date, there are at least 27 BTV serotypes (mainly determined by the VP2 outer capsid protein) circulating worldwide. In addition, high rates of reassortment involving all genome segments have been documented, complicating epidemiological studies and vaccination programmes.We have developed BTV-GLUE, a new bioinformatics sequence data resource for bluetongue virus. Sequences from the NCBI nucleotide database are curated along with complementary sequence metadata. These data are integrated together inside GLUE (http://tools.glue.cvr.ac.uk), a data-centric software package for capturing virus sequence data and organising it along evolutionary lines. The dataset also contains reference sequences with genome feature annotations, multiple sequence alignments and phylogenetic trees, for each BTV segment and clade. The beta version of the BTV-GLUE dataset is available via a public web server (http://btv.glue.cvr.ac.uk). The resource may also be used as an offline bioinformatics toolkit. BTV-GLUE will help the BTV community to study varying aspects of BTV biology and evolution and will facilitate the adoption of a nomenclature that more easily distinguished the properties of BTV strains circulating worldwide.


Rotavirus species tropism: new insights on P-type classification

Samuel Miño (1), Matías Adúriz (2), Viviana Parreño (2)

1: Instituto Nacional de Tecnología Agropecuaria, Instituto de Virología, Argentine Republic; 2: Instituto Nacional de Tecnología Agropecuaria, IncuINTA, Argentine Republic

Rotavirus group A (RVA) infects enterocytes of a broad range of species with high specificity between strain and host cells. VP4 is mainly responsible for virus internalization and cell tropism. To date, 49 P-types (VP4) were reported. This protein is composed of two domains VP5 and VP8, which possess a trimer structure in the basal region and a dimer structure in the distal region (spike), with the VP8 domains on the top of the spike. The objective of this work is to study the aminoacid regions that are involved in the VP8 pocket in order to find a molecular fingerprint for the virus species tropism.Sequences from all available P-genotypes were aligned and compared. A representative strain from each P-genotype was structurally modeled. The aminoacids involved in the pocket were defined and analyzed.The pocket is formed by two VP8 (70-74, 200-205), and one VP5 (333-336) regions. The aminoacid sequences on the pocket are conserved among strains from the same genotype and the clusters based on these regions grouped the P-types by animal host.The structure of the Rotavirus spike functions as the ligand of a cell receptor protein with the initial sialic acid dependant interaction located on the external part of the spike. In a second step for cell infection the aminoacids on the spikes pocket interact with another cell receptor which is specific for specie. This correlation between the sequence of the pocket and the host tropism could be useful for taxonomy classification.


Exploring the diversity and dynamics of the gut virome of infants during the first year of life

Leen Beller (1), Ward Deboutte (1), Chenyan Shi (1), Daan Jansen (1), Claude Kwe Yinda (1), Mark Zeller (2), Marc Van Ranst (3), Jelle Matthijnssens (1)

1: KU Leuven, Laboratory of Viral Metagenomics, Belgium; 2: The scripps Research Institute, La Jolla, CA, USA; 3: KU Leuven, Laboratory of Clinical and Epidemiological Virology, Belgium

Background: Our early life gut microbiota is of crucial importance for proper training of our immune system and achieving health later in life. We investigated the longitudinal dynamics of eukaryotic viruses in the gut of healthy infants. Methods: Fecal samples were collected almost daily for one year from eight healthy infants. Per infant, twenty-one samples at predefined time points were selected. Additionally, samples collected two weeks before and after disease, antibiotic treatment or vaccination, were analyzed. Between 36-44 samples of every infant were characterized. Results: On average 10% of the reads represent eukaryotic viruses mainly belonging to the Adenoviridae, Anelloviridae, Astroviridae, Caliciviridae, Circoviridae, Endorna-viridae, Luteoviridae, Partitiviridae, Parvoviridae, Picornaviridae, Polyomaviridae, Reo viridae, Totiviridae or Virgaviridae families. Members of the Reoviridae family include the Rotarix strain detected after vaccination and G9P[8] and G12P[8] rotaviruses detected in one and two infants, respectively, in the absence of any symptoms. The other dsRNA viruses detected are Phaseolus vulgaris alphaendorna-virus 1 (Alphandornavirus), Cucumis melo alphaendornavirus (Alphandorna virus) and Carrot cryptic virus (Alphapartitivirus), all known to infect mainly plants which as in accordance with the fact that they are only detected at time points where solid food was included in the infants’ diet. In addition, also one very divergent virus belonging to the Totiviridaefamily was detected (no blastn hits, Diamond hit to ‘Panax notoginseng virus A’).Conclusions :These results show that many more eukaryotic viruses are present in the gut of healthy infants in their first year of life than previously thought, even in the absence of clinical symptoms.


Piscine orthoreovirus (PRV); in vivo propagation in erythrocytes enables virus purification, causality to important disease in farmed Atlantic salmon and protection by inactivated vaccine

Øystein Wessel (1), Turhan Markussen (1), Torstein Tengs (1), Dhamotharan K (1), Salman Malik (1), Maria Dahle (2), Espen Rimstad (1)

1: Norwegian University of Life Science, Norway; 2: Norwegian Veterinary Institute

Piscine orthoreovirus (PRV) is the causative agent of heart- and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar), and the first fish virus to be classified in the orthoreovirus genus. Erythrocytes are the main target cells for PRV, but the virus is also found in other cell types including cardiomyocytes, macrophages and melanomacrophages. A PRV subtype was demonstrated to cause erythrocytic inclusion body syndrome (EIBS) in juvenile Coho salmon, while yet another PRV subtype is the cause of both anemia and HSMI in farmed rainbow trout. The PRV subtype in Atlantic salmon is thus referred to as PRV1.There is currently no susceptible cell line for PRV and no vaccine available. Piscine erythrocytes are nucleated and contain the transcriptional and translational machineries that enable virus replication. In this study, we propagated PRV in salmon and purified virus particles from red blood cells at peak of infection. Challenge with purified PRV1 reproduced the disease and provided causal evidence for HSMI. Furthermore, inactivated purified PRV1 vaccine were used in challenge trials, and both demonstrated significant protection.Finally, challenge with different PRV1 strains elucidated difference in virulence between strains. We cannot yet firmly link single gene segments or amino acid motifs to virulence. An association between genomic segment linkage and virulence should not be ruled out.


WORKSHOP 2: EVOLUTION AND EPIDEMIOLOGY

Multiple introductions and antigenic mismatch contribute to increased predominance of G12P[8] rotaviruses in the United States

Kristen M Ogden (1), Yi Tan (1), Asmik Akopov (2), Laura S Stewart (1), Rendie McHenry (1), Christopher J Fonnesbeck (1), Bhinnata Piya (1), Maximilian H Carter (1), Nadia B Fedorova (2), Rebecca A Halpin (2), Meghan H Shilts (1), Kathryn M Edwards (1), Daniel C Payne (3), James D Chappell (1), John T Patton (4), Natasha B Halasa (1), Suman R Das (1)

1: Vanderbilt University Medical Center, Nashville, Tennessee, USA; 2: J. Craig Venter Institute, Rockville, Maryland, USA; 3: Centers for Disease Control and Prevention, Atlanta, Georgia, USA; 4: Indiana University, Bloomington, Indiana, USA

Rotavirus is the leading global cause of diarrheal mortality for unvaccinated children under five years of age. The outer capsid of rotavirus virions is composed of VP7 and VP4 proteins, which respectively determine viral G and P type and are primary targets of neutralizing antibodies. Successful vaccination depends upon generating broadly reacting neutralizing antibodies to a limited number of G and P type antigens. Vaccine uptake has coincided with decreased rotavirus disease burden but also the emergence of uncommon G and P types. To gain insight into the recent predominance of G12P[8] rotaviruses in the U.S., complete rotavirus genome sequences and metadata from 142 clinical specimens collected in Nashville, TN from 2011-2013 through the New Vaccine Surveillance Network were evaluated. Circulating G12P[8] strains were found to share many segments with other locally circulating rotaviruses but to have distinct constellations. Phylogenetic analyses of G12 sequences and their geographic sources provided evidence for multiple separate introductions of G12 segments into Nashville, TN. Antigenic epitopes of VP7, but not VP4, proteins of Nashville, TN G12P[8] strains differ markedly from those of vaccine strains. Nearly half of G12P[8] specimen donors had received a complete rotavirus vaccine series, and mathematical modeling suggests increased vaccination probability for G12P[8]-positive children compared with those who test positive for other rotavirus types. Multiple introductions and significant antigenic mismatch may in part explain the recent predominance of G12P[8] strains in the U.S. and emphasize need for continued monitoring of rotavirus vaccine efficacy against emerging rotavirus types.


Epidemiology and evolution of Rotavirus C (RVC) in India

Yashpal S. Malik (1), Sharad Saurabh (1), Jobin Jose Kattoor (1), S. Shanmuganathan (1), Shubhankar Sircar (1), Kuldeep Dhama (1), Souvik Ghosh (2), Nadia Touil (3), Raj Kumar Singh (1)

1: ICAR- Indian Veterinary Research Institute, Izatnagar, Bareilly, 243 122, Uttar Pradesh, India; 2: Department of Biomedical Sciences, One Health Center for Zoonoses and Tropical Veterinary Medicine, Ross University School of Veterinary Medicine, Basseterre, St. Kitts, West Indies; 3: Laboratoire de Biosécurité et de Recherche, Hôpital Militaire d’Instruction Med V de Rabat, 110 000, Morocco

Rotavirus-C (RVC) is appearing as a growing concern of gastrointestinal infections in pigs with the observation of zoonotic transmission. Although its prevalence has been noted in several parts of the world, the epidemiology, genetic diversity, and phylogeny of RVC in humans or animals remain largely understudied in India. This work describes the RVC epidemiology and explicates the genetic relatedness of circulating porcine RVC strains in India. From 2012-2017, fecal samples (n=534) of symptomatic (n=406) and asymptomatic (n=128) piglets from different states of India were screened for RVC infection by reverse-transcription-PCR targeting the group-specific VP6 gene. Overall, RVC prevalence of 16.1% (86/534) was recorded, significantly higher among symptomatic (18.6%, 77/406) than asymptomatic (7.03%, 9/128) piglets. Except for few cases of dual (6.96%, 6/86) or multiple infections (4.65%, 4/86) with rotavirus A, astrovirus or picobirnavirus, nearly in all other cases, RVC was found as the single cause of diarrhea. Furthermore, the phyloanalysis based on complete VP6 gene revealed genetic heterogeneity among Indian porcine RVC strains and confirm the occurrence of I2, I7 and I8 genotypes in Indian porcine population. Analysis of VP7 and NSP4 genes also exhibit high genetic divergence and evolution of new G and E types among Indian porcine RVC strains. Of note, Indian porcine RVC strains show higher genetic relatedness to RVC strains of human-origin, signifying the circulation of two distinct RVC populations in India i.e., porcine and human-like porcine RVC strains. The findings provide new insights into the epidemiology, diversity, and circulating RVC genotypes in Indian porcine population.

73Oral and shotgun presentations Workshop 2: Evolution and Epidemiology

GENETIC DIVERSITY OF GROUP A ROTAVIRUS STRAINS CIRCULATING IN PORCINE FROM FIVE PROVINCES IN SOUTH AFRICA DURING 2007, 2008 AND 2015.

Thenjiwe Grace Ngomane, Peenze I, Seheri ML

Sefako Makgatho health Sciences university, South Africa

Background and objectives: Rotaviruses (RV) are the major cause of diarrhea in humans and animals worldwide. Group A rotavirus (RVA) has been credited to have an economic impact through loss in pig breeding and pork production. ProSystem, the porcine RVA vaccine has not been introduced to South African farmers, therefore practice of good biosecurity majors is the only method used to prevent RV infection. In this study the prevalence, migration patterns and genotypes were determined and compared to worldwide strains from the NCBI GenBank. Methods: from 238 samples collected from South African pigs during 2007, 2008 and 2015, RVA antigen was detected using EIA and EIA positive samples were subjected to PAGE and RT-PCR, where migration patterns and genotypes were identified. RT-PCR product was subjected to Sanger sequencing and Chromatograms were used to draw phylogenetic trees.Results: RVA was detected in 27.3% (65/238), 64.6% (42/65), which had long migration patterns. There was 17 genotype combinations, where G3 and G4 combined with P[6] to P[8] and P[23], G5 with P[6] to P[8], P[13] and P[23] and G9 with P[8] and P[13].Conclusion: The RVA prevalence inSouth Africa (27.3%) is within a common range when compared with Kenya (27.2%) and Uganda (25.1%).Introduction of the ProSystem vaccine would be protective to South African pigs, since it includes Five of the nine genotypes detected in this study


Intriguing successive (sub)clinical shedding patterns of different rotavirus genotypes in suckling and weaned pigs

Sebastiaan Theuns (1), Quinten Bernaert (1), Philip Vyt (2), Hans J Nauwynck (1)

1: Ghent University, Belgium; 2: Dialab, Belgium

Rotavirus A and C are commonly found in feces of suckling and nursery piglets. The pathogenesis has been well studied in gnotobiotic pigs but importance under field conditions has been poorly addressed.For the first time, rotavirus shedding was quantitatively assessed in suckling and/or nursery pigs of six conventional and one high health pig farms in Belgium.In suckling piglets, clinical RVA infections were observed in one litter of a conventional farm, likely complicated by low environmental temperature. A negative correlation between daily weight gain and RVC shedding in suckling piglets was observed on a high health farm, without diarrhea. In nursery pigs, shedding of RVA started around weaning on all farms, leading to two or three successive replication waves with different genotypes. Similar successive RVC waves were observed after weaning, mostly occurring in between or simultaneously with RVA shedding. Explosive diarrhea in weaned piglets was observed on one farm with coinfections (RVC and E. coli). Impact of rotavirus replication on growth in weaned piglets varied between farms. Strict cleaning and disinfection led to lower viral shedding in feces and less genotypes circulating in nursery barns, compared to farms using disinfection only.In conclusion, successive subclinical rotavirus replication waves were occurring in all pigs and farms studied. Severe rotavirus infections had an impact on growth in the suckling piglets, but only caused explosive diarrhea in weaned piglets during coinfections. Strict hygiene led to an overall lower rotavirus infection pressure and caused less circulation of different genotypes on the farms


Rotavirus among children aged 0-5 years in Nouakchott Mauritania

Mohamed Lemine Cb. AHMED (1), Abdellahi Weddih (2), Mohemed Abdellahi Boullah (3), Meriam Sidatt (4), Abdelkarim Filali-Maltouf (5), Mohammed Benhafid (6)

1: National Institute of Research on Public Health, Mauritania; 2: University of Nouakchott; 3: National Institute of Research on Public Health, Mauritania; 4: university of Nouakchott; 5: University Mohammed V; 6: INH

Background: Rotavirus vaccine was introduced in Mauritania December 6, 2014. The present study build on the absence of documented data describes the burden of rotavirus in Mauritania. Our objective is to investigate the prevalence of diarrhea associated to hospitalization before and after introduction of rotavirus vaccine.Methodology: we conducted a prospective study enrolling children aged 0-5 years at the Hospitals in Nouakchott from July 2009 to June 2010 and from December 1, 2017 to April 20, 2018. Children were included if they were hospitalized with AGE, defined as having 3 or more diarrheal stools per day lasting less than 7 days. Demographic (age, sex, residence) and clinical information presence of fever, vomiting and diarrhea duration were obtained. Stool specimens were tested for rotavirus using rapid chromatographic immunoassay (Vikia Rota-Adeno, Biomerieux France).Results: One hundred-nineteen (119) stool specimens were collected before rotavirus vaccine introduction and 86 stools were collected after vaccine introduction. The prevalence was 32% in prevaccine period and 17% in postvaccine period. The highest proportion of RV-positive was in children aged 0-5 month with 39% in prevaccine and 47% in postvaccine, as compared to 6-11 month olds with 31% in prevaccine and 33% in post vaccine, and those 11 months of age and older were 25% in prevaccine and 20% in postvaccine period.Conclusion: Rotavirus vaccine had a substantial impact on rotavirus associated hospitalization among young children in Nouakchott, Mauritania.


Genetic characteristics of porcine rotaviruses H detected recently in Japan

Tohru Suzuki

National Institute of Animal Health, NARO, Japan

Rotavirus species H (RVH) has been detected in pigs and humans. Especially, porcine RVHs have been often identified in several pig-producing countries worldwide. Recently, we identified nine porcine RVH strains from fecal samples of pigs of various ages, in five farms in Nagasaki prefecture, Japan, between 2013 and 2015. In addition, most of them were co-infected with other porcine rotavirus species, predominantly porcine rotaviruses C (RVCs). Despite their zoonotic impact, genetic characterization of RVHs is still limited. We aimed to understand genetic diversity of RVH via comparative sequences and phylogenetic analyses among classical porcine RVH identified in Japan in 1990’s, multiple porcine RVHs identified in some pig-producing countries in recent years, nine porcine RVHs identified in Japan from 2013to 2015, and human RVHs identified in China and Bangladesh. Porcine RVHs could be classified into multiple genotypes based on the definition recommended by the Rotavirus Classification Working Group. Particularly, we indicated that porcine RVHs belonging into multiple genotypes have been present in Japan since the discovery of the first porcine RVH, SKA-1 in 1990’s.The nine porcine RVHs analyzed in this study displayed genetic variations in NSP1 and NSP3 genes, compared with other previously reported porcine RVHs. Surprisingly, we found two different types (short and long-types) in RVH NSP3, and that the long-type RVH NSP3might have been derived from a reassortment between porcine RVH and RVC. These findings would provide valuable information in understanding evolution of RVH.


Genetic variability and implications for identifying neutralizing epitopes in swine rotavirus A strains

Frances Katen Shepherd (1), Michael Murtaugh (1), Douglas Marthaler (2)

1: University of Minnesota College of Veterinary Medicine, Department of Veterinary Biomedical Sciences, USA; 2: Kansas State University College of Veterinary Medicine, Veterinary Diagnostic Laboratory, USA

Rotavirus A (RVA) is one of the most significant etiological agents of diarrheal disease in pigs. The outer capsid proteins, viral protein (VP)7 and VP4, stimulate neutralizing antibodies and are sequenced to determine G and P genotypes, respectively. In swine, 12 G genotypes and 16 P genotypes are known, leading to lower vaccine efficacy in the presence of diverse RV strains circulating in suckling piglets. To correlate antigenic diversity and possible vaccine escape, the VP7 and VP4 genes were sequenced from 196 swine RVA strains collected from the United States. The five G genotypes (G3, G4, G5, G9, and G11) and five P genotypes (P[6], P[7], P[13], P[19], and P[23]) identified had marked phylogenetic differences from the commercial Prosystems® Swine RVA vaccine, suggesting possible low vaccine efficacy. Furthermore, we identified multiple hypervariable residues (HR) on VP7 and VP8* (the VP4 cleavage product). High variability did not consistently align with known neutralizing epitopes (NE); between 1-10 and 1-9 amino acid types were present at NE of VP7 and VP4, respectively. Furthermore, only 5 of 17 HR for VP7 and 6 of 25 HR for VP8* aligned with known NE. The discrepancies between HRs and known NE suggests that epitopes described in the literature, based primarily on human and monkey RVA, may not be relevant in swine and swine-specific neutralizing epitopes may exist. In conclusion, the genotypic diversity and variability of the HR on VP7 and VP4 proteins demonstrates the need for investigating swine-specific epitopes to develop an effective swine RVA vaccine.


Worldwide evolution of equine RVA, four voices, one story

Samuel Miño (1), María Barrandeguy (1,2), Viviana Parreño (3)

1: Instituto Nacional de Tecnología Agropecuaria, Instituto de Virología, Argentine Republic; 2: Cátedra de Enfermedades Infecciosas. Escuela de Veterinaria, Universidad del Salvador, Buenos Aires, Argentine Republic; 3: Instituto Nacional de Tecnología Agropecuaria, IncuINTA, Argentine Republic

Rotavirus group A (RVA) is classified into G- and P- genotype according to the nucleotide sequence of both VP7 and VP8 genes. Moreover, the VP6 and NSP4 nucleotide sequence are used to classify the I-type (subgroup antigen) and the E-type (toxin antigen). The full genome sequence of equine RVA strains demonstrate that the equine RVA possess a constant genome constellation. Phylogenetic studies showed that RVA strains from different continent are grouped in different lineages. In this work, we used a Bayesian approach, together with all the information that was available in databases, to study the evolution of equine RVA.The mutation rate was estimated for VP7 (G3= 1.69E-4, and G14=4.94E-4), VP8 (P[12]= 5.65E-4), VP6 (I2=3.93E-4, I6=3.56E-4) and NSP4 (E2=4.49E-4, E12=4.93E-4). The time to the most recent common ancestor (TMRCA) was estimated for VP7 (G3, G14), VP8 (P[12]), VP6 (I2, I6=) and NSP4 (E2, E12), where the value for E12=374.9 (198.6 – 580.7) years was coincident with the introduction of horses in South America. The maximum clade credibility trees showed groups with a 100% posterior-probability that group strains from continent with a 100% concordance. The NSP4 E12 genotype analysis showed a South American origin, with a Guanaco strain as TMRCA; all the other reported RVA strains from equine, bovine, goat, camelid and humans are related to it.Our results show that the phylogenetic lineages in equine RVA are consistent with its geographical distribution, and the genetic footprint of the NSP4 gene (E12) was able to reconstruct its evolutive path.


Partitiviruses and related unclassified viruses in plant pathogenic fungi and oomycetes

Eeva Johanna Vainio, Anna Poimala, Tuula Piri, Muhammad Kashif, Jarkko Hantula

Natural Resources Institute (Luke), Finland

Plant-associated fungi commonly harbor dsRNA viruses, which may be cryptic (asymptomatic), harmful or mutualistic. Conifer root rot pathogens of the genus Heterobasidion host diverse species of family Partitiviridae with small linear bisegmented genomes. These viruses mediate variable phenotypic effects, and one of them affects the expression of hundreds of host genes and mediates considerable growth reduction, and is therefore being investigated as a potential biocontrol agent against its host. Fungal dsRNA viruses have no extracellular phase in their infection cycle and are only transmitted via cellular contacts and spores, which restricts their host range. In Heterobasidion fungi, pre-existing viral infections may facilitate superinfections by other virus species, while mutual exclusion has been observed between conspecific viruses. Multiple viruses often accumulate in vegetatively spreading Heterobasidion clones that may infect tens of trees and persist for decades in the forest. The phenotypical effects of multiple virus infections are not always cumulative, and coinfections may reduce symptom severity compared to single virus infections. During the last decade, unclassified bisegmented dsRNA viruses related to partitiviruses have been detected in many plant-associated fungi, including the conifer pathogens Heterobasidion spp. and Gremmeniella abietina and ectomycorrhizal fungi of genus Lactarius, but the ecological role of these novel viruses remains unknown. We also recently detected a new monopartite virus in the oomycete Phytophthora. This member of proposed genus “Ustivirus” has a wide geographical distribution and occurs in diverse species of genus Phytophthora, which includes some of the most notorious plant pathogens in the world.


Analysis of the Impact of Rotavirus Vaccines on Genotype Diversity in Australia

Susie Roczo-Farkas (1), Celeste Michelle Donato (2), Carl Kirkwood (1,3,4), Daniel Cowley (1,3), Graeme Barnes (1,3,5), Ruth Bisop (1,3), Nada Bogdanovic-Sakran (1), Karen Boniface (1), Julie Bines (1,3,5)

1: Enteric Virus Group, Murdoch Children’s Research Institute, Australia; 2: Biomedicine Discovery Institute and Department of Microbiology, Monash University, Australia; 3: Department of Paediatrics, University of Melbourne, Australia; 4: Enteric and Diarrheal Diseases, Global Health, Bill & Melinda Gates Foundation, USA; 5: Department of Gastroenterology and Clinical Nutrition, Royal Children’s Hospital, Australia

Background: Introduction of rotavirus vaccines into national immunisation programs could result in strain selection due to vaccine-induced selective pressure. The aim of this study was to describe the distribution and diversity of rotavirus genotypes before and after vaccine introduction in Australia. Rotavirus vaccines were introduced in 2007 and the state-based vaccine selection facilitated a unique comparison of diversity in RotaTeq and Rotarix vaccine states in the absence of demographic and geographical differences. From July 2017, Rotarix is used Australia-wide. Methods: From 1995 to 2017, the Australian Rotavirus Surveillance Program conducted genotypic analysis on 13,401 rotavirus-positive samples from children <5 years of age, hospitalised with acute gastroenteritis using serological and hemi-nested multiplex reverse-transcription polymerase chain reaction assays.Results: In the pre-vaccine era (1995–2006), G1 strains were dominant (53.4%). Following vaccine introduction (2007–2016), genotype dominance fluctuated and greater genotype diversity was observed nationwide. Detection of G1P[8] decreased (25.1%), while G2P[4] (21.3%), G12P[8] (18.0%) and equine-like G3P[8] (9.7%) increased. Genotype distribution varied; with G12P[8] dominant in states using RotaTeq, and equine-like G3P[8] and G2P[4] dominant in states and territories using Rotarix. In 2017, G2P[4] (33.4%), equine-like G3P[8] (23.8%) and G8P[8] (14.5%) were dominant. Conclusions: In the vaccine-era in Australia, there has been a sustained increase in the diversity of genotypes with differences in genotype dominance between RotaTeq and Rotarix jurisdictions. These results suggest the vaccines exert different immunological pressures that influence the diversity of rotavirus strains circulating in Australia. Continued surveillance is required to determine the impact of nation-wide Rotarix implementation.


Lewis and Secretor phenotypes affect rotavirus vaccine shedding in Nicaraguan children

Filemón Bucardo (1), Yaoska Reyes (2), Ylva Rönnelid (2), Fredman González (2), Lennart Svensson (2,3), Johan Nordgren (2)

1: National Autonomous University of León, Nicaragua (UNAN-León), Nicaragua; 2: Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden; 3: Department of Medicine, Karolinska Institute, Stockholm, Sweden

Blood types, Lewis (Le) and Secretor histo-blood group antigens (HBGA) are susceptibility factors for rotavirus in a P-genotype dependent manner and have been found to affect IgA seroconversion rates following rotavirus vaccination. To investigate the association between HBGAs phenotypes and rotavirus vaccine shedding, a total of 141 children (Rotarix = 70 and RotaTeq = 71) provide 354 prospective stool samples distributed as follows: 112 at ≤3, 98 at 4 - 7 and 144 at ≥8 days post vaccination. One step NSP3 qPCR was used to detect rotavirus and genotypes were determined by G:P Multiplex PCR and/or sequencing. Blood types were examined by hemagglutination and LeA, LeB and Secretor by saliva based ELISA and allele specific qPCR. The percentage of rotavirus-positive samples in Rotateq children decreased rapidly over the 3 collection time frames (48%, 19%and 11%). The shedding in Rotarix children exhibited less change over later collection times (22%,12% and 13%). At late time frames (>4 days), indicative of viral replication in vivo, significantly less stools samples from children LeA (4%, 1/26, OR = 0.08) and Le-negative (11% 4/38, OR = 0.2) were rotavirus-positive in both cohorts, as compared to LeB (33%, 59/178). A similar association was observed after comparing Non-secretors and Secretors children (5%, 2/37 vs 15%, 30/205, OR = 0.3). This study reports, that Rotateq and Rotarix vaccine strains predominantly infected LeB and Secretors children but LeA, Lewis-negative and Non-secretors are less permissive. Interestingly, the bovine derived P[5] genotype included in Rotateq was able to replicate in LeB children.


A viral metagenomics view on the honeybee virome reveals a plethora of undescribed dsRNA viruses

Ward Deboutte (1), Leen Beller (1), Claude Kwe Yinda (1), Nádia da Conceição Neto (1), Piet Maes (2), Dirk C de Graaf (3), Jelle Matthijnssens (1)

1: KULeuven, Laboratory of Viral Metagenomics,Belgium; 2: KULeuven, Laboratory of Clinical and Epidemiological Virology, Belgium; 3: Ugent, Laboratory of Molecular Entomology and Bee Pathology, Belgium

Advances in sequencing technologies and viral particle enrichment methodologies have resulted in the discovery of numerous, previously undescribed viruses. We applied high-throughput sequencing on Belgian Apis melifera (honeybee) samples, treated according to the netoVIR protocol to enrich for viral-like particles. Reads were quality trimmed and after de novo assembly, viral sequences showing homology to dsRNA viruses were identified by BLAST-based approaches on the protein level. In total 557 contigs where identified belonging to the Partitiviridae (2 segments), Totiviridae (1 segment), Chrysoviridae (4 segments) and Cystoviridae (3 segments) families. Contigs/segments containing an RNA-dependent RNA Polymerase (RdRP) region where identified using profile hidden markov models. In total, we could recover nearly 300 putative RdRP-containing contigs, longer than 1500 nucleotides, showing homology to aforementioned viral families. In total 41% of all Chrysovirus contigs where identified as segments containing an RdRP region (66 contigs), while for Partitivirus contigs 56% of all contigs contained an RdRP region (202 contigs). These skewed numbers potentially reflect the evolutionary constrain on the RdRP protein, while capsid and other segments could be to divergent to recover with regular BLAST-based methods. For Totivirus contigs 81% of them contained an RdRP region (18 contigs). Surprisingly, no RdRP motifs could be recovered for the Cystovirus contig set, indicating another protein is more conserved than the RdRP. Subsequent phylogenetic analysis reveals that many of these sequences constitute novel clades, reflecting the wide diversity of multiple families within the dsRNA virus group, associated with honeybees.


Epizootic hemorrhagic disease virus identified in affected cattle in Japan during 2015−2017

Hiroaki Shirafuji, Natsumi Kobayashi, Katsunori Murota, Shogo Tanaka, Tohru Yanase

National Institute of Animal Health, Japan

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus in the family Reoviridae. The EHDV is transmitted among ruminant hosts by the bite of Culicoides biting midges. We previously characterized 11 Japanese strains of EHDV isolated in 1985−2013 and identified them as EHDV-1, EHDV-2, EHDV-7 and another serotype tentatively named ‘EHDV-10’ (Shirafuji et al., Infect Genet Evol 53: 38−46. 2017). EHDV infection in cattle does not usually result in clinical disease, but a strain of EHDV-2, Ibaraki virus, has been known to cause Ibaraki disease in cattle. Recently, EHDV infections with other serotypes than EHDV-2 have been observed in affected cattle in Japan during 2015−2017. Forty-six cattle were affected in Hyogo Prefecture in October−December 2015, and the causative agent was found to be EHDV-6. The clinical and pathological findings in the case were similar to those observed for Ibaraki disease in cattle. In September−October 2016, 13 cattle in a farm displayed fever in Fukuoka Prefecture, and EHDV-7 was isolated from 3 of the 13 affected cattle. In August 2017, EHDV-5 RNA was detected in an aborted bovine fetus in Ishigaki island, Okinawa Prefecture. We thus conducted phylogenetic analysis of genome segments 2 and 3, and all the EHDVs identified in 2015−2017 were closely related with Japanese or Australian EHDV isolates. Although bovine clinical cases caused by EHDV-6 or EHDV-7 have been reported mainly in the Middle East and Africa, our results suggest that the EHDVs identified in Japan in 2015−2017 derive from EHDVs circulating in Asia-Pacific region.


A proposal for the lineage framework for sub-genotype phylogeny of the backbone genes of DS-1-like Rotavirus A strains.

Chantal Ama Agbemabiese (1), Toyoko Nakagomi (2), Susan Afua Damanka (1), Francis Ekow Dennis (1), Belinda Larteley Lartey (1), Frederick Karikari Asamoah (1), Michael Fokuo Ofori (1), George Enyimah Armah (1), Osamu Nakagomi (2)

1: Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana; 2: Nagasaki University, Nagasaki, Japan

There has been a global explosion in the detection of unusual G1P[8], G3P[8] and G8P[8] Rotavirus A strains bearing the DS-1-like genetic backbone (I2-R2-C2-M2-A2-N2-T2-E2-H2;genotype 2). However, it is often difficult to understand how such strains are related to each other in their backbone genes because current genotype 2 lineage designation is mostly limited to the genes of G2P[4] strains. Thus, we systematically collected genotype 2 sequences and performed phylogenetic analysis on them aiming at proposing a reference framework for lineage designation of global genotype 2 genes from both human and animal rotaviruses.Over 8500 RVA genotype 2 genes were systematically retrieved from the rotavirus database within the NCBI Virus Variation Resource. Alignment analysis tool in Virus Variation was used to generate multiple sequence alignments. Downloaded sequences were curated, p-distances determined and robust phylogenetic inferences made using MEGA 6.06. Together with the bootstrap values, clades, lineages and sub-lineages were designated.A phylogenetic framework was established for the classification of genotype 2 genes at the lineage level and <80 reference sequences depending on the gene were selected to represent lineages. More diverse lineages were observed in genotypes detected in both human and animal rotaviruses e.g. R2,C2,I2,N2 and E2. The backbone genes of unusual G1P[8], G3P[8] and G8P[8] reassortant strains originated primarily from contemporary DS-1-like strains, indicating that intergenogroup reassortment events occurred in the recent past. The established lineage framework with selected reference sequences will help researchers to distinguish the sources of the genes of both common and unusual DS-1-like strains in circulation.


Predominance of norovirus in children hospitalized with gastroenteritis after rotavirus vaccine introduction in Burkina Faso

Isidore Juste O. Bonkoungou (1), Ylva Rönnelid (2), Nafissatou Ouedraogo (3), Nicolas Barro (1), Lennart Svensson (2,4), Johan Nordgren (2)

1: University Ouaga 1 Pr Joseph KI-ZERBO, Burkina Faso; 2: Linköping University, Sweden; 3: University of Dedougou, Burkina Faso; 4: Division of Medicline, Karolinska Institute, Sweden

In Burkina Faso, national rotavirus (RV) vaccination using RotaTeq was implemented in October 2013. Follow up studies have reported a reduction of RV gastroenteritis after vaccination. Studies before RV vaccination have shown norovirus (NoV) to be the second most common viral pathogen, with approximately half the detection rate compared to RV. The aim of this study was to investigate the detection rate and molecular epidemiology of NoV and RV after vaccine implementation.From January to December 2015, 146 children <5 years of age hospitalized with acute gastroenteritis was enrolled at Hôpital du District de Bogodogo in the capital Ouagadougou. Stool specimens were collected and screened for RV and NoV by ELISA and TaqMan real-time PCR, respectively. The positive samples were further genotyped by sequencing.RV was found in 14% (20/146) and NoV in 20% (29/146) of samples. RV predominated during January to May while NoV was detected year round. G2P[4] was the most prevalent RV genotype with 32% followed by G12P[6] 26% and G12P[8] 21%. A high diversity of circulating NoV genotypes was observed with 10 different genotypes detected during the study. Genotype GII.4 strains predominated with 41% followed by GII.6 at 10% and GII.3, GII.6, GII.9, GII.12,GII.14, GII.15, GI.3 GI.5 to a lesser extent.To conclude, after the introduction of RV vaccination in Burkina Faso NoV is detected at higher rates than RV in children with severe diarrhea, with a high diversity of circulating genotypes. This study highlights the need for further assessment of NoV following RV vaccine introduction.


Neutral Evolutionary Rate of Rhesus Rotavirus

Syun-suke Kadoya (1), Syun-ichi Urayama (2), Takuro Nunoura (3), Masaaki Kitajima (4), Satoshi Okabe (4), Toyoko Nakagomi (5), Osamu Nakagomi (5), Daisuke Sano (1)

1: Tohoku University, Japan; 2: University of Tsukuba, Japan; 3: Japan Agency for Marine-Earth Science and Technology, Japan; 4: Hokkaido University, Japan; 5: Nagasaki University

The introduction of two rotavirus vaccines has resulted in decrease in infant deaths from rotavirus disease, but simultaneously, the genotype-specific effectiveness of these vaccines has been reported. If the immune responses induced by vaccines act as selection pressure on a genetically diverse population of rotavirus, the current target genotypes of the vaccines may obtain vaccine-tolerance. The combination with effective drugs is thus important for the infection control of rotavirus. Phenotypes with slower evolutionary rate are favorable as the drug targets, but the neutral evolutionary rate of rotavirus under the absence of selection pressures has not been well investigated. In this study, we performed the whole genome analysis of rhesus rotavirus (RRV, G3P[3]) populations obtained from 5-time serial passages using MA104 cells at the MOI of 10-1. The dN/dS ratio calculated from NGS data showed that selection pressure did not significantly exert on any genome segments. The neutral evolutionary rate, estimated by BEAST2 software, ranged from 5.03 x 10-5 (VP1) to 44.4 x 10-5 (NSP4) nucleotide substitutions/site/passage. VP1 (3267 bp), VP2 (2664 bp, 9.20 x 10-5 nucleotide substitutions/site/cycle) and VP4 (2362 bp, 8.02 x 10-5 nucleotide substitutions/site/cycle) were relatively conservative among twelve rotaviral proteins. We also found that there was a significantly negative correlation (p < 0.01) between the evolutionary rate value and genome segment size. These results indicate that the neutral evolutionary rate is dependent on the genome size, and viral proteins encoded by longer genome segments such as VP1, VP2 and VP4 are more preferable as drug targets.


Comparative analysis of trivalent P2-VP8 subunit rotavirus vaccine strains and P[8], P[4], and P[6] human rotaviruses reported globally during 1974 and 2017

Daniel E Velasquez Portocarrero, Baoming Jiang

Centers for Disease Control and Prevention, Atlanta, GA, USA

Non-replicating rotavirus vaccine candidates are in development for parenteral administration. One of them is a truncated recombinant VP8* protein, expressed in E. coli from original sequences of the prototype human rotavirus genotypes P[8], P[4] or P[6] isolated before 1983.From the GenBank, we retrieved 6,366 rotavirus VP8* gene sequences of strains P[8], P[4] or P[6] isolated from fecal specimens of children between 1974 and 2017, in 77 countries. We compared them with the sequences of the three P2-VP8 vaccine strains: Wa (USA, 1974, G1P[8]), DS-1 (USA, 1976, G2P[4]) and 1076 (Sweden, 1983, G2P[6]).Phylogenetic analysis showed that 94.9% (4,328/4,560), 99.8% (1,141/1,143), and 100% (663/663) of the strains P[8], P[4] or P[6] reported globally between 1974 and 2018 belong to non-vaccine lineages. These global P[8], P[4] or P[6] rotavirus strains have a mean of 9%, 5%, and 6% amino acid difference with the corresponding vaccine strain, respectively. Additionally, in the USA, the mean percentage difference between the P[8] rotavirus strains and the Wa strain increased with the time: 4% [during 1,974 - 1,980], 5% [1988 – 1991], and 9% [2005 - 2013], respectively.Our analysis found high evolutionary changes in VP8* of the three major rotavirus P[8], P[4] and P[6] strains and their increasing variations from the candidate subunit vaccine strains over time. We therefore suggest the development of parenteral vaccines composed of either an inactivated whole virus or constructs with various antigens derived from different gene segments to more effectively control rotavirus diarrhea


WORKSHOP 3: MOLECULAR AND CELLULAR VIROLOGY

Reverse genetics systems for orthoreoviruses and rotaviruses

Takeshi Kobayashi

Department of Virology, Research Institute for Microbial Diseases, Osaka University

A reverse genetics system to engineer viable viruses that contain a specific sequence modification has provided platforms to study viral replication and pathogenesis, and for developing vaccines and viral vectors. The development of a reverse genetics system for the Reoviridae family has lagged behind that for other animal RNA virus families because of the technical complexities associated with the manipulation of multiple-segmented double-stranded RNA genomes. However, in recent years, entirely plasmid and RNA-based reverse genetics systems have been established for several members of the Reoviridae family, including non-fusogenic mammalian orthoreovirus (genus Orthoreovirus), bluetongue virus, African horse sickness virus, and epizootic hemorrhagic disease virus (genus Orbivirus). In contrast with the reverse genetics systems established for these viruses, other important pathogens in this family, including rotaviruses (RVs), which cause severe gastroenteritis in infants, have not been generated entirely from cloned cDNAs. Recently, we developed two entirely plasmid-based reverse genetics systems for RVs and fusogenic bat-borne Nelson Bay orthoreoviruses associated with acute respiratory tract infections in humans. We used the systems to obtain new insights into the roles of viral structural and nonstructural proteins in their replication and pathogenesis. We also used the technologies to establish efficient gene transfer systems for the studying of live-cell imaging and anti-viral screening. The reverse genetics approach can be exploited for Reoviridae virus biology and used to develop vaccines, diagnostics, and therapeutics.


From Bench to Bedside: Screening of Small Molecules to Better Understand Virus Biology and Develop Improved Therapeutics

Angela K Berger (1,2), Jameson TL Berry (1), Roxana M Rodriguez Stewart (1), Bernardo A Mainou (1,2)

1: Department of Pediatrics, Emory University, Atlanta, GA, USA; 2: Children’s Healthcare of Atlanta, Atlanta, GA, USA

Reovirus is a nonenveloped, segmented dsRNA virus that infects most humans during childhood but seldom causes disease. Reovirus attaches to cell-surface carbohydrate, junction adhesion molecule-A (JAM-A) and the Nogo receptor, and enters via receptor-mediated endocytosis. To identify host factors that modulate infection, we screened compounds in the NIH Clinical Collection for enhancement or impairment of reovirus infectivity. Loperamide, a mu-opioid receptor agonist that is used as an anti-diarrheal, impairs reovirus infectivity without affecting cell number. Doxorubicin, a DNA-damaging drug, reduced cell number while also increasing the number of infected cells. Loperamide does not affect reovirus attachment to cells, but delays viral disassembly during cell entry, impairs establishment of viral factories, and synthesis of viral RNA and protein. Infection with infectious subvirion particles (ISVPs), which use JAM-A to infect cells but do not require endosomal transport, is not affected by loperamide. Doxorubicin has been used as a chemotherapeutic for decades for its anti-neoplastic properties, but its use has been dampened due to off-target cytotoxicities, including cardiomyopathy from days to up to years after treatment. To enhance delivery of doxorubicin to target cells and minimize off-target cytotoxicity, we conjugated doxorubicin to reovirus particles using a chemical linker. Reovirus-doxorubicin has enhanced cytotoxicity and induces DNA damage to similar levels than unconjugated doxorubicin in breast cancer cells. Conjugation of doxorubicin to reovirus does not affect attachment to cells or overall replication kinetics. We show that high-throughput screening with reovirus can be used to better understand virus biology and develop therapeutics to enhance patient outcomes.

91Oral and shotgun presentations Workshop 3: Molecular and Cellular Virology

Generation of Recombinant Rotaviruses Expressing UnaG Fluorescent Protein Using the RNA Capping Enzyme of African Swine Fever Virus

Asha Ann Philip (1), Siyuan Ding (2), Heather E Heaton (3), Chantal A Agbemabiese (4), Maya Shmulevitz (3), Harry G Greenberg (2), John T Patton (1)

1: Indiana University, Indiana, USA; 2: Stanford University, California, USA; 3: University of Alberta, Alberta, Canada; 4: University of Ghana, Ghana

Recently, Kanai and his colleagues developed an all plasmid-based reverse genetics (RG) system for SA11 rotavirus. The system consists of eleven T7-transcription plasmids, each expressing a unique SA11 (+)RNA, and three CMV support plasmids, one expressing an avian reovirus fusogenic FAST protein, and the other two expressing the vaccinia virus D1R and D12L capping proteins. In attempting to simply and improve the RG system, we synthesized the gene encoding African swine fever virus (NP868R), a capping enzyme with RNA triphosphatase, guanylyltransferase, and methyltransferase activities. The NP868R gene was cloned into a CMV expression vector and used in the RG system instead of D1R and D12L plasmids. RG experiments indicated that the NP868R plasmid was significantly more efficient in promoting formation of recombinant virus than the D1R/D12L plasmids. The extent to which the improved efficiency resulted from a reduction in the number of plasmids required (13 > 12) or a more robust activity of the ASFV capping enzyme is not known. Using our modified system, we then re-engineered SA11 segments 5 (NSP1) and 7 (NSP3) to express UnaG, small bilirubin-inducible eel fluorescent protein, leading to the recovery of epifluorescent ‘green’ recombinant viruses. Through the use of 2A translational stop-start elements, we also re-engineered rotavirus segments such they not only expressed full-length wildtype viral protein, but also the UnaG protein. Thus, we have generated a recombinant rotavirus that expresses 13 proteins, 12 of viral origin and one representing a foreign protein, advancing the use of rotaviruses as plug-and-play expression vectors.


Measuring forces during the cellular uptake of mammalian reovirus: Physico-chemical regulation of early events of clathrin-mediated endocytosis of viral particles

Marta Fratini (1,2), Tina Wiegan (2), Joachim Spatz (2), Ada Calvacanti-Adams (2), Steeve Boulant (1)

1: Universtiy Hospital Heidelberg Department of Infectious Disease and DKFZ, Germany; 2: Heidelberg University, Institute of Physical Chemistry, Department of Biophysical Chemistry

While the importance of cellular receptors and endocytic routes to mediate viral entry have been thoroughly characterized, how the physical properties (shape and size) of viruses participate in endocytosis and what are the mechanical parameters (forces) of virus uptake by cells remains elusive. In this work, we exploited reovirus and applied click-chemistry to covalently immobilize viral particles on glass surfaces to study early host-virus interactions. We could show that while the recruitment of the clathrin-endocytic machinery is dependent on particle size, the further commitment that leads to productive clathrin-coated vesicles is independent of cargo internalization. Moreover, we unexpectedly found that the final size of the clathrin vesicle depends on the particle size and appears “imprinted” in the clathrin coat at the early cargo-cell interaction. By combining live-cell microscopy and surface chemistry approaches with the development of biosensors for single molecule force measurements, we established a method to study the physical interaction of single viral particles with the cell surface. We could show that the crucial steps for force generation by the cell on viral particles are initiated by membrane bending depending on particle size, and do not rely on specific interactions with receptors at the cell surfaces (integrins, JAM-A) and cytoskeleton contractility. This suggests that a fine regulation of local, rather than global, physical events is crucial for virus uptake. Our studies provide evidence that the early surface interactions of viruses with cells are essentially determined by a size-dependent change in membrane curvature that participate in triggering the internalization events.


The use of a soluble African horse sickness virus VP7 protein to study virus replication and vaccine development

Shani May Bekker (1), Christiaan Potgieter (2), Vida van Staden (1), Jacques Theron (1)

1: Department of Biochemistry, Genetics and Microbiology, University of Pretoria, South Africa; 2: Deltamune (Pty) Ltd, Centurion, South Africa

African horse sickness is a deadly and highly infectious disease of equids caused by African horse sickness virus (AHSV). Along with bluetongue virus (BTV), AHSV is one of the most economically important members of the Orbivirus genus. Structural protein VP7 is the major core protein of orbiviruses. A unique characteristic of AHSV VP7 is that, unlike any of the cognate orbivirus proteins, it is highly insoluble and spontaneously forms flat hexagonal crystalline particles when expressed recombinantly and in AHSV-infected cells. The aggregation of AHSV VP7 into these crystals present many problems in AHSV vaccine development and it is unclear if VP7 aggregation affects AHSV assembly or contributes to AHSV pathogenesis. We therefore set out to abolish AHSV VP7 crystalline particle formation by targeting surface-exposed residues of the VP7 trimer that play a role in VP7 trimer-trimer interactions. We found that disruption of trimer-trimer interactions by several amino acid substitutions converted VP7 to a fully soluble protein which abolished the formation of VP7 crystals. To examine the role of AHSV VP7 during replication, we used a plasmid-based reverse genetics system to generate a recombinant AHSV that does not form crystalline particles. We found that the presence VP7 crystals affect AHSV release and yield. This work provides insight into AHSV replication and possibly cytopathogenesis. We also found that substitution of VP7 with a soluble version increases core-like particle yields. Therefore, the availability of a soluble AHSV VP7 now unlocks new possibilities for AHSV vaccine development.


Viroplasms and remodelled stress granules and P-bodies: Union of the triad to promote progeny rotavirus production

Durga Rao Chilakalapudi, Poonam Dhillon

Indian Institute of Science, India

Rotavirus replicates in virus-induced unique cytoplasmic inclusion bodies called viroplasms (VMs). Recently we have demonstrated that rotavirus infection induces cytoplasmic re-localization and colocalization with VMs of several host proteins, which either promoted or inhibited virus growth. Based on the analysis of a few proteins, earlier studies reported that rotavirus infection inhibits stress granule (SG) formation and disrupts P-bodies (PBs). However, our recent observation of sequestration in the VM of a large number of AU-rich element-binding proteins (ARE-BPs), which are known components of SGs/PBs, suggested to the possibility that rotavirus might induce remodelling of SGs and PBs. This observation formed the basis to investigate a large number of their components to understnad the status of SGs and PBs in rotavirus-infected cells. Here we demonstrate that rotavirus infection induces molecular triage by selective exclusion of a few proteins of SGs and PBs and sequestration of the re-modelled/atypical cellular organelles containing majority of their components in the VM. The punctate SG and PB structures are seen at about 4 hpi, coinciding with the appearance of small VMs, many of which fuse to form mature large VMs with progression of infection. Employing siRNA-mediated knock-down and/or ectopic over-expression, majority of the SG and PB components, except ADAR1, are observed to inhibit virus growth. In conclusion, this study demonstrates that VMs are highly complex supramolecular structures and that rotavirus employs a novel strategy of sequestration in the VM and harnessing the remodelled cellular RNA recycling bins to promote its growth.


Mapping the RNA binding and packaging motifs of Bluetongue virus capsid protein VP6 by biophysical and molecular analysis

Po-yu Sung (1), Cheng Kao (2), Polly Roy (1)

1: London School of Hygiene and Tropical Medicine, UK; 2: Indiana University, IN, United States

Bluetongue virus (BTV) is an important animal pathogen and the prototype of Orbivirus, a genus of the Reoviridae family. Orbiviruses have a unique structural protein, VP6, located inside the viral inner capsid. During replication, genomic single-stranded positive sense RNA (ssRNA) segments are packaged into the assembling virus capsid, prior to double-stranded RNA (dsRNA) synthesis. VP6 is rich with charge residues and binds any nucleic acid. Since VP6 has both ssRNA and dsRNA binding affinity and plays a role in the ssRNA packaging process, we utilised bioinformatics and phylogenetic analyses, as well as RNA-cross-linking and peptide fingerprinting (RCAP) using both the recombinant VP6 (reVP6) and BTV capsid to identify the RNA binding sites of VP6. The importance of these regions in VP6 were then examined by introducing a series of site-specific mutations in the replicating genome, using BTV reverse genetics (RG), as well as reVP6 for in vitro binding activity. Accumulating data showed that VP6 has multiple RNA binding regions; that ssRNA and dsRNA bound to recombinant VP6 (reVP6) at similar regions, whilst the RCAP of BTV capsid showed a different interaction profile of interactions between VP6 and genomic dsRNA. Through the RG system, we showed that VP6-RNA interactions in the viral capsid are essential for virus replication. Further, in vitro completion assay revealed the RNA-binding motif within VP6 selectively binds BTV RNA indicating that this motif may be involved in RNA recruitment during capsid assembly.


Orbivirus replication and reverse genetics

Piet A. van Rijn

Department of Virology, Wageningen Bioveterinary Research (WBVR), Lelystad, The NetherlandsDepartment of Biochemistry, North-West University, Potchefstroom, South Africa

Bluetongue (BT), African Horse Sickness (AHS) and Epizootic Haemorrhagic Disease (EHD) caused by insect borne orbiviruses (Orbivirus, Reoviridae) are notifiable animal diseases according to the World Organization for Animal Health (OIE). Research on orbivirus is mainly focused on the prototype BTV and virus species AHSV and EHDV. Each of these orbivirus species consists of multiple serotypes showing no or low cross protection, and is transmitted by so-named competent insect vectors, specific Culicoides species. Spread of disease is almost completely dependent on virus transmission by competent midges. Thus, orbivirus must replicate efficiently in the susceptible mammalian host and the competent midge vector leading to viremia in the host and virus release in midge saliva, respectively, which drives virus transmission between both organisms. This complex life cycle and virus replication in completely different organisms make insect borne orbiviruses an intriguing subject to study virology for more than a century. In the last decade, a tremendous progress has been made by accomplishment of reverse genetics enabling research on viral proteins in infected cells from different origin. Similarly, virus mutants can be studied in vivo in infected hosts and in inoculated or fed insects. Using reverse genetics, genome flexibility and requirements of RNA sequences can be investigated in more detail through forcing the genome constellation by rescue of ‘synthetic’ reassortants and by genetic modification to generate virus mutants. Several examples of recent research using reverse genetics will be discussed, including the development of a new generation of promising vaccine candidates to combat these devastating animal diseases. Currently, molecular interactions between virus and its competent vector is poorly understood. New insights about the complex phenomenon ‘vector competence’ which is crucial in the life cycle of midge borne orbiviruses can be expected by use of reverse genetics. This technology will play a key role in future research to further unravel the multiple vector-virus-host interactions of these groups of successful orbiviruses.


Study of molecular interactions between BTV and its hosts

Damien Vitour, Grégory Caignard, Cindy Kundlacz, Aurore Fablet, Marie Pourcelot, Rayane Amaral, Corinne Sailleau, Emmanuel Bréard, Cyril Viarouge, Stephan Zientara

ANSES, France

Bluetongue virus (BTV), which belongs to the Orbivirus genus, is an arbovirus transmitted by Culicoides biting midges. More than 27 serotypes (BTV1-27) are recognized today with no or poor cross-neutralization in between serotypes. BTV infects a wide range of domestic and wild ruminants and provokes clinical manifestations ranging from asymptomatic infection to lethal haemorrhagic fever. This variability is due to several viral and host factors that have been only partially elucidated. In order to gain insight into BTV-host interactions at the cellular and molecular level, we aim to develop high-throughput screening approaches, notably based on the yeast two-hybrid (Y2H) system, to map interactions between viral and cellular proteins. A complete set of viral proteins from BTV8, a serotype that has recently re-emerged in France, was used as baits to screen two cDNA libraries originating from hosts naturally infected by BTV: Culicoides and cattle. These libraries have been chosen to provide a comparative view of protein interactions at the host level (mammalian host versus arthropod vector). Therefore, 28 Y2H screens were performed allowing us to identify 61 cellular interactors. A preliminary global analysis of these interactions has uncovered many signal transduction factors involved in the modulation of autophagy, apoptosis or the ubiquitin-proteasome system. Interactions are currently re-tested by several methods (Y2H, GST pulldown and GPCA (Gaussia princeps protein complementation assay). By targeting such protein functions, it should be possible to test or design drugs and peptides that are able to disrupt the interactions previously identified and potentially confer antiviral properties.


Dissection of rotavirus replication intermediates NSP5 and VP2: A step towards an antiviral

Catherine C Eichwald (1), Antonino Buttafuoco (1), Kevin MIchaelsen (1), Kurt Tobler (1), Stuart Sims (1), Ohad Medalia (2), Cornel Fraefel (1), Mathias Ackermann (1)

1: Institute of Virology, University of Zurich, Zurich, Switzerland; 2: Dept. of Biochemistry, University of Zurich, Switzerland

In the course of rotavirus (RV) replication, the core-shell protein VP2 is an essential component of viroplasms. VP2 spontaneously forms core-shell structures, however, when co-expressed with the viroplasm matrix protein NSP5, viroplasms-like structures (VLSs) are formed. The VLSs are cytosolic inclusions morphologically identical to replicative viroplasms but composed only of NSP5 with VP2 or NSP2. We hypothesized that the NSP5 interaction with VP2 disrupts spontaneous cores to allow the formation of viroplasms. By dissecting VP2 and NSP5, we have identified and characterized the protein-protein interaction partners for replication intermediates between these two proteins based on VLS formation. We found that VP2 (simian strain SA11) single point mutation to alanine of amino acid L124, V865 or I879 in co-expression with NSP5 impaired VLS formation as well as the triggering of NSP5 hyperphosphorylation. Additionally, we established that NSP5 and VP2 interaction, by binding assay and VLS formation, is independent of the NSP5 hyperphoshorylation state. On the other hand, NSP5 tail region (amino acids 180-198) is required to associate with VP2. In silico analyses revealed that VP2 L1224, V865, and I897 are highly conserved not only among members of group A but also in RV groups B to H as denoted by sequence alignments and 3D-projection from the VP2 crystal structure. Specifically, VP2 L124A concedes core-shell formation, and its over-expression in MA104 cells reduces rotavirus infectivity. The conserved interaction domains identified for both VP2 and NSP5, essentials for the replication initiation, make them excellent targets for the development of novel and broad-range antivirals.


Visualization of Ca2+ loss from rotavirus during cell entry

Eric N. Salgado (1), Brian Garcia Rodriguez (1), Nagarjun Narayanaswamy (3), Yamuna Krishnan (3,4), Stephen C. Harrison (1,2)

1: Children’s Hospital Boston, Harvard Medical School, USA; 2: Howard Hughes Medical Institute, USA; 3: University of Chicago, USA; 4: Grossman Institute of Neuroscience, Quantitative Biology and Human Behavior, University of Chicago, USA

The rotavirus triple-layered particle (TLP) contains the double-layered particle (DLP), an outer shell of Ca2+-mediated VP7 trimers, and 60 trimeric VP4 spikes anchored on the particle by the VP7 layer. We have previously demonstrated that these three components of rhesus rotavirus (RRV) TLPs can be individually purified, fluorescently labeled, and reconstituted into fully infectious virus particles (rcTLPs). Labeled rcTLPs can be tracked in living cells by fluorescence microscopy, allowing us to monitor the uncoating of the VP7 shell and release of the DLP into the cytoplasm.We report here the generation of rcTLPs containing a Ca2+ sensor, Rhodamine 5F (Rhod5F) azide, conjugated to a cysteine mutant of VP4, VP4 S469C. Live-cell imaging of these Rhod5F rcTLPs allows the sensor to report the loss of Ca2+ from the vesicular environment surrounding the particle in question. From these data we see that the loss of Ca2+ surrounding a given rcTLP is gradual and begins on average 2 min before loss of VP7 and 7 min before DLP release. Combined with further mutagenesis studies of VP4, and advances in cryoEM studies of the spike conformation in situ, these live-cell data modify and enahnce our structural understanding of RRV membrane interactions and the initialization of Ca2+ loss from the TLP.The talk will present the novel Ca2+ sensor data, integrating them into the picture of entry informed by the new cryoEM results.


Molecular determinants of bluetongue virus that drive replication in the insect-vector cell

Marc Guimera Busquets (1), Gillian Pullinger (1), Massimo Palmarini (2), Karin E. Darpel (1), Rennos Fragkoudis (1,3), Peter P. C. Mertens (1,3)

1: The Pirbright Institute, Woking, Surrey, UK. GU24 0NF; 2: MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, UK. G61 1QH; 3: The School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Leicestershire, UK. LE12 5RD

Bluetongue virus (genus Orbivirus, family Reoviridae) needs to replicate in its vectors, biting midges of the genus Culicoides, in order to be transmitted between susceptible vertebrate hosts, which include both wild and domestic ruminants. However, since 2008 novel BTV strains have been identified that are unable to amplify effectively in BTV-susceptible adult Culicoides sonorensis midges, or in a Culicoides-derived cell line (KC cells). These novel BTV serotypes, which include BTV-26, have been labelled as “non-conventional” and appear to use direct contact as a main route of transmission, changing our understanding of BTV evolution and epidemiology. Using reverse genetics, we have linked the inability of BTV-26 to replicate in vector KC cells with 4 viral genome segments/proteins: Seg-1/VP1 (the viral RNA dependent RNA polymerase); Seg-2/VP2 (major outer-capsid-protein); Seg-3/VP3 (sub-core-shell protein) and Seg-7/VP7 (outer-core protein) [Pullinger et al., 2016].In this study, we further characterised mechanisms involving these genes/proteins that can prevent or reduce BTV-26 propagation in KC cells. The ability of BTV-26 to bind and enter KC cells, along with other stages of the BTV replication cycle were investigated in insect cells, in comparison to other conventional BTV strains. We demonstrate that the propagation of BTV-26 is blocked at multiple stages in Culicoides cells, providing a better understanding of the replication of this serotype, as well as the transmission of BTV by these vector-insects. Altogether, these results shed light on BTV-Culicoides interactions, showing that these non-conventional strains are powerful tools for the study of BTV determinants that drive vector-competence and transmission.


Local implementation and optimisation of entirely plasmid-based SA11 rotavirus reverse genetics

Marno GJ Huyzers (1), Abraham Christiaan Potgieter (1,2), Louis Jeremia Cornelius Theart (1), Alberdina Aike van Dijk (1)

1: North-West University, Potchefstroom, South Africa; 2: Deltamune (Pty) Ltd, South Africa

The recent breakthrough of the development of the first entirely plasmid-based reverse genetics system for rotaviruses by the Japanese group has generated huge excitement. However, to date mixed results have been obtained in recreating the Japanese reverse genetic system in various institutions, showing that this RG system still needs further development and optimization. The first reports on improving the efficacy of the system and using it are starting to emerge. We will report on two entirely plasmid-based SA11 RG systems.We purchased the Japanese SA11-L2 based RG system from Addgene. However, we could not rescue by transfecting BHK-T7 cells and co-seeding with MA104 cells. We did have some success transfecting BSR-T7 cells that are used for African horsesickness virus rescue, using equimolar amounts of the SA11 plasmids instead of equimicrogram amounts, varying the ratio between the SA11 transcription and capping and FAST expression plasmids, co-seeding with and passaging once in swine testis (ST) cells before switching to MA104 cells.We also generated a set of plasmids containing consensus sequence SA11-N5 genome segments using the significantly smaller pSMART as backbone. Our results suggest that reducing the plasmid load improves the efficacy of rescue. Other modifications that we made include using the genomic HDV ribozyme instead of the antigenomic ribozyme, transfecting BSR-T7 cells and co-seeding with ST cells. This approach seems to improve rescue compared to the original Japanese system.


Expression of a structural protein of the mycovirus FgV-ch9 negatively affects the transcript level of a novel symptom alleviation factor and causes virus-infection like symptoms in Fusarium graminearum

Joerg Bormann (1,2), Cornelia Heinze (2), Christine Blum (2), Michael Mentges (2), Anke Brockmann (2), Arne Alder (2), Svenja Kim Landt (2), Brian Josephson (3), Daniela Indenbirken (4), Michael Spohn (4), Birte Plitzko (3), Sandra Loesgen (3), Michael Freitag (3), Wilhelm Schäfer (2)

1: University Bremen, Germany; 2: University Hamburg, Germany; 3: Oregon State University, USA; 4: Heinrich-Pette-Insititut, Germany

Infections of fungi by mycoviruses are often symptomless but sometimes also fatal as they perturb sporulation, growth, and, if applicable, virulence of the fungal host. Hypovirulence-inducing mycoviruses, therefore, represent a powerful mean to defeat fungal epidemics on crop plants. Infection with Fusarium graminearum virus China 9 (FgV-ch9), a dsRNA chrysovirus-like mycovirus, debilitates Fusarium graminearum, the causal agent of Fusarium Head Blight. In search for potential symptom alleviation or aggravation factors in F. graminearum, we consecutively infected a custom-made F. graminearum mutant collection with FgV-ch9 and found a mutant with constantly elevated expression of a gene coding for a putative mRNA-binding protein that did not show any disease symptoms despite harboring high amounts of virus. Deletion of this gene, named virus response 1 (vr1), resulted in phenotypes identical to those observed in the virus-infected wild type with respect to growth, reproduction, and virulence. Similarly, the viral structural protein coded on segment 3 (P3) caused virus-infection like symptoms when expressed in the wild-type but not in the vr1-overexpression mutant. Gene expression analysis revealed a drastic downregulation of vr1 in the presence of virus and in mutants expressing P3. We conclude that symptom development and severity correlate with gene expression levels of vr1. This was confirmed by comparative transcriptome analysis showing a large transcriptional overlap between the virus-infected wild type, the vr1 deletion mutant and the P3-expressing mutant. Hence, vr1 represents a fundamental host factor for the expression of virus-related symptoms and helps to understand the underlying mechanism of hypovirulence.


Molecular mechanism of polymerase activity and RNA-binding properties of E. coli expressed different domains of Antheraea mylitta cytoplasmic polyhedrosis virus RNA-dependent RNA polymerase

Anirban Kundu, Amit Kumar Das, Ananta Kumar Ghosh

Indian Institute of Technology Kharagpur, India

Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is responsible for morbidity of the Indian non-mulberry silkworm, A. mylitta. AmCPV belongs to the family Reoviridae and has 11 double-stranded (ds) RNA genome segments (S1-S11). Segment 2 (S2) encodes a 123-kDa polypeptide with RNA-dependent RNA polymerase (RdRp) activity and helps the virus to propagate its genome in the host cell. To examine the RNA-binding properties and for functional analysis, the full-length RdRp and its three domains [N-terminal (NTD), central polymerase (PD) and C-terminal domains (CTD)] were expressed in E. coli with hexahistidine and trigger factor tag fused at its amino terminus, and the soluble fusion proteins were purified. The PD showed nucleotide binding properties, but not the NTD and CTD. Isolated PD did not exhibit RdRp activity due to increased internal motion but the activity can be reconstituted when all three domains are included together. RNA binding assay showed that the purified full-length polymerase specifically bound to the 3’ untranslated region (3’-UTR) of a viral plus-sense (+) strand RNA but the isolated polymerase domain of the enzyme exhibited poor RNA binding ability. Further, the RdRp recognition signals were found to be different from the cis-acting signals that promote minus-sense (-) strand RNA synthesis, because different internal regions of the 3’-UTR of the (+) strand RNA did not effectively compete out the binding of RdRp to the intact 3’-UTR of the (+) strand RNA, but all of these RNA molecules could serve as templates for (-) strand RNA synthesis by the polymerase.


WORKSHOP 4: IMMUNITY AND PATHOGENESIS

Factories, Granules, and Hypoxia: Does stress granule disruption by mammalian orthoreovirus factory proteins contribute to hypoxia downregulation in tumor cells?

Cathy Miller, Bussiere Luke, Promisree Choudhury

Iowa State University, USA

Our lab has shown that mammalian orthoreovirus (MRV) induces, then disrupts stress granules (SGs). SG disruption is driven by interaction of the MRV replication protein, σNS, with SG effector protein G3BP, and viral factory matrix protein, μNS. SG formation and disruption correlates with induction of and release from translation inhibition. Moreover, G3BP knockout results in enhanced replication of viral strains that induce translational shutoff. Interestingly, SGs contain the ribosomal associated protein RACK1. RACK1 plays a critical role in the regulation of HIF-1, the master transcriptional activator of the cellular hypoxic response. Upregulation of HIF-1 drives tumor cell proliferation and metastasis, and is a prognostic marker of biochemical failure and mortality in cancer. Hypoxia-induced SGs sequester and inhibit RACK1 activity. As we have previously shown that MRV infection induces a massive downregulation of HIF-1 in a manner dependent on RACK1, we hypothesized that MRV-induced disruption of SGs may release RACK1 and drive HIF-1 degradation. We found that RACK1 was sequestered in SGs in hypoxic tumor cells, and was released from SGs upon MRV infection. Additional work examining the role of SG disruption and viral protein involvement in RACK1-dependent HIF-1 degradation in MRV infected hypoxic tumor cells will be discussed. These findings link interactions between the virus and host cell that impact tumor cell survival in a specific tumor microenvironment and shed light on potential treatment modalities for MRV as a cancer therapeutic.


Reovirus non-structural protein σ1s enhances viral resistance to type-1 interferons

Karl W. Boehme

Department of Microbiology and Immunology and Center for Microbial Pathogenesis and Host Inflammatory Responses, University of Arkansas for Medical Sciences, Little Rock, AR

Mammalian orthoreovirus (reovirus) spreads from the site of infection to every organ system in the body via the blood. Non-structural protein σ1s is a critical viral determinant of reovirus bloodstream dissemination. The mechanism by which σ1s promotes hematogenous spread remains undefined. We found that σ1s enhances reovirus resistance to type-1 interferon (IFN-1) responses to facilitate reovirus bloodstream dissemination. Previous work revealed that σ1s is required for efficient reovirus replication in multiple cell lines. We found that reovirus lacking σ1s replicated to wild-type levels in cells lacking the type-1 interferon receptor (IFNAR1). However, σ1s does not function as a canonical IFN-1 antagonist. Interferon regulatory factory 3 (IRF3) activation, IFN-β production and IFNAR1 signaling each were comparable between wild type and σ1s-null viruses. Wild-type reovirus spreads systemically following oral inoculation of neonatal mice, whereas the σ1s-null virus remains localized to the intestine. In contrast, the σ1s-deficient virus disseminated to target organs in IFNAR1-/- mice. These results indicate that σ1s functions to promote reovirus spread in the face of a functional IFN-1 response. We subsequently used tissue-specific IFNAR1 deletion in combination with the IFN-1-sensitivity of σ1s-null reovirus to define cell types that mediate dissemination. We found that IFNAR1 deletion from lymphatic endothelial cells enabled systemic spread of the σ1s-deficient virus. Together, our findings indicate that σ1s functions to promote efficient reovirus replication in an antiviral environment. Our data further suggest that the lymphatics are an important conduit for reovirus spread.

107Oral and shotgun presentations Workshop 4: Immunity and Pathogenesis

Neurotropic factors from enteric glial cells contribute to maintain the intestinal epithelial barrier during rotavirus infection.

Marie Hagbom

Linköping University, Sweden

Increased Intestinal permeability have been proposed as a mechanisms of rotavirus diarrhea. Studies in human and mice have however shown that rotavirus leave the intestinal permeability unaffected/or reduced at the time of diarrhea, possibly by contribution of the vagal nerve and/or enteric glia cells (EGCs).The enteric nervous system (ENS) is composed of neurons and EGCs with the latter being involved in the control of major gastrointestinal functions via crosstalk to enteric neurons and neighboring cell types. In this study, we investigated if vagus or EGCs contributed to maintaining the gut barrier intact during rotavirus infection. Using subdiafragmal vagotomized mice we found that the unaffected epithelial barrier during rotavirus infection is not dependent of the vagal nerve. Immunofluorescence analysis show that rotavirus infected enterocytes are in close contact with EGCs and enteric neurons. Moreover, rotavirus (p<0.001) and serotonin (p<0.001) activated EGCs, shown by an increase in expression of the EGC activation marker gfap and increase of intracellular calcium. By mounting biopsies of the small intestine in Ussing chambers we found that the glia derived neurotropic factors GDNF and GNSO contribute to maintain a tight gut epithelium in mice (p<0.001). Similar effect was shown in rotavirus infected Caco-2 cells, with GNSO contributing to keep the epithelial barrier unaffected up to 22 hours post infection with reduced passage of FITC-dextran (p<0.05), and increased expression of ZO-1 tight junction proteins (p<0.001). This is the first report to show that EGC derived neurotropic factors contribute to maintain the gut epithelial barrier during viral insult.


Species-specific restriction of Bluetongue virus replication correlates to host resilience

Alexandra Hardy (1), Meredith Stewart (1), Mariana Varela (1), Andrew Shaw (1), Sam Wilson (1), Richard Randall (2), Massimo Palmarini (1)

1: MRC - University of Glasgow Centre for Virus Research; 2: School of Biology, University of St Andrews

Bluetongue is a vector-borne disease of ruminants caused by bluetongue virus (BTV). BTV can infect essentially all domestic and wild ruminants but the clinical outcome of infection differs substantially between host species. Clinical disease induced by BTV, including haemorrhagic fever in severe cases, is normally evident only in sheep. Conversely, cattle are more resilient to BTV infection, as they develop high levels of viremia and can be reservoirs of infection, but rarely show clinical signs. Here, we concentrated on BTV-host cell interactions using primary cells as an experimental system. First, we determined that BTV reaches higher titres in ovine cells, compared to bovine cells although it induces comparable levels of antiviral cytokines in both cell types. Importantly, these differences are abolished by inhibiting the Jak/Stat pathway. In addition, pre-treatment with interferon (IFN) severely hampers BTV replication in bovine, but not in ovine, primary cells. These data suggest that bovine, unlike ovine, IFN-stimulated genes (ISGs) are effective in controlling BTV replication. Using a high-throughput flow cytometry approach, we screened an expression library of over 300 bovine ISGs to identify genes with antiviral properties against BTV. We have identified ~10 bovine ISGs that negatively impact BTV replication (by at least 50%). Currently, we are assessing the sheep orthologues to the bovine ISGs of interest in order to assess host-species differences. Our study provides novel insights on how bovine cells restrict BTV replication and could provide an intellectual framework to understand the host determinants involved in disease severity.


Interferon-stimulated gene SAMD9 restricts rotavirus replication and is counteracted by non-structural protein 1

Siyuan Ding (1,2,3), Peter K. Jackson (2), John T. Patton (4), Jia Liu (5), Harry B. Greenberg (1,2,3)

1: Department of Medicine, Division of Gastroenterology and Hepatology, Stanford School of Medicine, Stanford, CA; 2: Department of Microbiology and Immunology, Stanford School of Medicine, Stanford, CA; 3: VA Palo Alto Health Care System, Department of Veterans Affairs, Palo Alto, CA; 4: Department of Biology, Indiana University, Bloomington, IN; 5: Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR

Rotaviruses (RVs) are one of the most common causes of severe gastroenteritis and diarrhea in young children, resulting in an annual death of 215,000 people worldwide. Host interferon (IFN) signaling constitutes an important determinant of RV host range restriction and suppresses the replication of heterologous RVs in vivo. However, the identity of specific IFN-stimulated genes (ISGs) that restrict RV infection in the small intestine remains unknown. To bridge this gap, we have comprehensive profiled ISG induction in IFN-treated human intestinal epithelial cells (IECs), the cell type that RV predominantly infects in vivo. We constructed a lentiviral vector-based expression library consisting of 52 IEC-specific ISGs and examined individual ISG’s anti-RV properties. Our pilot screen identified one ISG, sterile alpha motif-containing domain 9 (encoded by SAMD9), as a potent RV restriction factor. CRISPR-Cas9 depletion of SAMD9 led to a 10-fold increase in both intracellular viral RNA and virus yield in the supernatant. In addition, we found that RV-encoded non-structural protein 1 (NSP1) co-precipitated with SAMD9 and induced its degradation during RV infection. Collectively, we have uncovered an intriguing and important interaction between a host restriction factor SAMD9 and its corresponding RV antagonist NSP1. These novel findings of SAMD9 inhibition of RV replication and NSP1 countermeasures represent a previously unknown component of an ever-evolving RV-host arms race. Our current study will shed light on the multifunctional RV NSP1 mechanism of action and the potential cell-intrinsic antiviral activity of SAMD9 against enteric viruses.


Polarised interferon-mediated immune response against mammalian reovirus reveal novel mechanisms of immune tolerance in the human gut

Megan Stanifer (1), Stephanie Muenchau (1), Dorothee Albrecht (2), Sina Bartfeld (3), Franck van Kuppeveld (4), Takashi Kanaya (5), Steeve Boulant (1,2)

1: Universtiy Hospital Heidelberg, Germany; 2: DKFZ, Heidelberg, Germany; 3: Univeristy of Wuerzburg, Wuerzburg, Germany; 4: Ultrecht Univeristy, Ultrecht, Netherlands; 5: Riken, Yokohama, Japan

Intestinal epithelial cells (IECs) lining the surface of our gastrointestinal tract have their apical sides in constant contact with the lumenal commensal flora. As such, IECs are faced with a major challenge as they must tolerate the presence of the microbiota while maintaining full responsiveness against enteric pathogens.Using human mini-gut organoids, we found that primary hIECs assemble a polarized immune response against the dsRNA virus, mammalian reovirus (MRV). hIECs mount a distinct immune response as a function of infection side. MRV infection of hIECs from their apical side (lumenal side) leads to an acute production of both type I and type III interferons (IFNs) which is quickly downregulated. On the contrary, infection from their basolateral sides (lamina propia side) triggers a stronger innate immune response characterized by prolonged production of type III IFNs. Importantly we showed that this response was not virus specific and was also found with additional pathogen associated molecular patterns (PAMPs). We identified the clathrin/AP1-dependent polarity program as a specific regulator of TLR3 signaling allowing hIECs to mount this polarized immune response. Interestingly, mice lacking AP1 display colitis due to an over-reaction to their microbiota.We propose that this polarized response represents a strategy to maintain gut immune homeostasis by avoiding excessive response against microbes located in the lumenal side while maintaining full responsiveness against invasive pathogens that have passed the epithelium barrier.


Bacterial products within Culicoides midge saliva enhance Bluetongue virus infection in bovine monocytes

Lyndsay Cooke (1), Katy Moffat (1), Laura Tugwell (1,2), Nicolas Locker (2), Geraldine Taylor (1), Karin Darpel (1)

1: The Pirbright Institute, UK; 2: University of Surrey, United Kindgdom

Bluetongue virus (BTV) causes an economically important disease of ruminants and is transmitted to its mammalian host during blood feeding of its biological vector Culicoides midges. Within its ruminant host BTV replicates within a wide range of target cells including endothelial cells, monocytes, lymphocytes and dendritic cells. Culicoides saliva contains a pharmacologically active mixture of biomolecules which can directly modify the structure of the BTV particle and its subsequent infectivity for certain cells. Furthermore, midge saliva may indirectly alter BTV infectivity through modulation of the cellular immune response.Here we demonstrate that the presence of Culicoides saliva increases BTV replication in bovine blood-derived monocytes but not in any other subpopulation of peripheral blood mononuclear cells (PBMCs). The infection enhancing effect of Culicoides saliva seems to be mediated by bacterial lipopolysaccharide (LPS) present within Culicoides saliva. Using flow cytometry, confocal microscopy and viral titration we detect an up to ten fold increase in the proportion of monocytes expressing the viral non-structural protein NS2, as well as an increase in progeny virus production following infection in the presence of vector saliva or just purified LPS. Furthermore, initial studies suggest that the observed LPS mediated enhancement of BTV infection in monocytes might be linked to cell entry mechanisms or early replication steps of the virus rather than modulation of cellular anti-viral responses.Overall our investigations highlight the exceptional complexity of arbovirus-insect vector-mammalian host interactions which might be further influenced by insect and host microbiomes in addition to other vector saliva components.


Evidence of a distinct mechanism for rotavirus acceleration of type 1 diabetes development

Barbara S Coulson

The University of Melbourne, Australia

Type 1 diabetes results from chronic progressive autoimmune destruction of insulinproducing pancreatic cells. Infection with certain RNA viruses including rotavirus may modulate diabetes development. Particular coxsackieviruses and rotaviruses accelerate type 1 diabetes in NOD mice. For RRV rotavirus, this relates to pancreatic lymph node (PLN) infection and a Th1-specific immune response. RRV induces bystander lymphocyte activation, with plasmacytoid dendritic cell (pDC) activation and strong upregulation of interferon-dependent gene expression within PLN. Comparison with RRV-infected NOD mice lacking a functional type I interferon receptor (NOD.IFNAR1-/-) shows interferon signalling is required for the Th1 bias, bystander activation and accelerated diabetes. TLR7 signalling mediates the pDC activation in RRV-stimulated NOD splenocytes. Here, the TLR7 role in RNA virus activation of NOD and C57BL/6 mouse splenocytes was analysed. TLR7/9 agonists, RRV, coxsackievirusB1 and X31 influenza virus each increased B cell and dendritic cell (DC) activation, probably dependent on pDC. NOD mice have heightened type I interferondependent responses to RRV infection over C57BL/6 mice, and NOD splenocytes showed the greater capacity for bystander activation. NOD.IFNAR1-/- splenocyte stimulations demonstrated the interferon signalling dependence of the TLR7/9 agonists, RRV and X31 but not coxsackievirusB1, suggesting X31 might induce bystander activation. However, X31 infection of NOD mice neither increased the pDC to cytoplasmic DC ratio and MHCI expression in PLN nor accelerated diabetes development. In our proposed mechanism, RRV activation of pDC via TLR7 in PLN leads to type 1 interferon production and bystander activation of pre-existing islet-autoreactive lymphocytes. These data suggest this mechanism may be rotavirus-specific.


Neuron-specific NF-κB-dependent genes mediate reovirus neuropathogenesis

Gwen M. Taylor (1), Andrea J. Pruijssers (2), Terence S. Dermody (1)

1: University of Pittsburgh School of Medicine, USA; 2: Vanderbilt University Medical Center, USA

Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germline ablation of NF-κB subunit p50. It is not known whether the pro-apoptotic function of NF-κB is mediated by neuron-intrinsic processes, NF-κB-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-κB signaling in reovirus-induced neuronal injury, we established mice that lack NF-κB p65 expression in neurons using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type mice succumbed to infection, whereas more than 90% of neuron-specific NF-κB-deficient mice survived. While viral loads in brains of wild-type and NF-κB-deficient mice were comparable, histological analysis revealed that reovirus antigen-positive areas in the brain of wild-type mice displayed enhanced cleaved caspase-3 immunoreactivity, a marker of apoptosis, compared with neuron-specific NF-κB-deficient mice. These data suggest that neuron-intrinsic NF-κB-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected cortices of wild-type and neuron-specific NF-κB-deficient mice suggests that ablation of NF-κB p65 expression in neurons dampens upregulation of genes involved in cell death, innate immunity, and inflammation following reovirus infection. A better understanding of the contribution of cell type-specific NF-κB-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations.


Transkingdom viral, bacterial and fungal microbiome correlation with rotavirus vaccine immunogenicity in rural Ghana

Scott Handley (3), George Armah (4), Lindsay Droit (3), Barry Hykes (3), Chandni Desai (3), Herbert Virgin (3), Vanessa Harris (1,2)

1: Amsterdam Institute for Global Health and Development, Netherlands, The; 2: Department of Medicine, Division of Infectious Diseases and Center for Experimental and Molecular Medicine (CEMM), Academic Medical Center, Amsterdam, The Netherlands; 3: Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri, USA; 4: Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghan

Rotavirus (RV) causes serious childhood gastroenteritis. Rotavirus vaccines (RVV) have lower effectiveness in developing countries. The intestinal microbiome correlates with RVV immunogenicity. This nested case-control study hypothesized that the composition of the bacterial, viral, and fungal microbiome correlates with RVV immunogenicity in developing country settings. We employed next generation sequencing to describe the bacterial, viral, and fungal microbiome in 460 stool samples collected from 122 infants in a RVV (Rotarix) vaccine trial in rural Ghana, comparing RVV responders to nonresponders (anti-RV IgA > 20 IU/mL) over time. Preliminary analysis shows that RVV seroconversion correlates with a diminished abundance of Bacteroidetes strains, an expansion of Gamma Proteobacteria, and decreased fungal abundance at the first vaccination dose. Enteric virome sequencing revealed the presence of a large diversity of eukaryotic viruses including numerous entero and anelloviruses. Strains of Picornaviridae correlate with decreased RVV seroconversion at every dose. Ongoing assembly of viral contigs suggest several Gammaproteobacteria phage correlate with non-response. This ongoing study provides a unique snapshot ot trans-kingdome microbiome dynamics in infants from a developing coutnry setting and support a role for the intestinal microbiome in RVV immunogenicity.


Antigenicity of simian and human reassortant rotaviruses generated by reverse genetics

Yuta Kanai (1), Takahiro Kawagishi (1), Misa Onishi (1), Pannacha Pimfhun (1), Ryotaro Nouda (1), Jeffery Nurdin (1), Keiichiro Nomura (1), Hiroshi Ushijima (2), Takeshi Kobayashi (1)

1: Department of Virology, Research Institute for Microbial Diseases, Osaka University; 2: Department of Pathology and Microbiology, Nihon University School of Medicine

Rotavirus (RV) is the leading cause of severe diarrhea among infants and young children worldwide. Safe and effective RV vaccines have been available since 2006, and have markedly reduced the number of deaths caused by severe gastroenteritis. However, RVs remain responsible for approximately 200,000 deaths annually worldwide. A major barrier to successful development of a RV vaccine is antigenic diversity of endemic RV strains. During the last year, we reported comprehensive reverse genetics (RG) systems able to generate recombinant RV strains with desired nucleotide mutations. We used RG technique to develop a next generation vaccine platform by modifying outer capsid proteins VP7 (G serotypes) and VP4 (P serotypes).To develop vaccine strains with divergent G and P serotypes, we generated reassortant RVs using the simian SA11 (G3P[2]) strain as a backbone; these RVs harbored the VP4 and VP7 genes from human RV strains. Human clinical isolates (G8P[8], G3P[8], G2P[4], and G9P[8]) were prepared. cDNAs generated from the VP4 and VP7 genes were co-transfected along with cDNAs encoding SA11. Replication of the VP4/VP7 mono-reassortant viruses was slower than that of the wild-type SA11 virus. A neutralization assay using a monoclonal antibody against SA11-VP4 demonstrated clear alterations in the antigenicity of VP4 mono-reassortants. Furthermore, mouse anti-SA11 antiserum neutralized VP4/VP7 mono-reassortants to varying degreesindicating that both VP4 and VP7 individually affected the viral antigenicity. This strategy for analyzing VP4/VP7 reassortant RV strains will increase our understanding of the complex antigenicity of RVs and help to establish a platform for developing future RV vaccines.


The African horse sickness virus NS4 counteracts the antiviral response and is a determinant of viral virulence

Yi Jin (1), Maxime Ratinier (1,2), Siddharth Bakshi (1), Marco Caporale (3), Aislynn Taggart (1), Meredith Stewart (1), Massimo Palmarini (1)

1: MRC-University of Glasgow Centre for Virus Research, UK; 2: Infections virales et pathologie comparée, EPHE, France; 3: IZS Istituto Zooprofilattico Sperimentale, Italy

African horse sickness is a major infectious disease of equids and is caused by African horse sickness virus (AHSV), a dsRNA virus with 10 genome segments encoding for 7 structural and 4/5 non-structural proteins. Here, we focused on the characterisation of the AHSV NS4, the latest protein found to be expressed by this virus. In silico analysis of available sequences confirmed the existence of two phylogenetically distinct AHSV clades: NS4-I and NS4-II. NS4-II is further divided into three subtypes (α, β and γ). Confocal microscopy demonstrated that all AHSV NS4 types localised in the cytoplasm of infected cells, unlike the BTV NS4 which, has a strong nucleolar localisation. The replication kinetics of reverse genetics derived AHSV NS4 deletion mutants (AHSVΔNS4) were similar to their wild type counterparts in insect (Kc) or interferon incompetent (BSR) cells. However, replication of AHSVΔNS4 mutants in primary horse endothelial cells was restricted, in comparison to wild-type viruses. Importantly, primary cells restriction to AHSV replication was dependent on the JAK/STAT pathway. Furthermore, AHSVΔNS4 mutants were not able to efficiently suppress the secretion of anti-viral cytokines from primary cells, while the wild-type viruses suppressed this response to varying degrees. Importantly, AHSVΔNS4 mutants were less virulent than their wild type counterparts in a murine model of AHSV infection. These results indicate that AHSV NS4 has a role in interferon IFN antagonism and a determinant of viral virulence. We are currently carrying out mass spectrometry analyses to identify the cellular proteins interacting with the AHSV NS4.


The VP4 protein from a very virulent IBDV strain antagonises type I IFN responses, in contrast to the VP4 from a classical strain

Katherine L Dulwich (1,2), Efstathios S Giotis (2), Michael A Skinner (2), Andrew J Broadbent (1)

1: The Pirbright Institute, UK; 2: Imperial College London, UK

Infectious bursal disease virus (IBDV) is a double stranded RNA virus belonging to the Birnaviridae family that is of economic importance to the poultry industry worldwide. IBDV infects B cells in the bursa of Fabricius (BF) causing morbidity, mortality and immunosuppression in infected birds. Classical strains (e.g. F52/70) cause 1-2% mortality whereas very virulent strains (e.g. UK661) cause over 60% mortality for reasons that remain poorly understood. We inoculated birds with either F52/70 or UK661, and found that the expression of pro-inflammatory and type I IFN-related genes was significantly down-regulated in UK661 compared to F52/70 infected birds (p<0.05), despite no statistically significant difference in peak virus titres between the two strains. This was also observed in vitro in an immortalised B cell line where UK661 caused significantly reduced IFNβ and Mx1 expression compared to F52/70 (p<0.05). The IBDV protease (VP4) has previously been reported to act as an IFN antagonist. Using a luciferase reporter assay, we compared the IFNβ production in DF-1 cells in response to polyI:C stimulation in the presence of eGFP-VP4 expression plasmids, finding UK661 VP4 was able to down-regulate IFNβ production to a greater extent than F52/70 VP4 (p<0.01). There are 9 amino acid differences between the two VP4 proteins and we are identifying those contributing to the observed phenotype. Taken together, our data suggest that the VP4 protein in very virulent IBDV strains evolved a greater ability to antagonise type I IFN responses than classical strains which may, in part, explain their enhanced virulence.


Bluetongue virus uses the lysosome-autophagy pathway to degrade STAT2 and block IFN signaling

Miguel Avia (1), Lisa Miorin (2), José Manuel Rojas (1), Elena Pascual (1), Verónica Martín (1), Adolfo García-Sastre (2), Noemí Sevilla (1)

1: CISA-INIA - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Spain; 2: Department of Microbiology and Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, USA

The mammalian interferon (IFN) signaling pathway is a primary component of the innate antiviral response. As such, viral pathogens have devised multiple mechanisms to antagonize this pathway and thus facilitate infection. In here we studied how Bluetongue virus (BTV), the prototype member of the genus Orbivirus, family of the Reoviridae interferes with IFN responses by degrading STAT2 and blocking STAT1 phosphorylation. BTV-NS3 protein which is involved in virion egress also participated in IFN signaling blockade. BTV-NS3 can bind to STAT2 and reduce its levels by an autophagy-lysosome-dependent mechanism. This STAT2 degradative process requires the recruitment of an E3-Ub-ligase to NS3 as well as NS3 K63 poly-ubiquitination. BTV-NS3 probably acts as a scaffolding protein that orchestrates STAT2 autophagic degradation. Our study identifies a new mechanism by which a virus targets STAT2 for degradation and blocks IFN signaling. The present work highlights the diversity of mechanisms employed by viruses to subvert IFN signaling.


Nelson Bay Orthoreovirus cell attachment protein σC determines strain-specific differences in viral replication and pathogenesis

Takahiro Kawagishi (1), Yuta Kanai (1), Yusuke Sakai (2), Ryotaro Nouda (1), Hideki Tani (3), Masayuki Shimojima (4), Masayuki Saijo (4), Yoshiharu Matsuura (5), Takeshi Kobayashi (1)

1: Research Institute for Microbial Diseases, Osaka university, Japan; 2: Laboratory of Veterinary Pathology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Japan; 3: Department of Virology, Faculty of Medicine, University of Toyama, Japan; 4: Department of Virology I, National Institute of Infectious Diseases, Japan; 5: Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Japan

Nelson Bay orthoreovirus (NBV), a member of family Reoviridae, was isolated originally from a flying fox. While NBV is not associated with any animal diseases, it causes acute respiratory tract infections in humans. The S1 gene segment of NBV encodes two non-structural proteins, FAST and p17, and one structural protein, σC. Sequence analyses indicate that viral proteins encoded by the S1 gene segment of NBV strains are less homologous than those of other viral proteins. However, the molecular mechanisms by which NBV becomes a zoonotic pathogen of humans are unclear. Here, we characterized NBV NelB (isolated from a bat) and MB (isolated from human) strains to understand how strain-specific differences in the S1 gene segment affect viral infectivity and pathogenesis.We inoculated mice with NelB and MB strains via the intranasal route and monitored survival. MB caused a severe respiratory illness that killed all mice. However, mice infected with NelB did not show any signs of illness. To clarify which viral protein is critical for NBV pathogenesis, we generated recombinant viruses expressing NelB-FAST, NelB-p17, or NelB-σC on a MB genetic background. None of the mice infected with NelB-σC virus showed any sign of illness. To better understand functional differences in σC between the NelB and MB strains, we generated chimeric σC mutant viruses and identified the functional domain within σC that is associated with strain-specific differences in viral infectivity and pathogenesis. The data suggest that biological properties of σC, including infectivity and pathogenesis, segregate in a strain-specific manner.


WORKSHOP 5: STRUCTURE

Structure and Function of Human Picobirnavirus

Aaron M. Collier* (1), Outi L. Lyytinen* (2), Jason Kaelber* (3), Yusong R. Guo (1), Yukimatsu Toh (1), Xiaoying Lei (1), Wah Chiu (3), Minna M. Poranen (2), Yizhi J. Tao (1)

1: Dept. of BioSciences, Rice University, 6100 Main Street, Houston, TX, 77005, USA2: Dept. of Biosciences, University of Helsinki, P.O.Box 56 (Viikinkaari 9), FIN-00014, Helsinki, Finland3: Graduate Program in Structural and Computational Biology and Molecular Biophysics and Verna and Marrs McLean, Dpartment of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030

* These authors contributed equally to this work

Picobirnaviruses (PBV) are small, non-enveloped dsRNA viruses that have been isolated from a wide range of mammalian and avian species. Human PBV (hPBV) has been identified on almost every continent, and has been associated with both acute and mild gastroenteritis. PBV has a bi-segmented genome with the genome segment two (PBV2) encoding the viral RdRP and the genome segment one (PBV1) encoding the CP and a protein of unknown function. Previous study of a rabbit PBV (rPBV) indicated an unique capsid organization that is somewhat different from the organization of the larger dsRNA viruses. To elucidate the mechanisms of RNA replication and transcription by this group of largely uncharacterized, small dsRNA viruses, we have determined the structure of a hPBV RdRP and systematically characterized its biochemical and enzymatic activities. Co-expression of the hPBV RdRP and CP resulted in the formation of ~35 nm VLPs that were incapable of sequestering the RdRP molecules. Using cryo-electron microscopy reconstruction, we have established the structure of hPBV VLP to atomic resolution. Superposition of the hPBV and rPBV structures reveals highly-conserved secondary structural elements proximal to the capsid interior and divergent structures at the outer surface. Results from glycan microarray analysis suggest conserved glycan motifs among strong binders. Regardless of whether the natural host of HBV is human intestine or a prokaryote of the gut microbiome, these glycans may be relevant to the virus entry and/or passive retention in appropriate microenvironment.


Nanoscopic organization of rotavirus replication machineries by super resolution microscopy

Yasel Garcés Suárez (1), José Luis Martínez Guevara (1), David Torres Hernández (1), Haydee Olinca Hernandez Avina (1), Juan Manuel Rendón Mancha (2), Adán Oswaldo Guerrero Cárdenas (1), Susana Lopez Charreton (1), Carlos Federico Arias Ortiz (1)

1: Institute of Biotechnology, Autonomous National University of Mexico, Mexico; 2: Institute of Basic and Applied Sciences, Autonomous University of the State of Morelos, Mexico.

The rotavirus replication cycle is a complex process that takes place in cytoplasmic non-membranous electron dense inclusions termed viroplasms (VPs), where the formation of new double layered particles (DLPs) occurs. Although there had been several studies about the intracellular distribution of rotavirus proteins and their organization in VPs, these observations had been done using traditional optic microscopic techniques whose limit of resolution is unable to resolve in detail the molecular organization of proteins (Rayleigh’s limit between [0.2, 0.4] microns). In this work, using super-resolution microscopy (SRM), we observed, for the first time, in nanoscopic scale, the viroplasms formed during rotavirus infection and describe quantitatively, by three different approaches, the structural organization of 7 viral proteins and the dsRNA in the VPs. The results obtained suggest that the VPs are organized as 6 concentric layers where NSP5 is in the central part of the VPs, followed by a layer of NSP2 and NSP4 proteins. In the outside of this layer, we found an intermediate zone composed by VP1, VP2, and VP6, followed by dsRNA. Finally, in the outermost layer, we observed a ring of VP4 protein and finally a layer of VP7. This study was supported by the analysis of approximately 350 SRM images with multiple VPs and more than 600 pre-segmented SRM images (isolated VPs).

123Oral and shotgun presentations Workshop 5: Structure

These can go up to eleven: shedding light on the mechanisms of multi-segmented genome assembly

Alexander Borodavka (1,3), Eric C Dykeman (2), Waldemar Schrimpf (3), Don C Lamb (3)

1: Astbury Centre for Structural Molecular Biology, University of Leeds, UK; 2: York Centre for Complex Systems Analysis, University of York, UK; 3: Department of Chemistry and Center for Nano Science, Ludwig-Maximilians-Universität, Munich, Germany

Genome segmentation offers certain evolutionary benefits to a number of pathogenic RNA viruses, including rotaviruses and influenza viruses. However, as the number of RNA segments per virion increases, the task of a non-random selection of a full set of distinct genomic RNAs poses a formidable challenge to maintaining the integrity of segmented genomes. Recently, have shown that binding of the rotavirus-encoded non-structural protein NSP2 to viral (+)ssRNA genome precursors results in the remodeling of RNA, which is conducive to formation of inter-segment sequence-specific contacts. These protein-RNA interactions result in the stabilisation of extended inter-molecular RNA-RNA contacts, potentially underpinning transient inter-segment interactions prior to genome encapsidation. Using this approach we have identified a number of RNA-RNA interaction sites in the rotavirus genome, which may be involved in genome segment assortment process. To unravel the mechanisms by which NSP2 controls the formation of inter-molecular RNA helices we have applied RNA structure probing methods that allowed us to monitor conformational rearrangements, which are prerequisite for the formation of the RNA-RNA contacts. Our findings offer further insights for understanding the challenges for further improvement of the recently developed fully plasmid-based reverse genetics systems for rotaviruses.References:1.Borodavka A., Dykeman E.C., Schrimpf W., Lamb, D.C. Protein-mediated RNA folding governs sequence-specific interactions involved in genome segment selection in rotaviruses. eLife, 2017.2. Bravo J.P.K., Borodavka A., Barth A. et al. Formation of intersegment RNA-RNA contacts in related reoviruses is dependent on cognate non-structural RNA-binding proteins.Nucleic Acids Res, 2018.


Structural basis of glycan specificity in human rotaviruses

Liya Hu (1), Wilhelm Salmen (1), Banumathi Sankaran (2), Sasirekha Ramani (1), Mary Estes (1), B. V. Venkataram Prasad (1)

1: Baylor College of Medicine, USA; 2: Berkeley Center for Structural Biology, Lawrence Berkeley National Laboratory

Initial attachment to host cells is a critical event in the life cycle of a virus. This involves recognition by the viruses of specific receptors on the cell surface, including glycans. Viruses typically exhibit strain-dependent variations in recognizing specific glycan receptors, a feature that contributes significantly to cell tropism, host specificity, host adaptation and interspecies transmission. Rotaviruses (RVs) are the leading cause of life-threatening gastroenteritis in infants and young children worldwide. Glycan recognition by RVs for cell attachment is mediated by the distal VP8* domain of the spike protein VP4. Although structurally well conserved with a galectin-like fold, sequence-wise, VP8* is the least conserved among RV structural proteins giving to rise to a phylogeny consisting of ~50 P genotypes which can be broadly grouped in 5 clades. While some animal RVs bind to sialo-glycans, most human RVs recognize histo-blood group antigens (HBGAs), which are the determinants of blood types. To elucidate the molecular mechanisms of how human RVs including sporadic, globally-dominant, and neonate-specific strains recognize specific cellular glycans, we have carried out glycan array, crystallography, and cellular biology studies. Glycan array analysis supported by infectivity assays show that human RVs bind to specific HBGAs, such as A-type, H-type and precursor HBGAs in a genotype-dependent manner. Our crystal structures of VP8* of human RVs in complex with HBGAs reveal remarkable variations in the glycan binding sites providing a possible structural basis for the global dominance, neonate-specificity, and interspecies transmission exhibited by human RVs. This work is supported by grants from NIH (AI36040) and Robert Welch Foundation (Q1279).


Near-atomic resolution cryo-EM structure of Rotavirus capping enzyme VP3

Dilip Kumar (1), Xinzhe Yu (1), Sue E Crawford (2), Rodolfo Moreno (1), Anish Ramakrishnan (1), Liya Hu (1), Mary K Estes (2), Zhao Wang (1), B.V.Venkataram Prasad (1)

1: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA.; 2: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA

Rotavirus is a triple layered particle containing a genome of 11 segments of dsRNA, which encodes 6 structural (VPs) and 6 non-structural proteins (NSPs). VP3 is a multifunctional enzyme that facilitates 5’-capping of nascent viral mRNA generated by the viral RNA-dependent RNA polymerase VP1. It is also implicated in evading the antiviral interferon-induced oligoadenylate synthetase-RNase L pathway in host cells. With the exception of VP3, atomic structures of the other structural proteins have been determined. Our goal is to determine the atomic structure of VP3 to provide mechanistic insights into its role in RV transcription, replication and assembly. We successfully expressed and purified VP3. The purified protein showed high-affinity binding to ssRNA and exhibited all the enzymatic activities necessary for RNA capping. The VP3 structure was determined by collecting cryo-EM data in a super-resolution counting mode at 30K magnification using JEM3200FSC (JEOL) equipped with an energy filter and direct electron detector K2 (GATAN), and using EMAN2 and RELION software. The final resolution of the map is 3.2 Å based on the Fourier shell correlation (FSC) using 0.143 criterion. The atomic structure of the whole protein was built de novo from this map and refined using PHENIX, COOT, and Rosetta. The structure revealed the atomic details of the phosphodiesterase domain and all the domains necessary for the capping activity, including novel features that may be important for understanding how VP3 coordinates capping of the nascent mRNA during endogenous transcription. This research is supported by grants from NIH and Robert Welch Foundation.


Atomic structure of translation regulatory protein NS1 of bluetongue virus in tubule form

Adeline Kerviel (1), Mason Lai (2), Jonathan Jih (3), Peng Ge (3), Hong Zhou (2,3), Polly Roy (1)

1: London School of Hygiene and Tropical Medicine (LSHTM), Department of Pathogen Molecular Biology, London, UK; 2: Electron Imaging Center for Nanomachines (EICN), and Dept of Microbiology, Immun. & Mol. Genetics, UCLA, USA; 3: California NanoSystems Institute (CNSI) and Dept of Microbiology, Immun. & Mol. Genetics, UCLA, USA

A unique hallmark of the Bluetongue virus (Reoviridae family) infection of host cells, is a large deposit of tubular structures formed by the non-structural protein NS1, known to be the up regulator of viral protein translation. The functional form of NS1, tubular or soluble, still remains unclear. We have recently obtained for the first time the 3D structure of tubules at the near atomic resolution (4.0Å) by cryo-electron microscopy. Our aim is to understand the structure-function relationship of NS1 during viral replication. The tubules are formed by NS1 dimers arranged into a helix containing 40 to 41.5 protomers/turn. Its architecture is stabilized by interactions between neighboring dimers of the same layer, as well as protomers from two different layers. The atomic model for the BTV NS1 monomer shows three domains—foot, body and head—with an extended C-terminal arm, and two metal-binding motifs characteristic of Zinc finger motifs. Using interdisciplinary approaches, we observed substitution mutations of the Zinc finger motifs impaired NS1 tubule formation, that virus recovery was either unsuccessful or highly attenuated and the ability of NS1 to specifically enhance viral mRNA translation was reduced. Deletion of the C-terminal arm also prevented tubule formation. However, these mutant viruses were recovered as efficiently as the wildtype virus and the NS1 remained functional. Thus, although NS1 does not need to form tubules to perform its function, its intramonomer zinc finger motifs are essential and are the first step to a better understanding of NS1-viral RNA interaction during the BTV replication cycle.


Imaging viral RNA genomes and RNA transcription in action by cryoEM

Z. Hong Zhou

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA 90095, USA

Viruses in the Reoviridae, like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented dsRNA inside the viral capsid and carry out endogenous mRNA synthesis through a transcriptional enzyme complex (TEC). Visualizing the 3D organization of the viral genome and the process of TEC-catalyzed transcription of the genomic RNA presents a major challenge in structural biology. Direct electron-counting cryoEM now offers opportunity to overcome this challenge. I will present the organization of the dsRNA genome inside quiescent CPV (q-CPV) and the in situ atomic structures of TEC within CPV in the quiescent and transcribing (t-CPV) states. We show that the 10 segmented dsRNAs are organized with 10 TECs in a specific, non-symmetric manner, with a dsRNA segment attached directly to each TEC. TEC consists of two extensively-interacting subunits: an RNA-dependent RNA polymerase (RdRP) subunit and an NTPase subunit, VP4. By comparing the TEC structures in q-CPV and t-CPV, we find that the bracelet domain of RdRP undergoes significant conformational change, leading to formation of the RNA template entry channel and access to the polymerase active site. The N-terminal helix from each of the two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the missing link between sensing of external cues by the CSP and activation of endogenous RNA transcription by the TEC inside the virus. The work highlights new opportunities offered by electron-counting cryoEM to obtain in situ structures of viral RNA and DNA genomes and genome replication for viruses and other organisms.


Importance of Residues Surrounding +RNA Exit Tunnel Interface of the Rotavirus Polymerase for Genome Replication

Courtney L. Steger (1), Crystal E. Boudreaux (2), Courtney A. Cohen (3), Sarah M. McDonald (4)

1: Translational Biology, Medicine, and Health Graduate Program, Virginia Tech, Blacksburg, VA, USA; 2: Department of Biomedical Sciences, West Virginia School of Osteopathic Medicine, Lewisburg, WV, USA; 3: Department of Virology, United States Army Medical Research Institute for Infectious Diseases, Fredrick, MD, USA; 4: Department of Biology, Wake Forest University, Winston-Salem, NC, USA

The rotavirus RNA polymerase, VP1, mediates transcription (+RNA synthesis) and genome replication (dsRNA synthesis) within the confines of subviral particles and while interacting with the core shell protein, VP2. VP1 is a globular, cage-like enzyme, and its buried catalytic center is permeated by four distinct tunnels. One of these tunnels facilitates egress of nascent +RNA products of transcription. In the double-layered particle (i.e, transcriptase complex), VP1 is located off-center from the icosahedral fivefold axes and is oriented such that its +RNA exit tunnel abuts the VP2 core shell. This position presumably coordinates release of transcripts through fivefold pores of the particle. Several surface-exposed VP1 regions surrounding the +RNA exit tunnel (e.g., 264-267, 968-980 and 1022-1024) make contacts with VP2 in this position. Here, we sought to test the importance of these VP1 regions for VP2-dependent genome replication using an in vitro dsRNA synthesis assay. We engineered recombinant (r) VP1 proteins that contain single or clustered alanine mutations on the +RNA exit tunnel interface. Then we assayed the rVP1 mutants for the capacity to synthesize dsRNA in vitro in the presence of rVP2. Most of the single alanine rVP1 mutants synthesized dsRNA at levels indistinguishable from wildtype rVP1. However, several multi-point rVP1 mutants exhibited significantly reduced dsRNA synthesis, possibly as a result of inefficient VP2 binding. These results suggest that surface-exposed regions of the +RNA exit tunnel interface, which are VP2 contact points in the transcriptase complex, may be also be important for VP2 interactions during genome replication.


Dissection of the assembly pathway of Mammalian orthoreovirus using focused ion beam milling and in situ tomographic analysis of native assembly intermediates in infected cells

David Stuart (1,2), Mark Boyce (1), Corey Hecksel (2), Abhay Kotecha (1), Xiaofeng Fu (1), Geoff Sutton (1), Peijun Zhang (1,2)

1: Oxford University, UK; 2: Diamond Light Source, UK

The assembly pathways of viruses have been traditionally described using negative stain transmission EM of infected cells complemented by the analysis of purified stable intermediates using crystallographic or cryo EM analysis. The resolution achieved for cellular images has been limited, and the purification of stable assembly intermediates limits what can be analysed to abundant particles with selected physico-chemical properties (ielong-lived and stable).Here we report a preliminary analysis of the assembly pathway of Mammalian orthoreovirus in situin frozen infected cells using cryo focused ion beam milling of cells flash frozen at 12 hours post-infection to generate lamellae of ~150nm thickness. Assembly intermediates are captured in their native form during vitrification and imaged in the lamellae using cryo electron tomography. Data were collected on a Gatan K2 detector attached to a Thermo Fisher Krios microscope. The data have been analysed by reconstruction of the 3D tomogram, followed by selection of particles at difference stages of assembly and averaging of these with appropriate symmetry (using EMclarity). This not only reveals structures corresponding to those determined previously from purified particles, but also reveals states previously inaccessible. For some the resolution is sufficient to resolve the secondary structural elements of the component proteins. We will summarise the results, and the implications for our understanding of the assembly of viruses of the family Reoviridae. These are preliminary results, we expect that in the future such in situmethods will be applicable to all stages in the replication cycle of dsRNA viruses.


Structure of replicative cores of dsRNA fungal viruses

José R. Castón (1), Carlos P. Mata (1), Daniel Luque (1,2), Nobuhiro Suzuki (3), Said A. Ghabrial (4)

1: Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), Campus Cantoblanco, Madrid, Spain; 2: Centro Nacional de Microbiología/ISCIII, Majadahonda, Madrid, Spain; 3: Institute of Plant Science and Resources, Okayama, Japan; 4: Department of Plant Pathology, University of Kentucky, Lexington, KY, USA

Most fungal dsRNA viruses lack an extracellular phase of transmission (except reoviruses). Mycovirus capsids are based on an unusual 60-dimer, T=1 capsid that remains structurally undisturbed during endogenous transcription, thus avoiding induction of host cell defense mechanisms.T=1 capsids are not inert containers to protect the genome, but are dynamic assemblies involved in RNA synthesis and/or packaging genome. These T=1 capsid have been resolved at near-atomic resolution in totivirus, partitivirus, chrysovirus and quadrivirus.The totiviruses have a T=1 capsid built from 120 capsid proteins (CP), in which the asymmetric dimer (A and B subunits) is approximately parallel, similar to the inner core in the reovirus and cystovirus. Each monomer adopts two conformations with notable structural differences. The structural unit of the partitivirus capsid is a quasi-symmetric CP dimer. A similar quaternary organization was found in a picobirnavirus. This dimer is stabilized by domain swapping within the shell regions of A and B subunits and by intradimeric interactions between equivalent protruding surface domains. Chrysoviruses have capsids that are authentic T=1 shells formed by 60 copies of a single polypeptide. The CP is formed by duplication of an α-helical domain. This organization is clearly reminiscent of the T=1 lattice in totivirus capsids. Quadrivirus, such as Rosellinia necatrix Quadrivirus 1, has a single-shelled T=1 capsid formed by 60 P2 and P4 protein heterodimers. P2-P4 heterodimers are organized in a quaternary structure similar to that of reovirus, chrysovirus and totivirus. Notably, mycovirus CP have acquired new functions by domain insertion at the capsid outer surface.


Multiple Liquid Crystalline Geometries of Highly Compacted Nucleic Acid in a dsRNA Virus

Serban Ilca (1), Xiaoyu Sun (2), Kamel El Omari (3), Jonathan Grimes (1,3), David Stuart (1,3), Minna Poranen (2), Juha Huiskonen (1,4)

1: Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Roosevelt Drive, OX3 7BN, Oxford, UK; 2: Molecular and Integrative Biosciences Research Program, Faculty of Environmental and Biological Sciences, University of Helsinki, Viikinkaari 9, 00014, Helsinki, Finland; 3: Diamond Light Source, UK; 4: Helsinki Institute of Life Science and Molecular and Integrative Biosciences Research Program, Faculty of Environmental and Biological Sciences, University of Helsinki, Viikinkaari 1, 00014 Helsinki, Finland

Characterising the genome of mature virions is pivotal in understanding the highly dynamic processes of virus assembly and infection. The packaged double-stranded nucleic acid, constrained by the rigidity of the double-helix, adopts a liquid crystalline arrangement by organising either in a spooled or non-spooled fashion. Current models suggest that dsDNA viruses predominantly display single-spooled conformations due to the lack of genome segmentation and the absence of transcriptional machinery. dsRNA genomes vary greatly in the degree of segmentation and for cypoviruses their organisation has been shown to be tightly associated to the viral RNA-dependent RNA polymerases (RdRPs). However, whether one of these two genome organisation models is applicable to dsRNA viruses in general remains unknown. Here we use cryogenic electron microscopy (cryo-EM) to show that dsRNA viruses can adopt dsDNA-like single-spooled genome organisations by resolving the in situ structure of the tri-segmented dsRNA genome of bacteriophage φ6 (cystovirus). We determine that in this group of viruses, the RdRPs are not instrumental in the genome arrangement and that the dsRNA can adopt multiple conformations. We build an atomic model for 90% of the entire genome that allows us to quantify the packing density across the inside of the capsid and to describe the different liquid crystalline states exhibited by the tightly packed nucleic acid. The results enable us to extend the canonical model for dsDNA packing to a model organism for the study of packaging and transcription in dsRNA viruses.


WORKSHOP 6: USING AND ABUSING HOST PATHWAYS

Structure, Function and Evolution of Reovirus Membrane Fusion Proteins

Roy Duncan

Dalhousie University, USA

The fusogenic reoviruses are the only well-characterized examples of nonenveloped viruses that induce syncytium formation. These viruses are members of two related genera in the family Reoviridae; aquareoviruses exclusively infect fish while orthoreoviruses infect a wide range of vertebrate hosts. As with the viruses that encode them, the proteins that mediate cell-cell fusion segregate into two distinct subgroups, the aqua- and orthoreovirus fusion-associated small transmembrane (FAST) proteins. Phylogenetic analysis suggests the fusogenic reoviruses evolved from an ancestral non-fusogenic precursor by at least two separate gain of function events, and FAST proteins continued to evolve via extensive genetic changes including recombination events. FAST proteins are the smallest viral fusogens and unlike their enveloped virus counterparts, are nonstructural proteins that evolved specifically to induce cell-cell, not virus-cell, membrane fusion. These diminutive virus fusogens share features with viroporins, a large group of small viral membrane remodelling proteins that promote virus replication, assembly and/or release. As the only known example of fusogenic viroporins, FAST proteins assembled a diverse repertoire of membrane-interactive motifs into small, modular fusion machines that function from both sides of the membrane to drive cell-cell fusion. These rudimentary membrane fusion machines function as “opportunistic” fusogens, converting sites of cell-cell attachment into fusion synapses that promote virus dissemination and pathogenicity. FAST proteins provide a very different perspective on how proteins can fuse membranes, they offer new insights into mechanisms of nonenveloped virus dissemination, and they are being exploited to develop improved intracellular drug delivery platforms and to enhance oncolytic virotherapy.


Inhibition of IRF and NF-κB nuclear translocation by rotavirus NSP1

Michelle M. Arnold

Louisiana State University Health Sciences Center, Shreveport, Louisiana, USA

To enhance viral spread from cell to cell, rotaviruses have evolved multiple ways in which to control the host innate immune response to infection. Group A rotaviruses encode the nonstructural protein NSP1 to prevent interferon (IFN) induction and signaling. NSP1 proteins prevent nuclear translocation of key transcription factors required for IFN induction. In some cases, IFN regulatory factors (IRF3, IRF5, IRF7) are targeted for degradation by certain NSP1 proteins, thus preventing IRF localization to the nucleus of infected cells. In other cases, the β-transducin repeat containing protein (β-TrCP) is targeted for degradation by NSP1 to prevent the nuclear translocation of NF-κB. NSP1s from different virus strains can be broadly grouped by their principal target of degradation, but there is conflicting data on the mechanism by which NSP1 induces host protein degradation. NSP1 has long been hypothesized to function alone as an E3 ubiquitin ligase, but recent data suggest that NSP1 may instead be utilizing host cullin-RING ubiquitin ligase complexes to induce protein degradation. I will summarize the data supporting the different possible mechanisms of NSP1-mediated host protein degradation. In addition, my lab is exploring if NSP1 proteins that induce IRF degradation, but not β-TrCP degradation, can block NF-κB nuclear translocation by a degradation-independent mechanism. These studies will allow us to determine if NSP1 has more than one activity in infected cells that contribute to IFN inhibition.

135Oral and shotgun presentations Workshop 6: Using and Abusing Host Pathways

African horse sickness virus non-structural protein NS4 suppresses host innate immunity

Gayle Wall (1), Christiaan Potgieter (2,3), Fourie Joubert (4), Vida van Staden (1)

1: Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Pretoria, South Africa; 2: Deltamune (Pty) Ltd, Lyttelton, Centurion, South Africa; 3: Department of Biochemistry, Centre for Human Metabolomics, North-West University, Potchefstroom, South Africa; 4: Centre for Bioinformatics and Computational Biology, Genomic Research Institute, University of Pretoria, Pretoria, South Africa

African horse sickness virus (AHSV) causes a fatal disease in horses. The determinants of AHSV virulence and attenuation, as well as the host cellular responses that result in disease or resistance, remain unclear. Non-structural protein NS4, the most recently discovered AHSV protein, is suggested to play a role in modulating the host antiviral response. To investigate the function of NS4 we used transcriptome sequencing to analyse differential gene expression (DGE) at 1, 2 or 4 days post infection, in horses infected with virulent AHSV-5 expressing NS4 (rFR) or attenuated AHSV-5 lacking NS4 (rFRminNS4). Genes involved in the innate immune response were upregulated on Day 1 in horses infected with rFRminNS4. This was confirmed by KEGG enrichment analyses. On Day 2, however, fewer genes related to innate immunity were differentially expressed and KEGG pathways related to immunity were no longer enriched. In contrast, few genes related to innate immunity were differentially expressed on Day 1 in the horse infected with rFR, and no evidence of innate immunity was seen in KEGG enrichment analyses. When NS4 is present a strong immune response is observed 24 hours later (Day2) than when the protein is absent (Day 1) and continues to Day 4. As the two viruses differ only with respect to the presence or absence of NS4 it appears that NS4 causes suppression of the immune response in the early stages of infection. This could give AHSV a replication advantage in the host which could have an impact on future vaccine development.


The TRiC chaperonin is required for reovirus assembly

Jonathan Knowlton (1,2), Isabel Fernández De Castro (3), Daniel Gestaut (4), Paula Zamora (1,2), Judith Frydman (4), Cristina Risco (3), Terence Dermody (1)

1: University of Pittsburgh School of Medicine, USA; 2: Vanderbilt University School of Medicine, USA; 3: National Center for Biotechnology, CNB-CSIC, Madrid, Spain; 4: Stanford University, USA

Protein folding encompasses the biological process by which a polypeptide achieves its functional conformation. Although some proteins spontaneously form their native shape, others cannot fold without the assistance of chaperones. Viruses replicate in the context of the host protein folding machinery, and many viruses co-opt cellular chaperones to productively infect cells. Mammalian reoviruses require the TRiC chaperonin to fold the σ3 outer-capsid protein for assembly onto progeny virions. Although σ3 exhibits an absolute requirement for TRiC to fold, the mechanism of TRiC-mediated σ3 folding and capsid assembly is unknown. We defined the minimal conditions required to fold and assemble σ3 onto the outer capsid. TRiC folds chemically-denatured σ3 into a conformation capable of assembling onto its cognate binding partner in the reovirus outer capsid, μ1, forming stable and soluble μ1/σ3 heterohexamers. This folding reaction is ATP-dependent and occurs rapidly after incubating σ3 with TRiC in the presence of μ1. Multiple regions within σ3 interact with TRiC, suggesting that independent binding events between TRiC subunits and σ3 influence the folding process. TRiC is incapable of solubilizing or refolding aggregated σ3, suggesting that σ3 must fold through TRiC immediately after translation to avoid entering thermodynamically favorable misfolded conformations. Our findings establish the steps involved in reovirus outer capsid assembly and provide insight into the mechanisms by which TRiC folds proteins into their native conformation.


Comparative Activities of the Phosphodegrons for beta-TrCP in Rotavirus NSP1 and IkappaB

Sarah Katen, John T. Patton

Dept. of Biology, Indiana University, IN, USA

Rotavirus NSP1 suppresses innate immune responses by directing E3 ubiquitin ligases to recognize and induce the proteasomal degradation of proteins necessary for interferon expression. For human and porcine rotaviruses, NSP1 contains a conserved C-terminal phosphodegron (PD) that recruits beta-TrCP to the E3 ligase. This NSP1 PD is a mimic of the PD present in inhibitor-of-NF-kB (IkappaB), a regulatory factor that must be recognized by beta-TrCP for NF-kB activation. An important question is how the NSP1 PD is able to outcompete the IkB PD for beta-TrCP in infected cells. To examine the possibility that the NSP1 PD has greater affinity for beta-TrCP than the IkappaB PD, we introduced mutations in the NSP1 PD (DSGIS), converting its sequence to match the IkappaB PD (DSGLDS). Through NF-kB reporter assays, we found that NSP1 with its wildtype PD was significantly more efficient in suppressing NF-kB activation than NSP1 with an IkappaB PD. Some co-immunoprecipitation assays indicated that suppression of NF-kB activation could be correlated with the degree of interaction between the PD and beta-TrCP. Although these results suggest that the NSP1 PD can prevent NF-kB activation simply by outcompeting the IkappaB PD for beta-TrCP, other factors may be at play. In particular, the NSP1 PD may be phosphorylated more efficiently than the IkappaB PD, and/or the phosphorylated form of the NSP1 degron may be more stable than that of the IkB degron. Work is underway to generate recombinant viruses that will allow us to compare the NSP1 and IkappaB PDs in context of infection.


Rotavirus Induces Intercellular Calcium Waves through Paracrine Purinergic Signaling from Infected Cells

Alexandra L. Chang-Graham, Jacob L. Perry, Melinda A. Engevik, Heather A. Danhof, Alicia C. Strtak, Jeanette M. Criglar, Mary K. Estes, Joseph M. Hyser

Baylor College of Medicine, USA

Rotavirus remains a major cause of severe diarrhea in children worldwide. During infection, rotavirus increases calcium signaling, which is necessary for replication and activation of secretory pathways in GI epithelium. A central premise of rotavirus-induced disease is that secretion of signaling molecules from infected cells activates fluid secretion from surrounding uninfected cells either directly or through the enteric nervous system. Candidate signaling molecules include the NSP4 enterotoxin, nitric oxide, and prostaglandins; however, signaling from infected to uninfected cells has not been directly observed. To characterize RV-induced calcium signaling dynamics and paracrine signaling, we conducted live-cell calcium imaging throughout the entire infection using cell lines and human intestinal enteroids (HIEs) engineered to stably express the genetically-encoded calcium indicator GCaMP. We found that rotavirus significantly increases steady-state and transient calcium signaling. Further, isolated rotavirus-infected cells generated multiple intercellular calcium waves (ICWs), which was the most prominent paracrine signal observed in both MA104 cells and HIEs. ICWs were abolished by blocking extracellular ATP/ADP signaling with apyrase or purinergic receptor inhibitors, but not by blocking extracellular NSP4, nitric oxide, or prostaglandin signaling. Knockdown of intracellular NSP4 attenuated ICWs, and blocking purinergic signaling significantly inhibited rotavirus replication. Rotavirus-induced ICWs were critical for multiple aspects of rotavirus pathogenesis, including fluid secretion and serotonin release, as well as regulating host responses, such as upregulation of IL-1α and mucin secretion. Thus, ICWs via purinergic signaling are a major contributor to rotavirus-induced paracrine signaling and represent a potent mechanism to amplify the pathophysiological signals underlying rotavirus diarrhea.


Reovirus uses macropinocytosis-mediated entry and fast axonal transport to infect neurons

Pavithra Aravamudhan (1), Jennifer Konopka-Anstadt (2), Krishnan Raghunathan (1), Amrita Pathak (2), Bruce D Carter (2), Terence S Dermody (1)

1: Department of Pediatrics, University of Pittsburgh School of Medicine and Children’s Hospital of Pittsburgh of UPMC; 2: Department of Biochemistry, Vanderbilt University

Mammalian orthoreoviruses (reoviruses) display serotype-specific patterns of tropism and disease in the central nervous system of newborn animals. Neurotropic reovirus serotypes spread through neural routes to cause lethal encephalitis. We used primary neuronal cultures from embryonic rat cortices or dorsal root ganglia (DRG) to study mechanisms of reovirus neuronal infection, which remain poorly understood. We also used microfluidic devices to separate neuronal soma from axonal termini and isolate treatments to the corresponding areas to study mechanisms of directional entry and transport. Inoculation of either neuronal soma or axonal termini of DRG neurons resulted in release of reovirus from the opposing terminus within 48 hours, suggesting that reovirus is capable of bidirectional transmission in neurons. Fluorescently labeled reovirus particles were observed to traffic predominantly in a retrograde direction with an average velocity of ~ 1 μm/s, suggesting a fast, motor-mediated transport mechanism. The particles traffic in non-acidic structures and retain their outer protein shell. However, endosomal acidification and proteolytic processing of virions are required for neuronal infection and presumably occur in the soma. Reovirus particles were found to co-traffic with markers of macropinocytosis and not clathrin-mediated uptake. Treatment with small molecule inhibitors revealed that cellular machinery mediating macropinocytosis is required for reovirus trafficking and infectivity in neurons. This entry mechanism differs from the dependence on clathrin-mediated endocytosis of reovirus in non-polarized cells. These results illuminate the host machinery used by reovirus for neuronal entry and trafficking and suggest directions for further investigation of molecular factors enabling these processes.


MicroRNAs, Autophagy, and Rotavirus Infection: New Insights in Pathogenesis and Therapy

Urbi Mukhopadhyay, Mamta Chawla-Sarkar, Anupam Mukherjee

ICMR-National Institute of Cholera and Enteric Diseases, India

MicroRNAs (miRNAs) have been implicated in virtually all known fundamental biological pathways like stress response, proliferation, differentiation, apoptosis, autophagy etc. Recent studies show that despite the ability of autophagy to act as an antiviral mechanism, some viruses use the autophagy machinery to favor viral replication, and Rotavirus (RV) is one of them. Therefore, considering the importance of autophagy in RV infection, study of autophagy-regulating miRNAs will allow a better understanding of viral pathogenesis and lead to the development of novel disease markers as well as therapeutic strategies. In the present study, we focused on the role of three miRNAs, miR-29b, let-7g and miR-99b, and analyzed their functions in context to RV infection and autophagy regulation. RV infection significantly downregulates miR-29b and let-7g expression. We found GSK3β and TSC1 as direct targets of miR-29b and let-7g, respectively. Downregulation of miR-29b and let-7g stabilizes the TSC1-TSC2 complex which ultimately leads to inhibition of mTOR activity. Moreover, following RV infection, miR-99b is upregulated and directly targets mTOR, therefore induces autophagy. Along with miR-99b, these three miRNAs cooperatively interact to regulate GSK3β-TSC1/2-mTOR pathway, the important factors of autophagy signaling. In addition, we observed that exogenous expression of mimic miR-29b and let-7g and/or knockdown of miR-99b resulted in inhibition of autophagy, therefore restricts RV replication. Taken together, our present study provides new mechanistic insight of cooperative interaction of three miRNAs in autophagy regulation and demonstrated an importance of combination therapy to restrict RV infection.


Phosphorylation cascade regulates the formation and maturation of rotavirus viroplasms

Jeanette Marie Criglar (1), Ramakrishnan Anish (1), Liya Hu (1), Sue Ellen Crawford (1), Banumathi Sankaran (2), B.V. Venkataram Prasad (1), Mary K Estes (1)

1: Baylor College of Medicine, USA; 2: Lawrence Berkeley National Laboratory, USA

Many RNA viruses replicate in cytoplasmic compartments (virus factories or viroplasms) composed of viral and cellular proteins, but the mechanisms required for their formation remain largely unknown. Rotavirus (RV) replication and genome packaging into nascent virus in viroplasms requires interactions between RV nonstructural proteins NSP2 and NSP5. We previously found two forms of NSP2 in RV-infected cells; a cytoplasmically dispersed dNSP2 that interacts with hypo-phosphorylated NSP5, and a viroplasm-specific vNSP2 that interacts with hyper-phosphorylated NSP5. Other reports indicate that hyper-phosphorylation of NSP5 by the cellular kinase CK1α requires NSP2 for reasons unclear. Here we show that silencing CK1α in cells prior to RV infection resulted in a >90% decrease in RV replication; disrupted vNSP2 and NSP5 interaction; resulted in a dispersed vNSP2 phenotype instead of localization into viroplasms; and reduced vNSP2 protein levels, indicating that CK1α directly affects NSP2. In vitro phosphorylation assays and crystallization studies showed that CK1α phosphorylates Ser313 of NSP2 which triggers NSP2 octamers to form lattice-like structures. Mass spectrometry and in vitro kinase assays indicated that NSP2 exhibits a novel autokinase activity that requires His225, and Cys8 which forms a disulfide bond with Cys85. Our studies show that a coordinated phosphorylation cascade involving CK1α phosphorylation of NSP2 controls viroplasm assembly. Using reverse genetics, we generated recombinant rotavirus with Ser313 mutations to directly evaluate CK1α phosphorylation of NSP2 and viroplasm formation. CK1α plays a role in the replication of other RNA viruses, suggesting that similar phosphorylation-dependent mechanisms may exist for other viruses requiring virus factories for replication.


WORKSHOP 7: APPLIED DSRNA VIROLOGY

Reovirus: Bench to Bedside and back again (this time with re-purposed therapeutics)

Don Morris

University of Calgary, Canada

Introduction: Oncolytic viruses (OV) are attractive therapeutics against multiple histologies given their direct cytotoxic effects and ability to prime therapeutic immune responses. Our group has previously shown utilizing in vitro, ex vivo and in vivo models that reovirus (RV), a dsRNA OV is an active monotherapy for the treatment of renal cell (RCC), breast carcinomas (BrCa) and multiple myeloma (MM) and have performed the first in man RV cancer clinical trials. To further optimize efficacy without increasing toxicity we investigated RV synergy with therapeutics such as Sunitinib, Bortezomib and immune checkpoint inhibitors. Experimental Design: In-vitro cytotoxicity, viral replication and chemokine production were assessed in a panel of human and murine cancer cell lines following exposure to reovirus in combination with other relevant therapeutics. In-vivo, RENCA (RCC), EMT6 (BrCa) and VK*Myc (MM) cells were implanted into syngeneic immunocompetent murine models. Tumor growth and overall survival were assessed following treatment with reovirus plus or minus re-purposed drugs. IFN-γ, myeloid-derived suppressor cells (MDSC), and protective immunity were assessed by ELISA, flow cytometry (Gr1+/CD11b+) and adoptive transfer experiments, respectively. PDL-1 and PD-1 expression on both tumour and immune cells were assayed. Results: In vivo, reovirus significantly reduced tumor burden and generated anti-tumor immune responses that were augmented by sunitinib, bortezomib and/or PD1 antagonism and resulted in improved overall survival and enhanced protective immunity leading in part to the development Phase II/III human clinical trials.


IgY from chicken egg yolk and Camelid derived nanobodies: Two strategies to control rotavirus diarrhea in animals and humans

Viviana Parreño

Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina

During the last decade our group of research has worked in the development of oral passive immune strategies to control Rotavirus A diarrhea based on IgY and VHH technologies.We will summarize the path from the inicial development and characterization steps to commercial registration and launch to the market of Bioinnovo IgY DNT, an alternative for the control of neonatal calf diarrhea. This product is based on specific IgY immunoglobulins from chicken egg yolk against the main infectious agents involved in neonatal calf diarrhea. The administration of IgY DNT reduces the severity and duration of scours, minimizing weight loss and improving the performance of calves. We have also obtained llama-derived nanobodies to Rotavirus. The versatility of these molecules allowed the development and validation of a sandwich ELISA for Rotavirus diagnosis that is being used by the Diarrhea Surveillance network of the Argentinean Ministry of Health since 2017. Two of the VHH clones -3B2 and 2KD1- showed broadly neutralizing activity in vitro and induced protection against diarrhea and virus shedding in a sucking mouse model. Furthermore, 3B2 also induced protection against human rotavirus in a gnotobiotic pig model. We are currently scaling up their production in different expression platforms in order to develop a functional food to reduce the risk of rotavirus circulation in the population and a pediatric drug as alternative or complementary tool to control Rotavirus diarrhea in Human infants.

145Oral and shotgun presentations Workshop 7: Applied dsRNA Virology

dsRNA bacteriophage as a tool for production of RNA-based treatments and crop protectants

Minna Poranen

Faculty of Biological and Environmental Sciences, University of Helsinki, Finland

The polymerase complexes of dsRNA viruses are highly efficient in dsRNA production. The key component of the polymerase complex is the viral RNA-dependent RNA polymerase (RdRp), which has specifically evolved to replicate dsRNA. We have used the purified RdRp of Pseudomonas phage phi6 to produce dsRNA from heterologous viral and non-viral sequences. Depending on the application, the long dsRNA molecules generated are further processed to pools of small interfering RNAs (siRNAs) using Dicer enzyme. The siRNA pools derived from herpes simplex virus (HSV) genes efficiently inhibit HSV infection in human cell cultures, and promising results have also been obtained using a mouse model of HSV keratitis. Similarly, siRNA pools representing conserved influenza A virus sequences significantly reduce the replication of different influenza A virus strains in human dendritic cells. For cost-effective dsRNA production, which is particularly needed in agricultural applications, we constructed a bacterial dsRNA production system using components derived from phage phi6. We demonstrate the utility of this system in the production of tobacco mosaic virus (TMV)-specific dsRNA molecules. When applied on Nicotiana benthamiana, the dsRNA generated protected the plants from TMV infection. The phi6-based dsRNA production systems pave the way for the development of novel therapeutics and crop protection strategies.


Puna: the first transgenic cow expressing RVA-specific VHH antibodies in milk

Celina Guadalupe Vega (1), Claudio Bernardo Santos (2), Lorena Laura Garaicoechea (1), Marina Bok (1), Lucía Rocha (1), Andres Wigdorovitz (1), Marcelo Criscuolo (2), Mauricio Berro (2), Viviana Parreño (1)

1: INTA, Argentine Republic; 2: Biosidus S.A.

Rotavirus (RVA) infections are a leading cause of diarrhea in children <5 years old worldwide, resulting in >200,000 deaths annually. It can be prevented through vaccination but treatment strategies are non-specific and involve electrolyte replacement and maintenance of nutrition. Live-attenuated oral RVA vaccines have variable degrees of efficacy, coverage and high cost. Other complementary therapies can help dealing with RVA. We developed a genetically modified dairy cow expressing a patented lama-derived single-chain antibody fragment (VHH) to RVA in its milk. VHHs are the smallest molecules with antigen-binding capacity and present distinctive outstanding properties, such as the ability to remain intact in the gastrointestinal tract during oral administration. The VHH selected, called clone 3B2, is a 15 kDa protein that was obtained from a lama immunized with VP6 protein. It neutralizes all RVA strains tested so far, in vitro and in vivo. On February 2016, the transgenic calf named Puna was born and artificially reared. To determine if the Puna could express VHH in the milk, at 9 months of age it was subjected to a hormonal treatment of milk induction. Colostrum and milk were collected daily and evaluated. Clone 3B2 VHH was detected in every sample, showing functional ELISA RVA-antibody titers ranging from 16 to 4096, and similar virus neutralization titers. This last titer was previously reported as protective in vivo. This suggests that a transgenic cow is an optimal way to produce large scale functional VHHs as a complementary strategy for the prevention and treatment of RVA-associated diarrhea.

147Oral and shotgun presentations Workshop 7: Applied dsRNA Virology

One nucleoside viral polymerase inhibitor that blocks the replication of norovirus, rotavirus and sapoviruses.

Jana Van Dycke (1), Francesca Arnoldi (2,3), Guido Papa (2), Justine Vandepoele (1), Oscar R. Burrone (2), Eloise Mastrangelo (4), Delia Tarantino (4), Elisabeth Heylen (1), Johan Neyts (1), Joana Rocha-Pereira (1)

1: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium; 2: International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.; 3: Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy; 4: CNR - Biophysics Institute, Università degli Studi di Milano, Milano, Italy

Noroviruses and rotaviruses are the two most important causes of severe childhood diarrhea, being responsible for ~800.000 deaths in children under the age of five, each year. The vast majority occurs in developing countries, where children have multiple episodes of diarrhea/year, leading to nutritional deficits and long-term consequences such as growth stunting. Both noro- and rotaviruses can easily spread causing large outbreaks in hospitals, schools, etc. Having one single highly efficient antiviral treatment to rapidly treat viral diarrhea is highly desirable.We here report that one single molecule can inhibit the replication of noro-, rota- and sapovirus by targeting the viral polymerase. 7-deaza-2’-C-methyladenosine (7DMA) inhibits the in vitro replication of human noro-, rota- and sapovirus with an EC50 of ~7 µM, ~1 µM and ~4 µM, respectively; 2’-C-methylcytidine (2CMC) also has broad-spectrum antiviral activity. The antiviral effect was confirmed by qRT-PCR, reduction of rotavirus induced viroplasms and rotavirus VP6-antigen staining. Furthermore, the 5’-triphosphates of 2CMC and 2’-C-methyladenosine (2CMA) inhibited the transcription activity of purified rotavirus SA11 double-layered particles (EC50 4 µM and 9 µM, respectively) confirming that these molecules target the viral polymerase.This is, to the best of our knowledge, the first time that a nucleoside analogue is shown to be active against a highly pathogenic dsRNA virus. Given the broad-spectrum antiviral activity, this study opens the door for the development of a truly broad-spectrum antiviral to treat and prevent viral diarrhea in children.


ROTAVIRUS SATELLITE SYMPOSIUM

Global Rotavirus Vaccine Introduction and Impact

Umesh D. Parashar

CDC, Atlanta GA, USA

Before the implementation of vaccines, rotavirus was the most common etiologic agent of fatal and severe childhood diarrhea, causing about one-third of diarrhea hospitalizations in children under the age of five years worldwide. Two new rotavirus vaccines showed good efficacy against severe rotavirus disease in large clinical trials and were licensed in 2006. Between 2006 and 2018, more than 90 countries have introduced rotavirus vaccination into national immunization programs. Following vaccine implementation, the burden of severe rotavirus disease in these countries has decreased substantially in vaccinated children. In addition to these direct health benefits, reduced gastroenteritis risk has been documented in unvaccinated groups, including older children and adults, suggesting indirect benefits (i.e. herd immunity). Rotavirus vaccination has also led to large, sustained declines in childhood deaths from diarrhea in Brazil and Mexico. In some countries, unexpected vaccine benefits such as reductions in childhood seizures have been observed. Post-licensure studies have provided new insights into the performance of these vaccines, such as the duration of protection, relative effectiveness in poor populations, and strain evolution after vaccine introduction. Early data from low-income countries on vaccine impact is promising. However, as rotavirus vaccines are rolled out more widely in Asia and Africa, continued monitoring to assess the performance of vaccine in these challenging settings is vital.


Lessons learned from long term rotavirus vaccine impact: Belgium data from 2007 to 2017

B. Standaert (1), B. Benninghoff (1), M. Raes (2)

1: GSK, Av. Fleming 20, B-Wavre;2: Jessa Hospital, Hasselt

Vaccines have been developed to control infections so that the disease burden is reduced as well as its related costs. Maintained disease control is an important finality for infectious diseases that we surprisingly rarely include in our evaluation assessments of new vaccines as a critical endpoint because of the difficulty to specify and quantify the control concept. However, if we are able to evaluate the impact flow of a new vaccine over time, we should identify more precisely how we get to the level of disease reduction/control and what needs to be done to improve over time. This is critical for those countries that haven’t yet started with some specific vaccination programs on how to introduce and maintain the ideal pathway of vaccine implementation. It may also help justifying the investment policy made in vaccination back to the decision makers.In a rotavirus vaccine impact study conducted in Belgium over a period of 11 years we were able to identify different forces that may influence the results of rotavirus disease control. Detailing those forces identified 9 of them of which 3 were disease-specific and 6 intervention-specific. They determine after 5 years of vaccine introduction the level of disease control reached in children less than 5 years old. The level reached determines and predicts how the disease control pattern will evolve after that period and what could potentially influence its course when proposing additional intervention options. The forces identified related to the disease specific situation are: the wild type circulation of the virus within and outside the child population (1), the pediatric force of infection in function of age and behavior (2), and the pronounced seasonality peak of the infection (3). The intervention-specific forces are the vaccine effect expressed through the reduction in medical care use (1), the vaccine coverage rate (2), the vaccine compliance and completion rate (3), the herd effect (4), the immune response change of the vaccine over time (5), and the catch-up scenarios (6) of the vaccine uptake. Those forces are not independent but interactive and time dependent. For instance the herd effect depends on the vaccine coverage rate among the transmitters especially during the peak season of the disease and is mainly present until the whole at risk group (children up to age of 5 years) is covered by the vaccine. In addition each force has no equal impact on the disease reduction or level of control reached after the 5 years of vaccine uptake. Vaccine coverage and vaccine effect are more critical to impact the control level than compliance and completion at high level of vaccine use, while the wild type virus circulation force is most critical when the vaccine coverage rate is low.

151Rotavirus Satellite symposium

Meanwhile we were able to demonstrate that the level of disease control reached is much dependent on the way the vaccination program has been initiated in the early years of its introduction. Once the 5 year period of vaccine implementation has been reached, major changes in control improvement are difficult to obtain unless additional intervention types are proposed such as earlier vaccination to capture the reduction of young-aged disease events, cocooning vaccination strategies to create better competition with the wild type virus circulation in closed environments, or priority vaccination of high risk groups such as young infants going to intensive care units. Applying the above analysis scheme of the 9 forces in the Belgium context we discovered that for a normal condition of rotavirus circulation with an annual peak season of disease from February to end of April (disease specific forces), the impact of first-year herd effect could have been much higher if the vaccine coverage rate among the virus transmitters of children (< 1y old) during the peak season was reaching levels above 85%. It was however around 49% at that time. A higher coverage could have been achieved if the start of the systematic vaccination program should have been as early as possible (June-July) prior the subsequent peak season. The vaccine coverage rate reached as quickly as possible a maintained rate above the 85%, while the compliance and completion rates have achieved the same numbers. Under such circumstances the observed disease control level in hospital care in Belgium was reduced to 77% (95%CI: 75%-80%) after 5 years reaching 79% (95% CI: 72%-86%) on average over a 10 year period. Model simulations indicated that with a higher herd effect caused by a better coverage of the early virus transmitters at initiation of the vaccine program the disease control results could have been more impressive in reaching and maintaining a level close to 90% at 5 and 10 years post vaccine introduction. In contrast low level of vaccine coverage –lower than 40%- will not result in much of an adequate disease control condition because the force of the wild type virus circulation will be too high.


Update on the Pentavalent Human-Bovine (WC3) Rotavirus Vaccine

Esmée de Wolde

Global Vaccines Medical Affairs, MSD

RotaTeq®, a live, oral, pentavalent rotavirus vaccine, has been routinely used in the US and other countries since February 2006. More than 226 million doses were distributed worldwide, and the vaccine is licensed in over 120 countries, most recently China, as of March 2018.The safety and efficacy of the vaccine were demonstrated in prelicensure clinical trials involving >70,000 infants. Several large-scale studies to assess vaccine effectiveness in routine pediatric use have been conducted. Hospital-based and laboratory-based studies assessed the vaccine’s impact on rotavirus and all-cause gastroenteritis disease burden, hospitalizations, and/or laboratory testing. Data from these studies have demonstrated that the vaccine is highly effective in routine practice. The effectiveness of a 3-dose regimen against combined hospitalizations and ED visits related to rotavirus gastroenteritis (RVGE) was 80-100% in US case-control studies. Reductions in RVGE-related hospitalizations were seen as early as 2008 and continue to be sustained. In addition, studies have demonstrated herd protection, as shown by reduced disease in unvaccinated, older children and adults. Similar effectiveness and impact have been demonstrated in other countries in which the vaccine has been routinely used. Several studies have supported vaccine use in the nursery setting before children become too old to be vaccinated.These data indicate that the vaccine is highly effective and has had a major public health impact on rotavirus disease starting early in life and within a few years of introduction.


ROTAVAC® (nHRV) – Product Development and Programmatic Implementation

Krishna Mohan, Raches Ella, Sai Prasad

Bharat Biotech International

ROTAVAC® (nHRV) is a neonatal Human Rotavirus 116E strain based vaccine, licensed in India and has received WHO Prequalification for supply to UN Agencies. ROTAVAC® was introduced into the Universal Immunization Program in India during 2016, with initial implementation in 9 states with more States set to follow during 2018 and beyond. ROTAVAC® was evaluated extensively in several controlled clinical trials prior to licensure. It was evaluated in a multicentre / multi-year Phase III efficacy trial with 6800 subjects resulting in a efficacy of 56.4% and 48.9% in the first year and second year of life respectively, showing continuous protection during the first two years of life and exhibiting one of the lowest reduction in efficacy between year 1 and 2 in currently licensed vaccines. Non-interference studies with UIP vaccines have also been completed. The effect of buffers was evaluated in ROTAVAC®, which showed its ability to seroconvert at the same levels with and without buffers, and it is the only vaccine to be administered without buffer. Currently a Phase IV study is under way in ~ 100,000 subjects to study the safety of ROTAVAC®, along with immunogenicity trials in Vietnam and Zambia (planned). With product and administration profiles very similar to that of OPV, and a cold chain foot print of ~ 3.2 cm3/dose, ROTAVAC® is ideally suited for introduction into immunization programs worldwide. A low cold chain foot print and multi dose presentations in 5 doses and 10 doses / vial, reduces the costs of international shipping, in-country transportation and cold chain storage. ROTAVAC® has a dose volume of 0.5 ml/ dose, the lowest in the world. This dosing volume assures delivery of 100% of the intended dose, without spit ups in infants. A next generation formulation of ROTAVAC® is undergoing licensure in India. This product will be available in single dose and multi dose presentations, making it conducive and cost effective for introduction globally.ROTAVAC® is currently undergoing registration in several countries. The project to develop ROTAVAC® has 12 publications and 5 patents have been granted globally in USA, EU, India, China, Japan, Russia, Australia, Mexico, etc. With more than 50 million doses delivered till date, ROTAVAC® is well accepted globally.


The pipeline of live, attenuated and non replicating rotavirus vaccines

Carl Kirkwood

Bill and Melinda Gates Foundations, Seattle, USA

Rotavirus vaccines (RVV) have had an enormous impact in improving child health. Three RVV have obtained WHO PQ, Rotarix (GSK), RotaTeq (Merck) and RotaVac (Bharat Biotech). Rotarix and RotaTeq are included in the national immunization programs or sub-national programs of 95 countries. RotaVac has been implemented in a staged roll-out in India, providing rotavirus vaccination to almost 50% of the birth cohort of India. Three RVV have been licensed by national regulatory agencies. In Vietnam, Rotavin (POLYVAC) was licensed in 2012 based on a phase 2 immunogenicity study. In India, the Serum Institute of India licensed their pentavalent bovine-human reassortant RVV (RotaSIIL) in 2017, following 2 large Phase 3 efficacy studies. The Lanzhou Lamb Rotavirus vaccine developed by Lanzhou Institute of Biological Products (China) was licensed in 2000. Several “new generation” RVV candidates utilising different strains, formulations and routes of administration are under clinical development. Of these, RV3-BB (BioFarma PT, Indonesia), based on an asymptomatic neonatal rotavirus strain has shown excellent clinical protection when administered in a neonatal schedule in a Phase 2 efficacy study. Several non-replicating parenteral vaccines are being evaluated, including the inactivated rotavirus particles (CDC) and the P2-VP8 subunit vaccine (PATH and SK chemicals, S. Korea). Of these the P2-VP8- P[8]/P[6]/P[4] vaccine has demonstrated excellent safety and immunogenicity in South African infants. New rotavirus vaccines, including live oral rotavirus candidates and non-replicating approaches continue to be developed, with the aim to improve the global supply and cost of rotavirus vaccines, and improve vaccine effectiveness in developing settings.


Progress towards the development of an inactivated rotavirus vaccine

Baoming Jiang

Centers for Disease Control and Prevention, Atlanta, Georgia, USA

Since rotavirus immunity is polygenic and cross-reactive, we have pursued an inactivated rotavirus vaccine (IRV) for parenteral immunization to improve low efficacy in developing countries and overcome severe adverse intussusception associated with live oral vaccines in young children. We have developed a human vaccine strain, CDC-9 by adapting and serial passages in Vero cells. This adaptation process led to several amino acid sequence changes in VP4 gene of the virus, which contributed to its structural stability and high growth in cell culture, and apparent attenuation in neonatal rats. In this presentation, I will review our progress in manufacturing process, stability, potency, and proof of concept studies of IRV in animals. I will highlight the immunogenicity and compatibility of standalone IRV and IRV-IPV combination vaccine in rats and guinea pigs. CDC-9 IRV or combined IRV-IPV when formulated with alum and administered intramuscularly induced comparable neutralizing antibody titers to rotavirus in animals. Similarly, IPV or combined IPV-IRV induced comparable neutralizing antibody titers to poliovirus types 1, 2, and 3. In addition, we observed similar antibody titers to rotavirus and polio in animals that received 1/4 of a full dose and a full dose of IRV and IPV alone or in combination. Our studies demonstrated a lack of interference between the two vaccines and an apparent antigen dose sparing for both IPV and IRV in rats, and thus established the proof of concept for a combination vaccine against rotavirus and poliovirus. I will provide an update on our preparation for clinical trials of IRV and IRV-containing combination vaccine.


Safety and immunogenicity of a parenteral trivalent P2-VP8 subunit rotavirus vaccine

Alan Fix (1), Michelle J. Groome (2,3), Lee Fairlie (4), Julie Morrison (5), Anthonet Koen (2,3), Maysseb Masenya (4), Nicola Page (6,7), Lisa Jose (2,3), Shabir A. Madhi (2,3), Monica McNeal (8), Len Dally (9), Iksung Cho (1), Maureen Power (1), Jorge Flores (1), Stanley Cryz (1)

1: PATH, Seattle, WA, USA; 2: Medical Research Council: Respiratory and Meningeal Pathogens Research Unit, University of the Witwatersrand, South Africa; 3: Department of Science and Technology/National Research Foundation: Vaccine Preventable Diseases, University of the Witwatersrand, South Africa; 4: Wits Reproductive Health and HIV Institute, University of the Witwatersrand, South Africa; 5: Family Clinical Research Unit, Stellenbosch University, South Africa; 6: National Institute for Communicable Diseases: a division of National Health Laboratory Service, South Africa; 7: Department of Medical Virology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa; 8: Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center, Cincinnati, USA; 9: The Emmes Corporation, Rockville, USA.

Background and aims: Although of great benefit to populations in resource limited settings, the efficacy of live-oral rotavirus vaccines in those settings is substantially lower than that in high-resource settings. It has been proposed that parenteral, non-replicating vaccines may overcome obstacles to optimal efficacy of oral vaccines in low resource settings. A monovalent P2-VP8-P[8] vaccine was recently found to be well-tolerated and immunogenic in infants. We evaluated the safety and immunogenicity of a trivalent formulation of the vaccine (P2-VP8-P[4]/P[6]/P[8]) in South African adults, toddlers and infants.

Methods: A multi-site, double-blind, randomized, placebo-controlled, dose-escalation, descending-age safety and immunogenicity trial was conducted in South Africa in 2016/2017. Two dose-levels (30µg and 90µg, total dose) of vaccine were tested first in healthy adults (three injections) and toddlers (single injection), and then three dose-levels (15µg, 30µg and 90µg) were assessed in healthy infants starting at 6-8 weeks of age (three injections four weeks apart). Local and systemic reactions within seven days and adverse events within 28 days after each injection, and all serious adverse events were assessed. Primary immunogenicity endpoints were assessed four weeks after the third injection. These included serum anti-P2-VP8 IgA and IgG responses (≥ 4-fold increase) to each of the three vaccine antigens


and neutralizing antibody seroresponses (≥ 2.7-fold increase from baseline) to each of the strains from which vaccine antigens were derived (Wa, DS-1 and 1076). IgG and neutralizing antibody responses were adjusted for maternal antibody. Fecal shedding of Rotarix® administered after completion of study vaccinations was assessed in a subset of infants.

Results: Thirty adults, 30 toddlers and 557 infants were assessed for safety. No significant differences were observed between the treatment groups in the proportions of participants with local reactions, systemic reactions or unsolicited adverse events. The proportions of infants with anti-P2-VP8 IgG seroresponses to all three antigens (P[4], P[6] and P[8]) in the placebo, 15 µg, 30 µg and 90 µg groups were 9, 99, 99 and 100%, respectively (p<0.0001 for all dose groups compared to placebo). Although significantly higher than in placebo recipients, the proportion of infants with anti-P2-VP8 IgA seroresponses to each individual antigen was between 20 and 34% across all three dose groups. In the 90 µg group, the proportion of infants with neutralizing antibody responses to each of the 3 strains was 78.4%, 80.6% and 81.3% for Wa, DS-1 and 1076, respectively, and the proportion of infants with neutralizing antibody seroresponses to all three strains was 8, 50, 61 and 62%, respectively, in the placebo, 15 µg, 30 µg and 90 µg groups (p<0.0001 for all dose groups compared to placebo). Compared to placebo, there was a trend to increasing reduction of Rotarix shedding with increasing dose-level, albeit not statistically significant. Conclusion: The parenteral trivalent P2-VP8 vaccine was well-tolerated, with very promising anti-P2-VP8 IgG and neutralizing antibody responses across the three vaccine P-types (and broader than previously demonstrated for the monovalent vaccine). On the basis of these promising results, assessment of clinical efficacy in a Phase 3 study is being planned for 2019.


8. ABSTRACTSPoster presentations

(in alphabetic order by topic and presenting author)

WORKSHOP 1: VIRAL DIVERSITY

Prevalence and circulating genotypes of rotavirus induced diarrhoea amongst under fives at the University of Ilorin Teaching Hospital (UITH), Ilorin, Nigeria

D. O Amadu (1), M.B Abdulkadir (2), S.A Saka (2), A Fadeyi (1), A.S Aderibigbe (3), D.B Tijani (1), C. E. Nwabuisi (1), A.A Akanbi ii (1), J.M. Nwenda (5), A. Isiaka. (4), Armah George (6)

1: Dept. of medical microbiology and parasitology, university of ilorin teaching hospital, ilorin, Kwara, Nigeria; 2: Dept of pediatrics and child health, university of ilorin teaching hospital;03: Dept of community health and epidemiology, university of ilorin teaching hospital; 4: Abuja office. world health organisation country office; 5: World health organisation,regional office. for Africa, Brazzaville, Congo; 6: Noguchi memorial institute for medical research,university of Ghana

BACKGROUND: Rotavirus is the most common aetiological agent associated with severe gastroenteritis leading to dehydration and death in young infants worldwide and is responsible for an estimated 527,000 deaths annually. This study sets out to describe the prevalence and circulating genotypes of rotavirus induced diarrhoea amongst under fives at the University of Ilorin Teaching Hospital (UITH), Ilorin, Nigeria METHOD: All children Under-5 with acute diarrhoea hospitalised at UITH, Ilorin, Nigeria were recruited consecutively between January 2013 and 2017. Stool samples of participants were examined for rotavirus by ELISA using OXOID prospecT kit according to manufacturer’s instruction.Stool specimens positive for rotavirus group A were sent tGhana for genotyping (G and P typing) by multiplex PCR. RESULT: Over the review period, 733 under five children were hospitalized with acute diarrheal disease with 437(59.6%) males.213(29.0%) tested positive for rotavirus of which 144(67.7%) were infants, 49(23.2%) were aged between 12 and 23 months; and 19 (9.1%) were older than 23 months. From the rotavirus ELISA positive samples, 153 were selected for rotavirus genotyping. The most prevalent rotavirus G-type and P-type variants were G1(25.5%) and P[6](30.1%). The Predominant single G/P combination was G2P[8] (24, 15.7%) and G2P[6] (18,11.6%). Predominant G-mix identified were G1G3(17.0%), G1G2 (2.6%), G1G2G3(2.6%) and G3G12 (1.9%), while predominant p-mix identified include P[6][8](16.9%), P[4][8](3.2%) and P[4][6](2.6%) and P[4][6][8] (0.7%). CONCLUSION: Rotavirus gastroenteritis is common among under five children with acute diarrhoeal disease. Wide variability exists in the G-types and P-types variants in this environment with predominance of G1, G2, P[6] and P[8].


Emergence of rare G2P[8] rotavirus genotype among children <5 years of age in the Philippines

Joseph Bonifacio, Mary Ann Igoy, Amado III Tandoc, Socorro Lupisan

Research Institute for Tropical Medicine, Philippines

BACKGROUND: Recent reports from several regions identified the circulation of rare G2P[8] strains in <1%. In the Philippines, G2P[8] genotype emerged as the third most common type of group A rotavirus in 2017. The increasing case of G2P[8] strain in the country highlights the need to characterize the whole genomic RNA constellation of rotavirus G2P[8] strain in the Philippines.

MATERIALS AND METHODS: Whole genome sequencing of specimens with G2P[8] genotype from the rotavirus surveillance were included in the analysis. Nucleotide sequences of the 11-segments of rotavirus G2P[8] strains were determined using dideoxynucleotide chain termination method. Multiple sequence alignments were constructed using lasergene software. Phylogenetic and molecular analyses were also conducted. Genotypes of each segment were determined using the RotaC 2.0 genotyping tool.

RESULTS: G2P[8] strains from the stool specimens exhibited a complete unique DS1-like genotype constellation of G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. This novel genotype constellation revealed a close relationship between the sample and Bangladesh strain by comparing each segment. Based on sequence similarities, the Philippines G2P[8] strains were closely related with 88.7% to 99.2% identity in all gene segments.

CONCLUSION: To date, this is the first report on whole-genome based characterization of G2P[8] strain in the Philippines. Findings provide evidence that the circulating DS-1 like G2P[8] strain in the Philippines and the DS-1 like G2P[8] strains that have emerged in other Asian countries originated from a common ancestor. Study results can provide important insights on the evolutionary dynamics of novel DS-1 like G2P[8] rotaviruses and provide essential information for vaccine development and implementation.

163Poster presentations Workshop 1: Viral Diversity

Emergence of G8P[8] rotavirus strains of apparent Asian origin in four US cities, 2016-17

Michael D. Bowen (1), Mathew D. Esona (1), Charity Perkins (1), Slavica Mijatovic-Rustempasic (1), Geoffrey A. Weinberg (2), Amy Kinzler (3), John Williams (3), Gail A. Bloom (4), Mary Allen Staat (5), Monica M. McNeal (5), Mary E. Wikswo (1), Margaret M

1: Centers for Disease Control and Prevention, USA; 2: University of Rochester School of Medicine and Dentistry, USA; 3: Children’s Hospital of Pittsburgh, USA; 4: Indiana University Health Pathology Laboratory, USA;5: Cincinnati Children’s Hospital Medical Center, USA

G8P[8] rotavirus A strains emerged in Thailand in 2013 and have been detected subsequently in other countries of south-east Asia and Japan. These strains possess the genotype constellation G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 and appear to be the progeny of a reassortment event(s) between bovine and human rotavirus strains. G8 rotavirus strains have been rare historically in the US. Genetic analysis of stool samples collected in 2016-17 through two surveillance networks in the US, the New Vaccine Surveillance Network (NVSN, active surveillance) and the National Rotavirus Strain Surveillance Network (NRSSS, passive surveillance), detected G8P[8] strains at 3 NVSN sites (Cincinnati OH, Pittsburgh PA, Rochester NY) and one NRSSS site (Indianapolis IN). A total of 42 G8P[8] strains were detected, representing 8.2% of the aggregate NVSN and NRSSS rotavirus strains genotyped for the 2016-17 season. Next generation sequencing of a subset of these strains revealed that they possess the same genotype constellation as the Asian strains, G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. BLAST searches and phylogenetic analyses of individual genes indicate that these US G8P[8] strains are very closely related to G8P[8] strains from Thailand, Vietnam, and Japan. This observation strongly suggests these G8P[8] strains were introduced into the US from Asia. Continued surveillance is needed to monitor G8P[8] and other genotypes in the US and ensure continued high efficacy of RotaTeq® and Rotarix™ vaccines against circulating strains.


Genetic diversity of bovine rotavirus in Uruguay: detection of common and uncommon genotypes

Matias Castells (1), Dario Caffarena (2), Laura Casaux (2), Carlos Schild (2), Federico Giannitti (2), Daniel Castells (3), Franklin Riet-Correa (2), Viviana Parreño (4), Rodney Colina (1)

1: Universidad de la República; 2: Instituto Nacional de Investigación Agropecuaria; 3: Secretariado Uruguayo de la Lana; 4: Instituto Nacional de Tecnología Agropecuaria

Diseases that affect neonate calves are a major cause of global economic losses due to deaths, expenses associated with treatments, delayed growth/development and difficulties generated by not obtaining enough calves to make an adequate cows replacement. Infectious neonatal diarrhea, one of the leading causes of calves’death, involves many infectious agents including Rotavirus as the most prevalent worldwide.Milk and meat production constitutes one of the main economic incomes for Uruguay; however, prevalence and genetic diversity of Rotavirus remain unknown. With these objectives, 759 feces from live and death calves with diarrhea, collected from dairy and meat farms between July 2015 to December 2017 were studied.NSP3 real time PCR sample screening showed a 61% of positivity, while VP4/VP7 genotypes demonstrate the circulation of a wide genetic diversity from both, dairy and meat farms. The most common detected G and P genotypes combinations were G6P[11] (43%), G6P[5] (38%), and G10P[11] (17%), with different genetic lineages. In addition, a non-common genotype G24P[33] was identified; only described in one case in Japan in 2007. Genome constellation was studied for 10 samples: VP6 and NSP1-5 genes (in addition to VP4 and VP7) were sequenced. Surprisingly, the strain G24P[33] showed the same genetic pattern than strain Dai-10 from Japan, with T9 for NSP3 another non-common genotype, but with E12 genotype for NSP4 gene. All the Uruguayan samples showed E12 genotype (independently of the different VP4, VP6-7, NSP1-3 and NSP5 combinations), the same detected for other south American samples, a genotype associated with this geographical region.


Molecular characterization of a new botybirnavirus that infects the phytopathogenic fungus Botrytis cinerea CCg427

Luis Eduardo Cottet* (1), Christiaan Potgieter (2), Antonio Castillo (1)

1: Universidad de Santiago de Chile, Chile; 2: Centurion, Lyttelton, South Africa, Deltamune (Pty.) Ltd., Lyttelton

Cottet, Luis Eduardo

Botrytis cinerea is a phytopathogenic fungus that causes the gray mold disease in a wide host plants range. The fungal strain B. cinerea CCg427 showed low virulence in pathogenicity assays performed with leaves of bean plants and vines, besides presenting low lacasse enzyme activity. This strain contains at least nine dsRNAs with molecular sizes of about 1.0 to 10 kbp. The viral particles were purified by sucrose gradient, obtaining at least three types of isometric particles with diameters of about 18 to 50 nm, which would be associated with four different polypeptides of about 72, 75, 83 and 86 kDa. Electron microscopy of ultrathin sections from fungal mycelium, revealed the presence of the different types of viral particles in the same hyphal compartment.The most abundant dsRNAs were purified and the corresponding cDNAs were synthesized. The sequence analysis of two cDNA molecules with sizes of 5.6 and 6.2 kbp, revealed that each contains an ORF that encodes for polypeptides of 1611 and 1828 aa, respectively. The BLAST analysis showed that both polypeptide sequences have similarity with proteins of the viruses ABRV1 and SsBRV1, both classified as botybirnavirus that infects Alternaria and Sclerotinia species, respectively. This result was confirmed with a phylogeny analysis that showed that the new Botrytis virus was associated with others botybirnaviruses.The results suggest that B. cinerea CCg427 present a hypovirulence phenotype that could be conferred by mycoviruses, and that one of them correspond to a new botybirnavirus named BcBRV1.Supported by FONDECYT 3160278 Postdoctoral project.


VP4 Gene Polymorphism of human rotavirus and its impact on P-genotyping by RT-PCR

Susan Afua Damanka, Francis Ekow Dennis, Belinda Larteley Lartey, Chantal Ama Agbemabiese, Frederick Asamoah, George Enyimah Armah

Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana

Background: Rotaviruses (RVs) circulate in vast diversity both in humans and animals as a result of mutations, reassortment and interspecies transmissions. Indeed, rotavirus surveillance networks within the WHO have documented and shown eclectic geographic and temporal diversity in circulating G- and P- genotypes identified in children <5 years of age. To effectively monitor vaccine performance and effectiveness it remains vital to apply robust molecular methods to identify prevalent circulating genotypes and the emergence of novel strain types. This study sort to investigate and understand why a proportion of rotavirus strains remained untyped even though the methods and primer sets described by the WHO manual were duly followed. Methods: VP4 gene was amplified, sequenced and characterised using BLAST and RotaC v.2.0 web-based genotyping tool. Multiple sequence alignments were performed using CLUSTALW algorithm within the Mega v6.06 software.Results: Of the (n=80) rotavirus-positive samples, 71.3% (n=57) were successfully sequenced and characterized. Forty-three (43/57; 75.4%) of these were characterized as the globally common P[8]a genotype. Mutations were identified in the primer binding region of the VP8* fragment of the VP4 gene of the Ghanaian P[8]a strains, when aligned with P[8]-specific primers, which may be responsible for genotyping failure. The VP4 genes of all the study strains clustered in P[8]-lineage III, mainly with those from Africa, Asia, Australia and Europe. However, they appear to be genetically divergent to rotavirus vaccine strains (Rotarix and RotaTeq).Conclusion: The study highlights the need for regular evaluation of the multiplex PCR typing method especially in the post-vaccination era.


Detection and full genome characterization of human Group C rotavirus in Ghana

Francis Ekow Dennis (1), Yen Hai Doan (2,3), Chantal Ama Agbemabiese (1), Belinda Larteley Lartey (1), Susan Afua Damanka (1), Frederick Karikari Asamoah (1), George Enyimah Armah (1), Kazuhiko Katayama (3)

1: Noguchi Memorial Institute for Medical Research, University of Ghana; 2: National Institute of Infectious Diseases, Tokyo, Japan; 3: Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan

Background and aims: Group C rotaviruses (RVCs) are enteric pathogens detected in either sporadic cases or outbreaks of gastroenteritis mainly in human, bovine and porcine populations. Previous detection of RVC strain in Southern Ghana was by Polyacrylamide Gel Electrophoresis during a hospital-based burden of disease study in 2004. In March 2017, two RVC strains were detected during routine rotavirus surveillance in Northern Ghana, 9 days apart, in children aged 3 and 7 months respectively. This study sought to completely characterize the genomes of these sporadic RVC strains.Methods: Samples were collected from diarrhoeic children <5 years old reporting at the War Memorial Hospital in Navrongo, Northern Ghana as part of routine surveillance. Of the 59 samples collected between November 2016 and October 2017, two RVA EIA-negative samples displayed a typical RVC PAGE electropherotype. RNA extracts of these samples were subjected to Illumina MiSeq Next Generation Sequencing to characterize RVC genomes. Identified RVC genes were genotyped by phylogenetic analysis using MEGA v7.026.Results: The two RVC strains revealed a typical human RVC genotype constellation G4-P[2]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic analysis of all 11 gene segments of both strains confirmed their human origin. The Ghanaian RVC strains were nearly identical, with all genes displaying type II genome configuration, and being closely related to the Italian RVC strain PR713 isolated in 2012.Conclusion: The detection and characterization of human RVC strains of type II genome configuration extends the diversity of RV strains circulating in Ghana, and contributes to knowledge of the genetic diversity of human RVCs.


Comparative genomic analysis of genogroup 1 (Wa-like) and genogroup 2 (DS-1-like) rotaviruses circulating in seven US cities, 2013-2016.

Mathew D Esona (1), M. Leanne Ward (1), Mary E Wikswo (1), Rashi Gautam (1), Naga S Betrapally (1), Slavica Mijatovic-Rustempasic (1), Charity Perkins (1), Eric Katz (1), Jose Jaimes (1), Rangaraj Selvarangan (2), Christopher J Harrison (2), Julie A Boom

1: CDC, USA; 2: Kansas City Children’s Mercy Hospitals and Clinics, United States; 3: Texas Children’s Hospital, United States; 4: Seattle Children’s Hospital, United States; 5: Cincinnati Children’s Hospital Medical Center, United States; 6: Vanderbilt University Medical Center, United States; 7: University of Rochester School of Medicine and Dentistry, United States

Group A rotaviruses (RVA) are double-stranded RNA viruses associated with acute gastroenteritis (AGE), resulting in an estimated 200,000 deaths annually. For over a decade, the New Vaccine Surveillance Network (NVSN) has conducted active RVA strain surveillance in the US. The effects of possible vaccine pressure on contemporary circulating strains in the US and their genetic make-up are still under investigation. Here we report the whole-gene characterization (ORF regions) for 156 RVA strains collected at 7 NVSN sites during the 2013-2016 rotavirus seasons, including 100 collected from children who had received Rotarix™ or RotaTeq® vaccines. The strains included 56 G1P[8], 43 G12P[8], 21 G9P[8], 23 G2P[4], 5 G3P[6], 2 G6P[8], 2 RotaTeq (G6P[5] and G1P[5]), 1 G2P[8], 1 G3P[8], 1 G4P[8], and 1 G9G12P[8]. The majority of the single and mixed strains had a Wa-like backbone with consensus genotype constellation of G1/G3/G9/G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, while the G2P[4], G3P[6], and intergenogroup reassortant strains G2P[8] and G1P[8] displayed a DS-1-like genetic backbone with consensus constellation of G2/G3/G1-P[4]/P[6]/P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Phylogenetic and similarity matrices analysis revealed 2-9 and 4-7 subgenotypic allelic clusters among the Wa- and DS-1-like strains, respectively. Most study strains clustered into previously defined alleles, but we defined 3-5 new alleles. In addition, this study reports 3 double intergenogroup reassortant strains (G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) possessing Wa-like VP7 and VP4 gene segments on a DS-1-like genetic backbone.These sub-allelic constellations may enhance our understanding of RVA evolution under vaccine pressure and help identify a possible mechanism of immune escape, resulting in rotavirus-associated diarrhea in vaccinated individuals.


Epidemiology and Genetic Diversity of Group A Rotavirus in Acute Diarrhea Patients (<5 Years) in Pre-vaccination Era in Delhi.

Shipra Gupta (1), Vasundhara Razdan Tiku (2), Satender Aneja (3), Praveen Kumar (3), Mohammad Islamuddin (1), Pratima Ray (1, 2)

1: Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India; 2: Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India; 3: Department of Pediatrics, Kalawati Saran Children’s Hospital, New Delhi, India

India accounts for 22% (98,621) of 453,000 global rotavirus deaths among children (<5 years) annually and has recently introduced its indigenous rotavirus vaccine, ROTAVAC® into UIP. The present study aimed to estimate the molecular epidemiology of rotavirus strains circulating in Delhi (2013-2016), prior to vaccine implementation. A total of 883 fecal specimens collected from diarrheal children admitted at KSCH, Delhi were screened for Group-A rotavirus (RVA) by antigen ELISA. G/P genotyping was performed using multiplex reverse transcription PCR. Overall, 47% (n=415) cases were found positive for RVA. G/P typing could be performed for 320 (77.1%) samples. Among G-types, G1 was the most prevalent (53.4%) followed by G9 (10.6%), G12 (7.8%), G2 (4.7%) and G3 (2.8%). G4 (1.3%), G8 (1.0%) and G10 (0.3%) strains were rarely detected. P[8] was most predominant (42.8%) P-type followed by P[4] (16.3%), P[6] (17%), P[11] (1.9%) and P[9] (1.6%). Year 2016 marks the detection of rare P-type P[9]. Importantly, this is the first report of P[9] from India. In total 20 different G-P combinations, G1P[8] was most prevalent (35%) strain followed by G12P[6] (5.6%), G1P[6] (4.1%), G2P[4] (2.5%) and G9P[6] (1.6%). G4P[6], G8P[8], G12P[8], G3P[8] and G9P[8] strains were very rarely detected. Interestingly, the present study documents a high incidence (15.4%) of unusual G/P combinations viz. G9P[4], G1P[4], G3P[9], G2P[6], G1P[11], G2P[11], G1P[9], G3P[6], G12P[11] and G12P[4]. Our pre-vaccination data on rotavirus prevalence and strain diversity form the baseline to compare the post-vaccination data and would be helpful in gauge the impact of vaccine in Delhi.


Changes in Amino Acid Profiles in Antigenic Epitopes of the VP7 Protein of circulating Rotavirus Strains in Ghana

Belinda Naa Larteley Lartey, Susan Afua Damanka, Francis Ekow Dennis, Chantal Ama Agbemabiese, Frederick Asamoah, George Enyimah Armah

Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon, Accra, Ghana

Background: Due to the high burden of rotavirus associated gastroenteritis in children, two vaccines, Rotarix® and RotaTeq® have been licensed and introduced into national immunization programmes in many countries. Knowledge of circulating RV strains in a population before the introduction of RV vaccine into its immunization program is useful in evaluating the effect of the intervention. A comparative analysis study was performed to understand diversity occurring within circulating Ghanaian VP7 gene segments and vaccine strains pre-vaccination era. Methods: VP7 gene was amplified by one-step RT-PCR. Amplicons were purified, sequenced and genotypes determined using RotaC genotyping tool. Sequences were aligned using CLUSTALW algorithm and Phylogenetic analysis performed using the Maximum likelihood method in MEGA6 software.Results: Study strains were characterized as G3. Phylogenetically, they clustered in sub-lineage IIId of lineage III together with AU-1-like reference strain, SA188DGM from South Africa and Ito from Japan. Comparison of neutralizing epitopes of VP7gene of prototype and vaccine strains with that of study strains revealed amino acid changes in 7-1b (Ala146?Val146), and 7-2 (Val218?Ile218 and Ala221?Asp221) regions. The influence of the change in amino acid profiles in antigenic epitopes of circulating Ghanaian RV strains to vaccine effectiveness however remain yet to be elucidated.Conclusion: This study showed considerable changes of human rotavirus G3s detected in Ghana in comparison with the Prototype AU-1 strain, incorporated in the pentavalent licensed rotavirus vaccine. Detailed molecular analysis of circulating VP7 and VP4 gene segments needs to be performed regularly in order to track RV strains with altered antigenic epitopes.


Complete genome sequence of two cucumis melo endornavirus strains discovery during deep sequencing investigation in human stool samples in Brazil

Antonio Charlys da Costa (1,2), Elcio Leal (3), Flavio Augusto de Padua Milagres (4,5,6), Shirley Vasconcelos Komninakis (7,8), Rafael Brustulin (4,5,6), Maria da Aparecida Rodrigues Teles (5,6), Márcia Cristina Alves Brito Sayão Lobato (5,6), Rogério Togisaki das Chagas (5,6), Maria de Fátima Neves dos Santos Abrao (5,6), Cassia Vitória de Deus Alves Soares (5,6), Xutao Deng (9,10), Eric Delwart (9,10), Ester Cerdeira Sabino (1,2), Adriana Luchs (11)

1: Sao Paulo Institute of Tropical Medicina, Brazil; 2: University of São Paulo, Brazil; 3: Federal University of Para, Brazil; 4: Federal University of Tocantins, Brazil; 5: Public Health Laboratory of Tocantins State (LACEN/TO), Brazil;6: Secretary of Health of Tocantins, Brazil; 7: Faculty of Medicine of ABC, Brazil; 8: Federal University of Sao Paulo, Brazil; 9: Blood Systems Research Institute, USA; 10: University of California San Francisco, USA; 11: Adolfo Lutz Instute, Brazil

Endornaviruses are endogenous dsRNA viruses that cause persistent and symptomless infections in plants. The complete genome sequence of two Cucumis melo endornavirus (CmEV) strains were obtained using next-generation sequencing (NGS) while investigating fecal samples for the presence of gastroenteritis viruses. CmEV was recently reported in Cucumin melo in USA (CL-01) and South Korea (SJ1), being considered as a possible novel species in the genus Endornavirus. During a survey conducted throughout the state of Tocantins, in North Brazil, from 2010 to 2016, NGS techniques for a complete genome determination of rotavirus was used for samples BRA/TO-23 and BRA/TO-74, in which the CmEV were also detected. The Brazilian CmEV BRA/TO-23 and BRA/TO-74 isolates were nearly identical to the reference CmEV CL-01 (USA) and SJ1 (South Korea) strains, showing 97% and 98% of nucleotide and amino acid identity, respectively. The genome obtained from BRA/TO-23 and BRA/TO-74 samples was determined to be 14,737 bp and 15,004 bp in size, respectively, and contains a single large ORF as reported to Endornaviridae family. Characteristics endornavirus domains with enzymatic functions were also recognized: one viral RNA dependent RNA polymerase 2, two glycosyltransferase (GT) domains from capsular polysaccharide protein family, one UDP-glucoronosyl/UDP-glucosyl transferase domain, and one UvrD-like helicase C-terminal domain. Endornaviruses are not known to be associated with human disease and their presence may simply reflect recent dietary consumption. In Brazil, investigations


addressing the identification and genome characterization of endornavirus are virtually absent. Metagenomic offered an opportunity to identify these newly described endornavirus species.


Epidemiology of Rotavirus Strains Circulating in Niger, Africa from 2014 to 2016

Monica Malone McNeal (1), Amy Singh (1), Victoria Fabry (1), Nicole Meyer (1), Rebecca Grais (2), Celine Langendorf (2), Shelia Isanaka (2), Ousmane Guindo (3)

1: Cincinnati Children’s Hospital Medical Center, USA; 2: Department of Research, Epicentre, France; 3: Department of research, Epicentre, Niger

A randomized, placebo-controlled trial to evaluate the efficacy of a live oral bovine rotavirus pentavalent vaccine (BRV-PV, Serum Institute of India) was conducted in Niger beginning in August of 2014. Stool samples were collected when subjects experienced gastroenteritis,which was defined as three or more looser-than-normal stools during a 24-hour period with or without vomiting. Stools were tested for the presence of rotavirus antigen using an enzyme immunoassay (Premier Rotaclone kit, Meridian Bioscience). Rotavirus positive samples were characterized by molecular methods to determine G and P type and confirmed by sequencing. A total of 797 rotavirus positive stools were characterized by genotyping for VP7 and VP4. Eighty-four isolates had multiple G and or P types. No G type was determined for 5 isolates and 30 isolates did not have a P type determined either by genotyping or sequencing. Seven isolates did not type or sequence for either a G or a P type. Out of the 7 isolates, 4 were negative when run in a realtime PCR assay using primers for the NSP3 gene indicating a possible false positive signal in the EIA assay. The most predominate genotype was G2P4 with 373 isolates followed by G1P6 (94), G12P6 (93), G3P6 (54), and G9P8 (41). Much lower numbers of G12P6 (11), G2P6 (4), G2P8, G3P8 (2), G9P4 (3) and G9P6 (11) were determined. The large number of rotavirus strains containing P4 and P6 along with the different G types shows the extent of the diversity of circulating strains in Niger.


Identification of novel reassortant mammalian orthoreovirus serotypes 1, 2, 3 from bats in Slovenia

Tina Mikuletič (1), Danijela Rihtarič (2), Peter Hostnik (2), Nataša Toplak (3), Simon Koren (3), Urška Kuhar (2), Urška Jamnikar-Ciglenečki (2), Denis Kutnjak (4), Andrej Steyer (1)

1: University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia; 2: University of Ljubljana, Veterinary Faculty, Ljubljana, Slovenia; 3: Omega d.o.o., Ljubljana, Slovenia; 4: National Institute of Biology, Ljubljana, Slovenia

Recently, first report on mammalian orthoreovirus (MRV), strain SI-MRV01 with high similarity to newly described bat MRVs, was described in a child with severe diarrhoea. Genetically similar strains have also been reported from other mammals, which reveals their wide host distribution. The aim of this study was to retrospectively investigate the occurrence and genetic diversity of MRVs in bats in Slovenia, from samples obtained throughout the country in 2008 to 2010, and in 2012.Broad-range RT-PCR and specific bat MRV real-time RT-PCR were used for detection of MRVs in bat guano. MRV isolates were obtained from propagation in LLC-MK2 cell culture. Detailed whole-genome molecular characterisation was performed using Ion Torrent PGM platform.Overall, bat MRVs were detected in 1.9% to 3.8% of bats in 2008, 2009 and 2012. However, in 2010 the prevalence was 33%, which defined an outbreak of the single SI-MRV01 strain. Here, we report on the identification of five reassortant MRV isolates that were designated as SI-MRV02, SI-MRV03, SI-MRV04, SI-MRV05 and SI-MRV06. There is high genetic variability between these characterised isolates, with evident genome reassortment seen across their genome segments.In this study, we provide the first epidemiological overview that includes the molecular variabilities among the newly described bat MRV phylogenetic clusters. These show that all three MRV serotypes were present in bat population. This study generates new information about the epidemiology and molecular characteristics of emerging bat MRV variants, and provides important molecular data for further studies of their pathogenesis and evolution.


GENOMIC INVESTIGATION OF THE FAECAL RNA VIROME IN CHILDREN FROM OUKASIE CLINIC, NORTH WEST PROVINCE, SOUTH AFRICA

Milton Tshidiso Mogotsi (1,2), Peter Nthiga Mwangi (1), Hester Trudi O’Neill (2), Martin Munene Nyaga (1)

1: Next Generation Sequencing Unit, Faculty of Health Sciences, University of the Free State, South Africa; 2: Microbial, Biochemical and Food Biotechnology, Faculty of Natural and Agricultural Sciences, University of the Free State, South Africa

Establishing a diverse gut microbiota after birth is essential in preventing illnesses later in life. However, limited knowledge exists about the infant gut virome during this developmental stage, with RNA viromes being generally overlooked. A metagenomics study was performed to characterize and compare the enteric RNA viromes in infants below one year old in South Africa.Faecal samples were collected from four healthy infants at three different time points (6, 24 and 36 weeks old). Samples were treated using NetoVIR protocol to enrich for viral particles. RNA extraction and RT-PCR was performed, followed by library construction using Nextera XT DNA library preparation protocol. Sequencing was done on Illumina MiSeq for 500 cycles (2x251 bp paired-ends). Using an in-house analysis pipeline, raw reads were subjected to quality control using FastQC and de novo assembled on Metaspades. Contigs were BLAST searched against a bacterial/viral database.Diverse human enteric viruses were found in all faecal samples. Reoviridae sequences were the most detected, attributed to rotavirus A, followed by viruses within the Picornaviridae family such as parechoviruses, echoviruses, coxsackieviruses, enteroviruses and polioviruses. Astroviruses (Astroviridae) and norwalk viruses (Caliciviridae) were detected in only a few samples.Evidently infants’ gut is colonized by distinct viral populations irrespective of their health state. It would be interesting to follow up on their virome composition and diversity over a few years. Although the human enteric virome remains less studied, advancements in sequencing technology continuously allow characterization of the previously neglected virome, especially the largely unexplored RNA viromes.


Whole genome characterization of rotavirus strains from National Rotavirus Surveillance of acute diarrhea in Mozambique

Eva Dora João (1,2), Amy Strydom (3), Benilde Munlela (1), Martin Nyaga (4), Assucênio Chissaque (1), Jerónimo Langa (1), Ezequias Sitoe Sitoe (1), Jorfélia Chilaúle (1), Elda Anapakala (1), Júlia Sambo (1), Esperança Guimarães (1), Diocreciano Bero Bero

1: Instituto Nacional de Saúde, Mozambique; 2: Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Lisbon, Portugal; 3: Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa; 4: Next Generation Sequencing Unit, Department of Medical Virology, Faculty of Health Sciences, University of the Free State, Bloemfontein, South Africa

In Mozambique, the rotavirus vaccine (Rotarix®) was introduced in September 2015 as part of the National Immunization Program. To date there are few reports of the whole genome analyses of RVA strains in Mozambique. The aim of the present study was to determine the genotype constellations of rotavirus as part of National Surveillance of Acute Diarrhea (ViNaDia).Stool samples that tested positive for RVA by ELISA and RT-PCR, obtained from children <5 years of age, hospitalized with AGE, and collected at five sentinel posts of ViNaDiA in Mozambique, were identified.Ten pre-vaccine (2014-2015) and 13 post-vaccine (2016) rotavirus strains were selected for Whole Genome Sequencing (WGS). The dsRNA was extracted with TRI-reagent (Sigma) following precipitation with lithium chloride (Sigma). The self-priming PC3-T7loop primer was ligated to dsRNA and cDNA was synthesised using the Maxima H Minus double-stranded cDNA kit (Thermo Scientific).The WGS was performed using an Illumina Miseq sequencing platform and NGS data analyzed using CLC Bio Genomics Workbench (9.0). The genotyping tool, ViPR was used to determine genotype constellation. Phylogenetic analyzes of each rotavirus genome segment are ongoing.Eighteen strains (eight G1P[8], three G9P[x], two G9P[8] and five G2P[6]) were successfully sequenced. Rotavirus genotype constellation were determined for all strains: 13 presented with Wa-like genetic backbone (eight with G1P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, five with G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E-H1) and five had a DS1-like genetic backbone (G2-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2).Preliminary data analysis indicates that the pre- and post-vaccination genotype constellations of rotavirus strains circulating in Mozambique are similar to strains characterized in the rest of the world.


Whole-genome sequencing and analyses identifies high genetic heterogeneity and diversity of rotavirus genotype P[6] strains circulating in Africa

Martin Nyaga (1), Mapaseka Seheri (2), Duncan Steele (3), Jason Mwenda (4), Jeffery Mphahlele (2)

1: Next Generation Sequencing Unit, Department of Medical Virology, Faculty of Health Sciences, University of the Free State, Bloemfontein, South Africa.; 2: South African Medical Research Council/Diarrhoeal Pathogens Research Unit, Faculty of Health Sciences, Sefako Makgatho Health Sciences University, Medunsa, Pretoria, South Africa.; 3: Enteric and Diarrhoeal Diseases Programme, Global Health Program, Bill and Melinda Gates Foundation, Seattle, Washington, USA.; 4: World Health Organization, Regional Office for Africa, Brazzaville, People’s Republic of Congo

Little is known about the genetic composition of African rotavirus P[6] strains. Possible evolutionary mechanisms leading to genetic diversity of the African rotavirus P[6] strains were investigated in this study.Whole genome sequencing of 57 P[6] rotavirus strains from 13 African countries was completed using a high-throughput next-generation rotavirus sequencing pipeline. Phylogenetic analysis was performed on 167 full-length P[6] VP4 sequences, including 104 publically available full-length P[6] VP4 sequences isolated from humans and 6 known porcine-origin P[6] sequences. The strength of association between the phenotypic features and the phylogeny was also determined.Reassortment and mixed infection of rotavirus P[6] strains were found in Africa. Our phylogenetic analyses showed the extensive genetic diversity that exists among P[6] strains, defined porcine-like strains and clades/subclades, estimated that P[6] VP4 gene has a high substitution rate with the mean of 1.05E-3 substitutions/site/year, and suggested that there was endemic infection of human rotavirus A P[6] VP4 strains in Africa, which is characterized by the extensive genetic diversity and long-time local evolution of the viruses. This was also supported by phylogeographic clustering and G-genotype clustering of P[6] strains by BaTS analysis, which suggested the viruses evolved locally instead of spatial mixing among the regions.Overall, the results demonstrated that multiple mechanisms such as reassortment events, various mutations, and possibly interspecies transmission account for the enormous diversity in P[6] strain in Africa. These findings highlight the need for continued surveillance of rotavirus diversity and the potential impact on the effectiveness of the current group A rotavirus vaccines.


G and P Types of Circulating Rotavirus Strains in Himachal Pradesh, India During 2013-2016: Three Years of Prevaccine Data

Shipra Gupta (1), Sanjeev Chaudhary (2), Pratima Ray (1,3)

1: Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard University, India; 2: Department of Pediatrics, Rajendra Prasad Government Medical College, Himachal Pradesh, India; 3: Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, India

India has recently launched its indigenous rotavirus vaccine, Rotavac® in Himachal Pradesh (HP). We report G and P types rotavirus strains associated with severe AGE in 487 children prior to vaccine introduction in HP(2013-2016). Fecal specimens were screened for Group-A rotavirus by antigen ELISA. Viral RNA was extracted by TRIZOL method and analyzed by RNA-PAGE. G and P typing was performed by multiplex reverse transcription PCR. Rotavirus was detected in 52.0% diarrheal children, highest being observed among 6-11 months age group (44.3%). Vomiting was found more frequently associated with RV-infection. Among RV G types, G12 was found most prevalent (38.1%) followed by G1 (30.9%), G9 (10.3%) and G2 (7.9%). G10 (1.6%), G3(0.8%) and G4 (0.4%) strains were rarely detected. Among P-types, P[6] was the most prevalent (50%) followed by P[8] (25.4%) and P[4] (12.7%). Of note, P[9] were detected first time in India. Among G/P combinations, G12P[6] was most prevalent (36.1%) followed by G1P[8] (19.8%), G1P[6] (6.7%), G2P[4] (4.4%) and G9P[6] (1.2%). Interestingly, our study observed high percentage of unusual strains(12.7%) namely G9P[4], G2P[6], G2P[8], G12P[4], G1P[9], G1P[11] and G9P[9]. G3P[8], G4P[6], G9P[8], G10P[6], G10P[8] and G12P[8] strains were very rarely detected. Interestingly, RNA migration pattern of G1P[8] was DS-1 like and genomic heterogeneity was observed within G12P[4] strains with both long and short electropherotypes. Our study highlights rich genetic diversity with emergence of rare rotavirus strains in HP and provides base line data prior to introduction of ROTAVAC that will help to gauge the impact of vaccine in HP.


Characterization of human gut virome among populations in four regions of Peru with different urbanization level

Chenyan Shi (1), Raul Yhossef Tito Tadeo (2,3), Leen Beller (1), Ward Deboutte (1), Claire Belkhou (2,3), Jeroen Raes (2,3), Jelle Matthijnssens (1)

1: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Viral Metagenomics, Leuven, Belgium; 2: KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Molecular Bacteriology, Leuven, Belgium; 3: Center for Microbiology, VIB, Leuven, Belgium

Comparative studies of the gut microbiota across communities with different subsistence strategies and in geographical locations have been focused mainly on bacteria, whereas the virome has been studied rarely. To explore the gut virome variation in such human populations, viral metagenomics was performed on 148 human fecal samples collected in four regions of Peru. The four regions include the capital of Peru (Lima), the metropolis of the Peruvian Amazon region (Iquitos), one village located in the Peruvian Amazon region (Angamos) as well as one tribe in the Peruvian Amazon Jungle (Remoyacu). The analysis of eukaryotic virome show that Lima presents the highest eukaryotic viral richness compared to other three regions. A significant higher proportion of plant virus was identified in Lima and Iquitos compared to Angamos and Remoyacu. In particular, the most abundant plant virus family was the Virgaviridae, which was present in Lima, Iquitos and Angamos. Furthermore, the family Partitiviridae is the most abundant plant virus in Remoyacu. Viruses belonging to the family Picobirnaviridae are the most prevalent, present in 70% of the samples. Finally, also members of the family Caliciviridae, associated with gastroenteritis, were identified. Noroviruses were most prevalent in Lima, Iquitos and Angamos, whereas Remoyacu showed a high abundance of Sapoviruses. Our study shows a great diversity of eukaryotic viruses in different Peruvian populations, including a significant number of dsRNA viruses.


PICOBIRNAVIRUS IN HUMANS AND LIVESTOCK ANIMALS FROM AREAS OF ENVIRONMENTAL CHANGE IN BRAZIL

Julia Rezende Silva, Elaine Hellen Nunes Chagas, Renato da Silva Bandeira, Bruno de Cássio Veloso Barros, Ceyla Maria Oeiras Castro, Delana Andreza Melo Bezerra, Joana D’Arc Pereira Mascarenhas

Evandro Chagas Institute, Brazil

The State of Pará represents some of the areas in Brazil where the highest rates of Amazon forest deforestation are concentrated. In these areas, the strong anthropic pressure and the high level of environmental degradation have led to a close relationship between the local wildlife and the rural population, contributing for transmission of several zoonotic pathogens and the emergence of new viral strains and infectious diseases, such as enteric disorders caused by Picobirnavirus (PBV), which are among the main causes that affect productivity rates in livestock animals, resulting in economic implications. The goal of this study was investigate the occurrence of PBV in fecal samples from humans and livestock animals from areas of environmental change. A hundred fecal samples were submitted to viral nucleic acid extraction, Polymerase Chain Reaction preceded by Reverse Transcription (RT-PCR) and automatic sequencing followed by phylogenetic analysis. Overall 10% (10/100) of positivity was found by RT-PCR. Of these, 9 samples of livestock animals were positive for genogroup I PBV, 8 from porcine and 1 from bovine. One sample from human was also genogroup I PBV positive. The phylogenetic analysis indicated an intense diversity between the strains studied, presenting high nucleotide and aminoacid similarity with several human and different animals species from many locations of the world, evidencing the PBV zoonotic potential of interspecies transmission. Finally, continuous monitoring of this virus is required to obtain information about these genetically diverse viruses, mostly in areas of environmental change.


MOLECULAR ANALYSIS OF G3P[6] ROTAVIRUS COLLECTED FROM CHILDREN WITH ACUTE GASTROENTERITIS IN THE BRAZILIAN AMAZON REGION

Ana Carolina Silva Serra, Delana Andreza Melo Bezerra, Jonas França Cruz, Patrícia Santos Lobo, Sylvia de Fátima dos Santos Guerra, Joana d’Arc Pereira Mascarenhas, Luana da Silva Soares


Group A rotavirus (RVA) is the most common cause of severe acute viral gastroenteritis among children in both developed and developing countries. The most prevalent RVA strains found in humans are G1, G2, G3, G4, G9 and G12 genotypes in combination with P[4], P[6] and P[8]. Therefore, G3P[6] genotype is considered an uncommon strain, although this combination has been detected with a high frequency in Brazilian Amazon region. This study aimed to perform genetic characterization of G3P[6] RVA strains isolated in Brazilian Amazon region during 2012 and 2017. Fecal samples were collected from children with acute gastroenteritis as a part of the National Network Surveillance of Rotavirus Gastroenteritis. Viral RNA of 19 G3P[6] RVA samples were extracted, followed by reverse-transcription polymerase chain reaction (RT-PCR) and nucleic acid sequencing. All Brazilian RVA strains revelead a DS-1-like genotype constellation: G3-P[6]-I2-R2-C2-M2-A2-T2-E2-H2. Phylogenetic analysis showed that all strains were clustered closely with human RVA isolated from the Americas, Europe, Asia and Africa. For VP7 gene, two strains from 2017 were similar to contemporany equine-like human G3P[8] strains. These results suggest that G3P[6] RVA equine-like strains have been circulating in the Brazilian population. Detection of recombinant G3P[6] RVA strains highlights the evolutionary relationship os this genotype and may represent a challenge to vaccination strategies. Further studies are necessary to obtain more knowledge on G3 RVA genetic and molecular epidemiology.


Whole genome characterisation of Rotavirus A circulating in southern Mozambique during 2012-2013 reveals diverse strains and several reassortment events

Amy Strydom (1), Eva D Joao (2,3), Martin M Nyaga (4), Christiaan A Potgieter (5), Assa Cuamba (6), Inacia Mandomando (2,7), Marta Cassocera (7), Nilsa de Deus (2,7), Hester G O’Neill (1)

1: Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa; 2: Centro de Investigação em Saúde de Manhiça (CISM), Manhiça, Mozambique; 3: Institute of Hygiene and Tropical Medicine, Lisbon, Portugal; 4: Next Generation Sequencing Unit, Department of Medical Virology, Faculty of Health Sciences, University of the Free State, Bloemfontein, South Africa.; 5: Department of Biochemistry, Centre for Human Metabolomics, North-West University, Potchefstroom, South Africa; 6: Faculdade de Medicina, Universidade Eduardo Mondlane, Maputo, Mozambique; 7: Instituto Nacional de Saúde, Maputo, Mozambique

Rotavirus surveillance was introduced in southern Mozambique in 2012 and indicated a prevalence higher than 40% before the introduction of rotavirus vaccination. Here we report the first whole genome constellations of Mozambican rotavirus A strains detected between 2012 and 2013 at the Mavalane General Hospital in Maputo city and Manhiça District Hospital in the Manhiça district. Ten DS-1-like strains and four Wa-like strains were identified using RNASeq on an Illumina Miseq platform targeting cDNA synthesised from enriched dsRNA extracted from stool, without adaptation to cell culture. A mixed infection containing a Wa-like strain and seven genome segments of an additional strain of possible animal origin was identified in one additional sample. The seven genome sequences of the partial strain clustered with a diverse group of strains, indicating that it might be a unique strain. Phylogenetic analyses indicated diversity among the G2P[4] Mozambican strains, reassortment between G2P[4] and G8P[4] Mozambican strains, as well as the intragenogroup reassortment of all the genome segments encoding the double-layer particle for strain RVA/Human-wt/MOZ/0045/2012G8P[4]. The G12P[6] Mozambican strains were more closely related to G12P[8] strains, although the G and P types were related to a G12P[6] Asian strain. The NSP4 amino acid sequences was not highly conserved within E1 and E2 genotypes but supported reassortment between G2P[4] and G8P[4] strains, determined by phylogenetic analyses. The reassortment events and mixed infection identified in this study indicate a diverse rotavirus population in southern Mozambique prior to the introduction of Rotarix® in Mozambique in 2015.


Rotavirus: Genotype distribution and changing trends in children with acute gastroenteritis: A study from western India

Gopalkrishna Varanasi

National Institute of Virology, India, and Investigators of NRSN West zone, ICMR, India

Group A rotaviruses (RVA) remains as major cause of gastroenteritis among children and contributes significantly to the disease burden in India. The study was conducted under National hospital based rotavirus surveillance network (NRSN), Indian Council of Medical Research (ICMR), New Delhi, India, with the aim to estimate the rotavirus disease burden, to identify the circulating RV strains and their changing trends in children hospitalized for acute gastroenteritis (AGE) in western India. A total of 4336 stool specimens were collected from hospitalized children aged <5 years, enrolled for AGE (n=5005) during September 2013 to December 2016 in six clinical recruitment sites (Pune, Mumbai, Ahmadabad, Surat, Karad and Belgaum) of NRSN west zone, India. Demographic details were recorded in case history forms (CRF). Rotavirus (RV) positivity was detected at 33.3% level in all sites with highest in Pune (48.3%) and lowest in Ahmadabad (23.0%) sites. RV infected children showed significantly more severe (75.1%) disease as compared to RV negative children (66.3%). Over all, G1P[8] genotype was predominant (50%) followed by G2 P[4] 12.9%, G9 P[4] 11.9%, G1P[6] 5.3%, G3P[8] 4.7%, G9P[6] 2.3%, G9 P[8] 2.1%. RV G12 strains with P[11], P[6], P[8], P[4] combinations were observed at 4.7% with rare genotypes of G8 P[8] 0.2%, G3 P[4] 0.2% level. Over the years, changing pattern in circulation of RV genotypes was observed in Mumbai, G3P[8] 35.3%- 100%, G9P[4] 39.3%, 45.5% in Surat, Belgaum and G1P[6],35.7% in Belgaum sites. Changing trends in circulation of Rotavirus strains will have major concern while assessing the impact of rotavirus vaccination.


WORKSHOP 2: EVOLUTION AND EPIDEMIOLOGY

Molecular epidemiology of rotavirus gastroenteritis in Kashmir Himalaya. Tourism and climatic disturbance -A potent platform for transmission.

Irfan Ahmad (1), Anjum Afshan (2), Aasifa Jan (3), Taniya Kirmani (3)

1: Div. of Genetics & Biotechnology, Fofy, SK-University of Agricultural Sciences & Technology of Kashmir, India; 2: Department of Anaesthesia & Critical Care, King Fahads Specialist Hospital, Dammam, KSA, Formerly Deptt. of Anesthesia, GB Panth Children Hospital, Srinagar, Kashmir, India.; 3: Department of Commerce, Govt. JK-School Education Deptt. Srinagar. India.

The Kashmir has a temperate climate. The study was conducted from Jan 2014 to Jan 2016. Incidence of disease under 4 years of age was about 45%. Rotavirus was present throughout the year with winter peak from December to March (Mean 56.6%) followed by summer peak from April to June (Mean 42.4%) and autumn peak from August to November (Mean 23%). Incidence of disease was: from 13 to 18 months (62.5%) followed by 7-12 m (59.12), 0-6 months (48.57%), 19-24 months (28.5%), 25-36 months (26%) and 37-48 months (22.83%). G& P types detected were G1,G2,G9 & G12 and P[4],P[6] & P[8] respectively. No case of vaccine induced intussusceptions was reported. Kashmir being land locked has a high tourist influx (domestic, national and global) and annual pilgrimages. Climatic disturbances, natural disasters like earthquake, floods and pollution in freshwaters have contributed to the spread of virus to some extent. Fresh waters have been contaminated by the influx of domestic sewage contributing the viral particles in good numbers. Religious tourism on the banks of main fresh water streams and lakes has been a contributing vehicle in polluting the waters and genomic persistence and survival of the virus. Diagnosis of the rotavirus is rarely, if ever made at local level inspite of the fact that physicians & policy makers may appreciate that diarrhea is the first or second leading cause of death in children ≤ 5 years of age.


Increased Detection of G3P[6] Human Rotaviruses after Introduction of Rotavirus Vaccination in Kenya

Ernest Wandera Apondi (1), Satoshi Komoto (2), Shah Mohammad (1), Martin Bundi (3), James Nyangao (4), Cyrus Kathiiko (1), Amina Galata (1), Sora Guyo (1), Gabriel Miring’u (1), Tomihiko Ide (2), Koki Taniguchi (2), Yoshio Ichinose (1)

1: KEMRI/Nagasaki University, Institute of Tropical Medicine, Kenya Research Station, Nairobi, Kenya, Kenya; 2: Department of Virology and Parasitology, School of Medicine, Fujita Health University, Japan; 3: National Biosafety Authority, Nairobi, Kenya; 4: Centre for Virus Research, KEMRI, Nairobi, Kenya

Background: A monovalent rotavirus vaccine (RV1) was introduced in Kenya nationally in July 2014. We examined the distribution of rotavirus genotypes following the vaccine introduction. Furthermore, we sequenced and characterised the complete genomes of representative G3P[6] strains isolated. Methods: Data on rotavirus G and P genotype distribution were derived from a five-year pre-vaccine and a two-year post-vaccine hospital-based surveillance in Central Kenya. The genotypes were determined by semi-nested RT-PCR. Whole genomes of two representative G3P[6] strains were sequenced using the next generation sequencing Illumina MiSeq platform. Data were analyzed using CLC Genomic Workbench v8.0.1. Contigs were assembled by de novo assembly. Maximum-likelihood phylogenetic trees were constructed using the Jukes-Cantor substitution model with MEGA7.0.26.Results: There was increased detection of G3P[6], G3P[8] and G2P[4] in Kenya following the vaccine introduction. Notably, the frequency of G3P[6] increased considerably from 0.2% in pre-vaccine to 7% in post-vaccine period. Whole genomic analysis of the two G3P[6] strains revealed a rare genotype constellation: G3-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2. The strains showed very high nucleotide sequence identities (99.4-99.9%) in all the 11 genes. Phylogenetic analysis revealed that each of the 11 genes of the two strains was closely related to that of African G3P[6] and/or G8P[4] human strains.Conclusion: Our observations provide important insights into the evolutionary dynamics of unusual G3P[6] strains. Since the currently licensed rotavirus vaccines contain neither the P[6] genotype nor strains with the complete human DS-1-like genotype constellation, continued surveillance of this G/P genotype is necessary to determine its implication on the vaccines.

187Poster presentations Workshop 2: Evolution and Epidemiology

DISTRIBUTION OF NSP4 GENOTYPES OF GROUP A ROTAVIRUS STRAINS CIRCULATING IN TUNISIAN CHILDREN OVER A 10 YEARS PERIOD (2006 to 2016)

Mouna Ben Hadj Fredj (1,2), Haifa Bennour (1,3), Asma Bouazizi (1,3), Amira Jerbi (1,3), Meriam Ben Hamida-Rebai (1,3), Besma Abdi (1,3), Samia Lakhal (1,3), Saoussen Kacem (1,3), Zouhour Fekih (1), Imene Fodha (1,3), Jelle Matthijnssens (4), Abdelhalim

1: University Hospital Sahloul,LR14SP02, Laboratory of Microbiology, Tunisia; 2: Faculty of Sciences and Techniques, University of Kairouan, Kairouan, Tunisia; 3: Faculty of Pharmacy, University of Monastir, Monastir, Tunisia; 4: Laboratory of Clinical and Epidemiological Virology, Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium

Background: Non-structural protein 4 (NSP4), encoded by group A rotavirus (RVA) genome segment 10, is a multifunctional protein and the first recognized virus-encoded enterotoxin. Recently, a new classification system for RVAs was proposed and a total of 27 NSP4 genotypes (E1 to E27) are currently described.Methods: A total of 2697 faecal specimens collected from children less than 5 years old were screened by ELISA for the presence of RVA antigen. NSP4-encoding genes of the RVA positive strains were analyzed using a semi-nested RT-PCR.Results: Genotypes E1 and E2 were identified in 371 (80.3%) and 91 (19.7%) samples, respectively. Tunisian RVA strains analysed in the present study belonged to 2 different genotypes: E1 and E2. Such a result is concordant with literature data: indeed, although 27 RV NSP4 genotypes have been identified to date, previous molecular characterization of the NSP4 region has shown that most of the diversity in the NSP4-encoding gene among human RVAs lies in genotypes E1 and E2. Other studies, however, have detected unusual strains carrying genotypes E3, E6, and E13. Moreover, a predominance of NSP4 genotype E1 was observed over the entire period of study, from 2006 to 2016. Such a result was also quite expected as previous investigations have also shown that NSP4 genotype E1 was largely predominant among children worldwide.Conclusion: These results underline the need for further investigations to assess the validity of NSP4 as a suitable target for epidemiologic surveillance of rotavirus infections and vaccine development.


Characterization of group A rotavirus strains identified in Tunisian adults and children more than 5 years old (2015 to 2017)

Haifa Bennour (1,2), Imene Fodha (1,2), Asma Bouazizi (1), Amira Jerbi (1,2), Samia Lakhal (1,2), Mouna Ben Hadj Fredj (1,3), Meriam Ben Hamida-Rebai (1,2), Ouafa Kallala (1,2), Saoussen Kacem (1,2), Zouhour Fekih (1), Akila Mili (4), Noureddine Boujaafa

1: University Hospital Sahloul, Tunisia; 2: Faculty of Pharmacy, University of Monastir, Tunisia; 3: Faculty of Sciences and Techniques, University of Kairouan, Kairouan, Tunisia; 4: Laboratory of Parasitology, Farhat Hached University Hospital, Sousse, Tunisia

Background: Group A Rotaviruses (RVA) are the most important etiologic agents of acute gastroenteritis in infants worldwide. Nevertheless, its role as a potential pathogen in adults and older children needs to be clarified. The aim of the present study was to evaluate the rate of detection of RVA in Tunisian adults and children more than 5 years old, and to characterize VP7 (G) and VP4 (P) genotypes of RVA circulating in these particular populations.Material and methods: A total of 713 stool specimens were collected from adults (≥18 years; n=477) and children (5-17 years; n=236) hospitalized or consulting for gastroenteritis in Sousse between January 2015 and April 2017. All samples were prospectively screened by reverse transcription – polymerase chain reaction (RT-PCR) for the detection of VP6 gene specific of RVA. RVA-positive samples were further used for VP7 and VP4 genotyping using multiplex semi-nested RT-PCR.Results: Globally, 84 stool samples were positive for RVA, with 64 from adults (13.4%) and 20 from children (7.6%). Among the 54 strains that were typeable for both VP7 and VP4, 10 G/P combinations were detected: G9P[8] (29.8%), G2P[4] (20.4%), G1P[8] (16.7%), G4P[8] (3.7%), G9P[4] (3.7%), G1P[4] (3.7%),G3P[8] (1.8%), G12P[8] (1.8%), G2P[8] (1.8%), G3P[4] (1.8%), with mixed infections in 8 cases (14.8%).Conclusion: In the absence of national vaccination strategy, RVA remains the main etiological agent of infantile gastroenteritis in Tunisia. Adults are a significant source of RVA transmission to infants, highlighting the importance of monitoring the epidemiology of these viruses in this particular population.


Rotavirus genotype distribution after Rotavirus vaccine introduction in Cameroon

Angeline yvette BOULA (1), Rose NGOH (1), Mina NJIKI KINKELA (1), Grace KEMAJOU (1), Roger NGOYA (1), Eric NKOLO (1), Franky BAONGA BA POUTH (2), Marcellin NIMPA (3), Jason MWENDA (4), Paul KOKI NDOMBO (1)

1: Centre Mère et Enfant, Cameroon; 2: Expanded Progamme on Immunization, Minister of Health,Cameroon; 3: WHO, Country office, Cameroon; 4: WHO- AFRO, Brazzaville, Congo

Introduction: In Cameroon, a monovalent rotavirus vaccine based on an attenuated human G1P [8] strain, (Rotarix®, GSK Biologicals) was introduced into the Expanded Program on Immunization (EPI) in 2014. This study report the results of rotavirus A (RV-A) surveillance from January 2014 to December 2016Methods: A total of 1268 fecal samples were collected in four sentinel site of rotavirus surveillance. RV-A was detected by enzyme immunoassay, and genotyped through reverse transcription polymerase chain reactionResults: Out of 1268 samples, 279 (22%) tested positive for rotaviruses. Among children less than 5 years old, regardless the antirotavirus vaccination status, the rate of rotavirus detection was 32% in 2014, 19% in 2015, and 16% in 2016. Among positive samples, the most prevalent genotypes detected was in 2014 G1P [8] (41%), followed by G2P [6] (15%) and G3P [6] (14%). In 2015 G1P [8] (39%) followed by G3P [6] (33%) and G2P [6] (7.5%). In 2016 G3P [6] (24.5%), G2P [4] (22%), G2P [6] (12%), G1P [8] (3%)Conclusion: Our data demonstrate that the rate of rotavirus detection decrease and the emergence of G3P[6] and G2P[6] after the introduction of rotavirus vaccine from 2014 to 2016


Picobirnavirus and Reovirus in faecal samples of animals, mammals and birds, from areas of environmental change of Brazil”

Elaine Hellen Nunes Chagas, Júlia Rezende Silva, Bruno de Cássio Veloso Barros, José Wandilson Barboza Duarte Junior, Delana Andreza Melo Bezerra, Edivaldo Costa Sousa Júnior, Renato da Silva Bandeira, Sylvia de Fátima dos Santos Guerra, Ceyla Maria Oeir

Instituto Evandro Chagas, Brazil

Knowing the potential of enteric viruses deserves prominence because of the impact they have on animal health and possible economic losses. Thus, the Picobirnavírus (PBV) and Reovírus (REOV) are examples of pathogens that affect different species of animals, in a symptomatic or asymptomatic manner, acting as emerging agents, opportunists and zoonotic agents. This study aimed to detect picobirnavírus and reovírus in fecal specimens of animals, mammals and birds, collected from 2014 to 2016, from three cities in the state of Pará, Brazil. A total of 258 fecal samples were used. From the fecal suspensions, the extraction of the viral genetic material, the Polyacrylamide Gel Electrophoresis (PAGE), the quantification and characterization of the genetic material by means of the Polymerase Chain Reaction preceded by Reverse Transcriptase (RT-PCR) were carried out. Statistical analyses were performed using the G-test and Chi-squared, and nucleotide analysis for the construction of the phylogenetic tree. The results were negative for REOV and PBV by the PAGE technique. For the RT-PCR technique, the results for REOV were negative, and for PBV the positivity was 12.4% (32/258), where the highest values were for swine and felines, with 39.1% and 27.3%, respectively. The statistical test was significant for PBV in relation to the animal groups studied and in relation to the cities of origin of the samples, with value of p<0.05. From the nucleotide analysis it was observed that PBV are highly disseminated viruses and that can affect the most diversified types of host.


Unexpected emergence of novel Group A Rotavirus G9P[4] in Mendoza, Argentina

Juan Ignacio Degiuseppe (1), Vanina Sol Eibar (2), Sandra Fabiana Grucci (2), Juan Andrés Stupka (1)

1: INEI - ANLIS “Dr. Carlos G. Malbrán”, Argentine Republic; 2: Hospital Notti, Mendoza, Argentine Republic

Introduction. It has been suggested that rotavirus massive vaccination could produce the replacement of circulating genotypes by others not included in the vaccines or by new emerging strains. During routine surveillance in 2017, G9P[4] association was detected with high frequency in Mendoza, Argentina (95,8%). The aim of this study was to describe this unusual association assessed by complete genome analysis.Methods. To assess the possible origin of this emergent strain, we sequenced and analyzed the eleven genomic segments according to the recommendations of the Rotavirus Classification Working Group (RCWG).Results. The studied strain presented a G9P[4]-I2-R2-C2-M2-A2-N2-T2-E6-H2 genomic constellation. Although VP7 gene belonged to major lineage III, it clustered with G9 strains bearing P[4] genotype. Phylogenetic analysis revealed that these strains were more closely related to the G9P[4] strains detected in India during 2011-2013 than to the ones that circulated in Latin America in 2010. Deduced amino acid VP7 gene sequence comparison between G9P[4]-2017 and G9P[8]-2016 strains showed 10 changes, one of them located at antigenic site (S221N).Conclusions. These results indicate that the emergence of G9P[4] rotaviruses was due to the introduction of a new strain, rather than to a reassortment of the G9P[8] strains previously circulating in our country. Noteworthy, the high frequency of detection of this strain suggests more adaptability than other unusual G/P associations and even than other G9P[4] previously described. We consider important this kind of studies because they contribute to the knowledge of the complex evolutionary dynamics of rotaviruses, mainly in the post vaccination era.


High prevalence and changing genotyping pattern of rotavirus infection among Bangladeshi pediatric patients

Shuvra Kanti Dey (1), Sadia Farzana Sifat (1), Md. Almamun (1), Nadim Sharif (1), Nasir Uddin Nobel (1), Sadia Nowshin Sany (2), Md. Baki Billah (2)

1: Department of Microbiology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh; 2: Department of Zoology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh

Introduction: Acute gastroenteritis is one of the most common diseases in humans, and continues to be a significant cause of morbidity and mortality worldwide. Globally most cases are caused by rotaviruses along with noroviruses, adenoviruses, astroviruses and sapoviruses.Materials: A total of 341 fecal samples were collected between October 2013 and December 2016 from children below 5 years old with acute gastroenteritis from Enam medical college (Dhaka) & Mother and Children Hospital (Chittagong). All samples were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR) for the presence of rotavirus. PCR product of specific genes of rotaviruses was selected for sequence analysis.Results and Discussion: RT-PCR of all the samples revealed the presence of 31 rotavirus A positive samples (18.78%) in 2013 and 88 rotavirus A positive sample (50%) in 2016 with absence of rotavirus B and rotavirus C. Majority (64%) of the participants were aged between 7 and 18 months. Genotypic characterization by sequencing methods revealed that G2P[4] being the most common strain type of rotavirus in 2013 (Dhaka) and G1P[8] being the most common strain type in 2016 (Chittagong). Various other G/P combination (G2P[8], G12P[8], G3P[8], G10P[8], G9P[8] and G11P[25]) were also detected during this study. Bangladeshi rotavirus strains displayed highest similarity (98.2-99.4%) to previously studied Bangladeshi, Italy, India and Thailand strains.Conclusion: We found that the prevalence of rotavirus infection is increasing among Bangladeshi pediatric patients. Changing pattern of genotype distribution is also alarming. Such information will aid in seeking advocacy for rotavirus vaccine introduction in the country‘s national immunization program.


Tunisian Rotavirus Surveillance (1995-2017): A Brief Overview.

Imene Fodha Bouzgarrou (1,3), Anissa Chouikha (2), Mouna Ben Hadj Fredj (3), Amal Moussa (3), Haifa Bennour (3), Meriam Ben Hamida (3), Asma Bouazizi (3), Abdelhalim Trabelsi (1,3), Andrew Duncan Steele (4)

1: University Hospital Sahloul / Faculty of Pharmacy of Monastir, Tunisia; 2: Pasteur Institute of Tunis, Laboratory of Virology; 3: University Hospital Sahloul, LR14SP02; 4: Bill & Melinda Gates Foundation

Background: Group A Rotaviruses surveillance was implemented in Tunisia since 1995, and a Tunisian Rotavirus Network supported by WHO was created in 2001 in view to bring reliable information on rotavirus diseases. Material and methods: From 1995 to 2017, stool specimens were collected from children <5 years of age hospitalized or consulting for acute diarrhoea in sentinel hospitals participating in the surveillance. All samples were prospectively screened using either an ELISA for the detection of VP6 antigen specific of RVA, or a reverse transcription–polymerase chain reaction (RT-PCR) for the detection of VP6 gene. RVA-positive samples were further used for VP7 and VP4 genotyping using multiplex semi-nested RT-PCR. Results: From 1995 to 2017, 4202 stool specimens were collected. A total of 916 (21.8%) specimens tested positive for RVA during the entire period, with annual rates of RVA detection ranging from 14.8% (in 2017) to 26.2% (in 2007). Although G1P[8] was globally the most prevalent rotavirus strain, it was temporarily replaced by G3P[8] in 2005/2006, and by G2P[4] in 2007. G9P[8] strains were first detected in 2004, and remained consistently circulating since then. G12P[6]and G12P[8] strains emerged in 2009 and remain in circulation to date, whereas G6P[9] and G8P[8] were detected only in 2002 and 2008, respectively. Mixed profiles and uncommon combinations were also frequently reported. Conclusion: The permanent emergence of unconventional types highlights the need for a continual survey of local circulating strains. Epidemiological knowledge of rotavirus is critical for the development of effective preventive measures.


Molecular characterization of a group A rotaviruses in Mexico during 2018, emergence of G3P[8] strain variants.

Fabián Gómez-Santiago, Atenea Estela Andrés, Sergio Issac de la Cruz, Heidi Terán, Héctor Méndez, Herlinda García

Instituto de Diagnóstico y Referencia Epidemiológicos, Mexico

Background: Nowadays, two rotavirus vaccines have been licensed and are being administered in many countries of Latin America including Mexico, however little is know about the molecular epidemiology of novel strains variants that these vaccines probably may cause.Methods: Stool samples (n=437) were collected as a part of the Public Health Laboratories National Network surveillance from January 2018 till date. From these, 78 positive rotaviruses specimens were genotyped by RT-PCR. Thereafter, sequencing of 20 selected samples in both VP4/VP7 genes of G3P[8] strains variants was performed.Results: Our results showed that G12P[8] was the most prevalent genotype, accounting for 71.79% (56/78) followed by G3P[8] and G2P[4] with 23% and 5% respectively. Phylogenetic analysis of partial sequencing of the VP4/VP7 genes showed a single lineage of G3 Rotavirus and multiple sublineages of P[8] strains have been circulating at the beginning of this year, and might become in the near future the dominating human rotavirus genotype in our country after G12P[8] genotype.Conclusions: For the first time, we identified a novel rotavirus strain G3P[8] in Mexico. The study highlights the genetic drifts in VP4/VP7 genes year over year and suggests a potential event of selective-pressure given the genetic background of the vaccine, which is currently administered in our health system. These findings emphasize the significance to continue careful monitoring these emerging strains, in order to detect the emergence of possible uncommon and novel types, as well as to assess their potential impact on the effectiveness of vaccines.


Rotavirus C: Genetic evolutionary dynamics based on VP3 and VP7 Genes, and Detection in Fecal Specimens of Pigs from Western India

Madhuri Shantanu Joshi, Shalu Akash Arya, Atul Madhukar Walimbe, Varanasi Gopalkrishna

National Institute of Virology, Pune, India, India

Rotavirus (RVC) is a common cause of sporadic or large outbreaks of gastroenteritis in pig farms. RVC an emerging zoonotic infection with evidences of cross species transfer needs continuous monitoring. To elucidate the evolutionary relationship, time scale stasis or dynamics existing in RVC strains circulating in different hosts globally, Bayesian Markov chain Monte Carlo (MCMC) method was implemented in BEAST v1.8.4. Bayesian analysis showed porcine species to be the origin of VP3 and VP7 genes in humans and evolutionary rates suggested emergence of RVC in 15th and 9th century with respect to VP3 and VP7 genes. Retrospective stool specimens of asymptomatic pigs for diarrhea collected from pig farms of Nagpur and Shirwal (Maharashtra state), western India India were tested for RVC using VP6 based RT-PCR followed by nucleotide sequencing. RVC detection in asymptomatic pigs is being reported for the first time from India with detection rate of 16.7 % and is the highest rate reported till date. Majority of the study strains remained unclassified due to their clustering closer to I7 genotype strains with a low bootstrap support value with exception of a single strain of I8 genotype during phylogenetic analysis. The high RVC detection rate observed in asymptomatic pigs indicates possibility of perpetuation of the virus in the environment through such pigs and needs further studies. With a preliminary effort, this study throws light on evolution of human RVC from porcine species. To confirm this observation remaining nine gene segments of RVC are being analyzed.


Etiology of non-rotavirus-associated gastroenteritis among hospitalized children under five years of age in the Philippines, 2015-2016

C. Eures Iyar Garcia Lazaro, Joseph Bonifacio, Mary Ann Igoy, Amado Tandoc III

Research Institute for Tropical Medicine, Philippines

INTRODUCTION: In the Philippines, rotavirus infection remains to be one of the leading causes of child morbidity and mortality. Nearly forty percent of confirmed rotavirus cases were identified from the National Rotavirus Surveillance in 2013 to 2016. To assess the burden of disease caused by non-rotavirus-associated gastroenteritis and co-infection to rotavirus in hospitalized children in the Philippines, confirmed negative and positive for rotavirus stool specimens were tested for norovirus and enteric adenovirus.MATERIALS AND METHODS: Randomly selected samples collected in 2015 to 2016 were included in the study. Stool specimens were tested for the presence of norovirus and enteric adenovirus (40/41) using real-time polymerase chain reaction (qPCR).RESULTS: Enteric pathogens were detected in 65% (561/864) of the samples tested. A single pathogen infection was observed in 391 (45.25%) samples, 78 (9.03%) samples tested positive for two pathogens, and 4 (0.46%) samples were confirmed positive for all pathogens. Both norovirus and adenovirus yielded a positivity rate of 14%.DISCUSSION: This data showed that enteric viruses are the most common cause of acute gastroenteritis (AGE) in children less than 5 years of age in the Philippines. Such finding implies that continued and strengthened surveillance monitoring is essential in planning the disease control strategies for diarrhea. Data from this study can provide policymakers with accurate facts to help in the nationwide health agenda.


SPREAD OF THE EMERGING EQUINE-LIKE G3P[8] DS-1-LIKE GENETIC BACKBONE ROTAVIRUS STRAIN IN BRAZIL

Adriana Luchs (1), Antonio Charlys da Costa (2,3), Audrey Cilli (1), Shirley Cavalcante Vasconcelos Komninakis (4,5), Lais Boen (1), Rita de Cassia Compagnoli Carmona (1), Simone Guadagnucci Morillo (1), Ester Cerdeira Sabino (2,3), Maria do Carmo Sampai

1: Adolfo Lutz Institute, Brazil; 2: University of São Paulo, Brazil; 3: São Paulo Institute of Tropical Medicine, Brazil; 4: Faculty of Medicine of ABC, Brazil; 5: Federal University of São Paulo, Brazil

In 2013, equine-like-G3P[8]-DS-1-like rotavirus (RVA) strain emerged worldwide. In 2016, this strain was reported in Northern Brazil, but exhibiting N1 genotype. The aims of the study were to conduct a genetic retrospective investigation in order to identify the possible entry of equine-like-G3P[8] in Brazil, describe its distribution across the country, and help the understanding of potential implications in RVA vaccine programs. From 2013 to 2017, a total of 4227 fecal samples were screened for RVA by ELISA, PAGE, RT-PCR and sequencing. Six G3P[8]-DS-1-like samples were selected for whole genome investigation. G3P[8] represented 23.4% (187/800) of all RVA-positive samples; further divided as equine-like-G3 (10.6%;85/800) and wild-type-G3 (12.7%;102/800). Whole genome sequencing confirmed the DS-1-like backbone I2-R2-C2-M2-A2-N2-T2-E2-H2. During 2013-2014, wild-type-G3P[8] was dominant and no equine-like-G3P[8] was detected. Equine-like-G3P[8] was first identified in Paraná in March/2015, suggesting that the strain stepped into Brazil through Southern region. Equine-like-G3P[8] rapidly spread across the country, reaching Goiás in July/2016, Federal District in September/2016, and São Paulo in August/2017. No wild-type-G3P[8] was detected since August/2014, suggesting that equine-like-G3P[8] completely replaced wild-type-G3P[8] strains. Equine-like-G3P[8] strains exhibited high level sequence conservation (>99% identity) among each other, and clustered with contemporary DS-1-like-G3P[8] detected after 2013. Equine-like-G3P[8] strains described here possess a distinct NSP2 genotype (N2) compared to the previously equine-like-G3P[8] reported in Northern Brazil (N1), indicating that two different lineages could be circulating in the country. Equine-like-G3P[8] strains pose a challenge for RVA surveillance since G/P typing by RT-PCR is unable to identify this intergenogroup reassortant without inclusion of sequencing analysis.


Surveillance of Group A Rotavirus in Buenos Aires, Argentina after the introduction of the monovalent vaccine and the study of a rare strain in an unusual immune environment.

Marcelo Gastón Mandile (1), Estefanía Peri Ibañez (1), Marcelo Argüelles (1), Facundo Temprana (1), Dalila Silvestre (1), Alicia Mistchenko (2), Graciela Glikmann (1), Alejandro Castello (1)

1: Universidad Nacional de Quilmes, Argentine Republic; 2: Children’s Hospital Dr. Ricardo Gutiérrez, Argentine Republic

Group A rotaviruses (RVA) are the most frequent etiological agents causing severe diarrhea in infants. Since 2006, two RVA vaccines were licensed in many countries. Within the first years following vaccine introduction in national schedules, substantial declines in rotavirus gastroenteritis hospitalizations have been observed. Argentina was one of the last countries in America introducing rotavirus vaccination since January 2015 in the National Immunization Schedule.In this study we analyzed the RVA strains frequency after the introduction of the vaccine (Rotarix®) to the national immunization schedule. First, based on the information kindly provided by Alicia Mistchenko (director of the Virology Laboratory of the Children’s Hospital “Dr. Ricardo Gutiérrez”) we observed a sharp decrease in the number of children hospitalized with diarrhea due to RVA. Then, from the analysis of genotypes conducted in our laboratory we observed the strains with the highest frequency of appearance were G2 and G12, replacing G1 strains that were dominant in 2014 and causes severe cases and several death.In addition to what has been described above, in this work we want to add another interesting finding discovered by genotyping samples from the aforementioned hospital, which we believe is highly relevant for the evolutionary aspects of RVA. We found a rare strain (G3P[4]) in a hospitalized immunosupressed patient. This strain was found in the stool samples of this person over a year and we found it interesting to study its evolution in a different immune environment than normal by analyzing the vp4, vp7 and nsp1 genes.


Differences in VP6 Predict Restriction on the Reassortment of Co-circulating AU-1-like and Wa-like Rotaviruses Strains

Iara Magaly Martinez (1,2), Maria Eugenia Galeano (2), Graciela Russomando (2), Gabriel Ignacio Parra (1)

1: Division of Viral Products, Food and Drug Administration, Silver Spring, MD, USA; 2: Health Sciences Research Institute, National University of Asuncion, Paraguay

Group A rotaviruses (RVAs) are one of the most important pathogens associated with gastroenteritis in humans and animals. RVAs genome comprises 11 segments of double-stranded RNA that encode six structural (VP1-4, VP6and VP7) and six non-structural proteins (NSP1-6). Genome analyses have shown that most human RVAs present two genetic constellations, namely Wa-like (genotype 1) and DS-1-like (genotype 2). During 2006-2007, the strain G12P[9], presenting an AU-1-like (genotype 3) genome, circulated among children and adults in Paraguay. Notably, during 2008-2009, G3P[9] co-circulated with G3P[8] strains. Genomic analyses revealed that although G3P[9] strains presented the same genomic constellation (I3-R3-C3-M3-A3-N3-T3-E3-H6) as G12P[9], they were not related or a product of reassorment between G3P[8] and G12P[9] strains. To better understand rules of reassortment, we performed structural modeling of all structural proteins to determine differences between AU-1-like and Wa-like strains that mapped in their interacting regions. Eight VP6-residues were found at the VP6:VP7 interphase, four at the VP6:VP4 interphase, and eight VP6-residues and 28 VP2-residues at the VP6:VP2 interphase. Moreover, only two differences were detected at the VP7 inner face from AU-1-like (G3 and G12) and Wa-like (G3) strains, and six differences were detected in regions of VP4 that interacts with VP7 and VP6. Together, these differences could be responsible for the distinct genomes of G3P[9] and G12P[9] strains and the reassortment-restriction between co-circulating strains. Overall, this data supports the notion that genome constellations are strongly influenced by the structural constraints, probably linked to the central role of VP6 in the structure of the virion.


G1P[8] Rotavirus in childrens with acute diarrhea after vaccine introduction in Brazil: evidence of reassortments and structural modifications in VP7 and VP4 genes

Fabiolla da Silva dos Santos, Edivaldo Costa Sousa Junior, Sylvia de Fátima dos Santos Guerra, Patrícia dos Santos Lobo, Edvaldo Tavares Penha Junior, Ana Beatriz Figueiredo Lima, Caio Breno Gomes Vinente, Maria Cleonice Aguiar Justino, Alexandre da Costa Linhares, Luana da Silva Soares, Joana D’Arc Pereira Mascarenhas


Annually, rotaviruses A (RVA) are responsible for approximately 197.000 deaths of children aged less than 5 years. RVA genotype G1P[8] is associated with more than 50% of infections. Currently, two licensed vaccines are recommended worldwide and comprised of G1P[8] strain. This study aimet to describe the genetic variability of G1P[8] strains detected in children with acute diarrhea in Belém, Pará, Brazil, after RVA vaccine introduction. Viral genome was extracted from 40 fecal samples of RVA G1P[8] strains and submitted to nucleotide sequencing. Sequences obtained from VP4 and VP7 genes were assembled using CAP3 software, alignments were carried out using the MAFFT v. 7.221 program andcompared to other sequences available in GenBank.. Protein modeling was performed using Modeller 9.15 software and three-dimensional homology structures selected from the Protein Database Bank (PDB): 3FMG (VP4) and 2DWR (VP7). Three-dimensional models were validated using PROCHECK and VERIFY3D. Visualization and production of the images was realized using the PYMOL software. Phylogenetic analysis of VP4 and VP7 genes showed that G1P[8] Brazilian strains grouped into different lineages of vaccine strains. Genomic constellation demonstrated reassortments between genogroups Wa-like and DS-1-like (G1-P[8]-I1-R2-C1-M1-A1-N1-T2-E1-H1) and Wa-like and AU-1-like (G1-P[8]-I1-R3-C1-M1-A1-N1-T1-E1-H1). Antigenic epitopes of VP7 and VP4 proteins evidenced amino acid differences and changes to the electrostatic charges when compared with vaccines strains. Despite of RVA cases reduction after vaccine implantation, studies on genetic variability and antigenic characteristics of RVA G1P[8] strains are important to detect the emergence of novel variants that could have an impact on current vaccination strategies.


Isolation and Molecular Characterization of Bluetongue Virus Isolates from Southern India

Sarita Yadav (2,3,4), Kyriaki Nomikou (1,3), Sushila Maan (2), Narender Maan (2), Massimo Palmarini (3), Peter Paul Clement Mertens (1,4)

1: University of Nottingham, UK, UK; 2: Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal SciencesHisar, Haryana, India; 3: Institute of Infection Immunity & Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland, UK. G12 8QQ; 4: Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, UK. GU24 0NF;

BT is endemic in southern India, with twelve BTV-serotypes (BTV-1, 2, 3, 4, 5, 9, 10, 12, 16, 21, 23 and 24) isolated since 2001 and a pentavalent inactivated vaccine, containing serotypes 1, 2, 10, 16 and 23, in current use. Field samples (n=447, EDTA blood, spleen, lymph node and saliva) were collected from small ruminants and cattle, during disease outbreaks in Telangana, Andhra Pradesh, Karnataka and Tamil Nadu states in 2014-2015. Positive results for BTV RNA were obtained by serogroup-specific qRT-PCR for 236 samples. A total of 141 BTV virus isolations were made in KC and BHK cells.Sixty two isolates were serotyped by qRT-PCR, confirming co-circulation of BTV-1,-2,-4,-5,-9,-10 and -12. Dual BTV-infections were detected in 22 isolates and triple infections in 4 isolates. None of the serotypes present in the pentavalent vaccine were identified in the 22 isolates from vaccinated animals, suggesting that vaccination was effective against homologous serotype field-strains.Sequence analysis of Seg-2 from 46 isolates (BTV-1,-2,-4,-5,-9,-10 and -12), identified four serotypes BTV-1, BTV-2, BTV-4 and BTV-9, as eastern topotypes. However, three serotypes, BTV-5,-10 and -12 were western topotypes, reflecting introduction of western field/vaccine strains into the Indian sub-continent. Full-genome sequencing and analyses is ongoing, to determine the origins of all ten genome segments of each isolate and identify reassortment events during their recent ancestry. These data will improve our understanding of BTV epidemiology in India and help to ensure the use of appropriate BTV serotypes and strains in the BT vaccines used in the region.


Whole genome sequence-based characterisation of bluetongue virus serotype 5 from India

Sushila Maan (1), Narender Singh Maan (1), Sunil Kumar Mor (2), Nitish Bansal (1), Deepika Chaudhary (1), Panduranga P. Rao (3), Peter PC Mertens (4)

1: Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), India; 2: University of Minnesota, Veterinary Population Medicine Department, St. Paul, MN 55108, USA; 3: Ella Foundation, Hyderabad, Telangana, India; 4: The School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Leicestershire UK, LE12 5RD

Bluetongue (BT) is an arboviral disease of ruminants and is caused by different serotypes of bluetongue virus (BTV). The disease is endemic in tropical areas including India. Twenty-seven recognized serotypes of BTV have been reported so far. Thirteen BTV strains belonging to eleven different serotypes (BTV-1e, BTV-2e, 2w, BTV-3w, BTV-4e, BTV-5w, BTV-9e, BTV-10w, BTV-12w, BTV-16e, BTV-21e, BTV-23e and BTV-24w) had been isolated in India since 2001.BTV-5 was initially identified in South Africa and has subsequently been isolated from Cameroon, Nigeria, the United States of America, Israel, Caribbean island of French Guadeloupe and very recently from China and Southern India. Although there is previous serological evidence for antibodies to BTV-5 in India, this serotype was regarded as ‘exotic’ until its isolation from Southern India in 2016. Analysis of suspected samples from Northern India also revealed the presence of BTV-5. Full-genome sequencing and analyses to determine the origins and evolution of all ten genome segments of Indian strains of BTV-5 and identify any reassortment events during their evolution will be presented. These data will be beneficial for understanding the BTV epidemiology and improving diagnostic assays and control measures against bluetongue in India and its neighbouring countries.


EPIDEMIOLOGICAL AND CLINICAL ASPECTS OF GASTROENTERITIS IN THE PEDIATRICS DEPARTEMENT OF TEACHING HOSPITAL OF YOPOUGON ONE YEAR AFTER INTRODUCTION ROTATEQ VACCINE (ABIDJAN RCI)

Amani Rebecca N’GUESSAN -KOUAME (1), Marie -Helene AKE-ASSI-KONAN (1), catherine BONI (2), Flore ZABA (2), hamidou KONE (3), Nicaise AKA (3), Laurence ADONIS-KOFFY (1)

1: Teaching Hospital of Yopougon, Côte d’Ivoire; 2: central laboratory of yopougon Côte d’Ivoire; 3: Epi program Côte d’Ivoire

Introduction: Gastroenteritis are common pathologies in children. Since March 2017 Rotateq is given to infants. We will present the profile of patients with gastroenteritis during this first year.Material and methods: This is a descriptive cross-sectional study that took place in the pediatric department of teaching hospital of Yopougon ward from 2017 to 2018, as part of the WHO / AFRO networkAll children aged 1 to 5 years hospitalized, meeting the criteria for defining suspicious and enlisted cases. A stool sample was collected and sent to the laboratory for microbiological analysis.Results: We enrolled 115 children. Sex ratio of 1.4. The age group from 6 to 12 months. Rotavirus was isolated in 32.5% of the stool samples. The majority of patients showed signs of dehydration. The morbid states associated with Rotavirus infection were associated with malaria, pyelonephritis, acute malnutrition, pneumonia. 6 death cases, a mortality rate of 5, 1%.Conclusion: Rotavirus infection is a reality and can be very serious.Vaccine integration is an opportunity to reduce morbidity and mortality


Norovirus Co-infection with Rotavirus in Yaoundé,Cameroon

Gaston Eric Nkolo Mviena (1), Angeline Boula (1), Rose Ngoh (1), E. Mugyia Akongnwi (2), Mina Njiki Kinkela (1), Grace Kemajou (1), Roger Ngoya Ebiguide (1), Ba Pouth Baonga (3), Marcellin Nimpa (4), Jason Mwenda (5), Paul Koki Ndombo (1)

1: Mother and Child Center Chantal BIYA Foundation, Cameroon; 2: Faculty of Science, University of Buea, Cameroon; 3: Expanded Progamme on Immunization, Ministry of Public Health, Cameroon; 4: WHO, Country office, Cameroon; 5: WHO- AFRO, Brazzaville, Congo

Rotavirus and Norovirus are leading viral causes of diarrhea in children. This study is conducted to determine the prevalence of rotavirus and Norovirus and also to establish the circulating strain of rotavirus and norovirus in a community in Yaounde Methods: A total of 902 stool were collected among children aged


BTV-8 in Europe: direct sequencing of the full viral genome from clinical samples

Kyriaki Nomikou (1,3), Ana Da Silva Filipe (1), Roman Biek (1,2), Joseph Hughes (1), Sreenu Vattipally (1), Stephan Zientara (4), Emmanuel Breard (4), Damien Vitour (4), Bernd Hoffmann (5), Giovani Savini (6), Piet van Rijn (7), Peter Mertens (3,8), Mass

1: MRC-University of Glasgow, Glasgow, UK, UK; 2: Institute of Biodiversity, Animal Health and Comparative Medicine, Boyd Orr Centre for Population and Ecosystem Health, University of Glasgow, Glasgow, UK; 3: The School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, Leicestershire, UK; 4: Laboratoire de Santé Animale d’Alfort, Université Paris Est, ANSES, ENVA, INRA, UMR 1161 VIROLOGIE, Maisons-Alfort, France; 5: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany; 6: Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM), Teramo, Italy; 7: Department of Virology, Wageningen Bioveterinary Research (WBVR), Lelystad, The Netherlands; 8: The Pirbright Institute, Pirbright, Woking, UK

Bluetongue is an economically important disease of domestic and wild ruminant species.The disease is caused by bluetongue virus (BTV), an orbivirus within the Reoviridae family, transmitted by biting midges (Culicoides spp.). The BTV genome is composed by ten segments of dsRNA encoding 7 structural and 4/5 non-structural proteins.In recent years, bluetongue has emerged as an economically important disease in sheep and cattle in geographical areas that had not experienced the disease before.The first BTV outbreak ever recorded in north Europe started in the Netherlands and Belgium in 2006 and then spread to the whole of Europe in the following years. This was the largest BTV outbreak recorded causing extensive losses to animal health and the European economy. The BTV-8 outbreak was contained by massive vaccination but in 2015 the virus re-emerged in central France spreading subsequently to the whole country. We have developed high throughput strategies to successfully sequence BTV full genomes directly from clinical samples. One of these strategies include the use of bait capture probe enrichment allowing full genome sequencing of clinical samples with Ct values >30. We sequenced over 100 BTV-8 field samples from cases from 2006 up to 2017 from different European countries. Analyses from these studies are currently under way.The data generated will allow to study the evolution and transmission dynamics of BTV-8 in Europe.


Association of rotavirus gastroenteritis with bacterial infection in poultry birds.

Oluwole Oyetunde Oni (1), Ademola Amubieya Owoade (2), Christopher Adeyinka Adeyefa (2)

1: FEDERAL UNIVERSITY OF AGRICULTURE ABEOKUTA, NIGERIA; 2: DEPARTMENT OF VETERINARY MEDICINE, UNIVERSITY OF IBADAN, NIGERIA

Introduction: Avian rotaviruses cause gastrointestinal diseases of birds worldwide. However, its co-infection with other micro-organisms remain largely under-investigated in Africa.Materials and Methods: Four experiments were conducted to reproduce enteritis in broiler birds using rotavirus and Salmonella pullorum. Fifty-two broiler birds were obtained and randomly divided into 4 groups. Group A chicks were inoculated with 1 x 106 pfu/ml of rotavirus, group B chicks were inoculated with 1 x 106 cfu/ml of Salmonella pullorum, group C chicks were inoculated with 1 x 106 pfu/ml of rotavirus and 1 x 106 cfu/ml of Salmonella pullorum, while group D birds were given 1ml of PBS alone. Results and Discussion: Significant (P ≤ 0.05) growth retardation was observed in chicks given rotavirus and Salmonella pullorum combination when compared with other groups. Histological changes were characterized by swollen villus tips and constricted villus bases, proliferation of enterocytes and necrotic villi. At 7 dpi, nuclei were enlarged and irregularly positioned within the cells in infected groups. There was goblet cell hyperplasia with focal areas of necrosis and exfoliated enterocytes. In addition, severe lymphocytic infiltration, necrosis of villi and syncytial formation was observed in the rotavirus and Salmonella pullorum infected group. At 1week interval all cells in the intestine were dead and crypts with no villi observed. Conclusion: The present study reveals rotavirus co-infection complicates gastroenteritis in birds leading to diarrhea, significant growth depression and death. It is thus important to continuously screen and prevent against organisms infecting the gastrointestinal tract for optimal performance of birds.


Rotavirus and enterovirus: through irrigation waters to green vegetables.

Verónica Emilse Prez, Laura Cecilia Martínez, Gisela Masachessi, Miguel Oscar Giordano, Patricia Angélica Barril, Silvia Viviana Nates

Instituto de Virologia Dr JM Vanella, Argentine Republic

The link between vegetable crops and fecally contaminated irrigation water establishes an environmental scenario that can result in a risk to human health. In Argentina there is no legislation mandating enteric viruses monitoring in foods. The aim of this study was to assess rotavirus (RV) detection frequency and G-types diversity as well as the presence of infective enterovirus (iEV) in raw vegetables and the corresponding irrigation water used for primary production. The study was conducted in a leafy green vegetable farm located in Colonia Caroya, in the center of Argentina. During a six month period (June-December 2014) a total of 15 water samples and 46 raw vegetables were collected. Water samples and elution buffer from vegetables were concentrated (100X) and RT-nested PCR was applied for RV detection and characterization. Also the concentrates were inoculated in CaCo2 cells to monitor the occurrence of iEV. RV was detected in 26% (4/15) of the irrigation water and in 13% (6/46) of the vegetable samples. The genotypes found in water were G3 60% (3/5) and G9 40% (2/5) and in vegetables G2 22% (2/9), G3 66% (6/9) and G9 12% (1/9). iEV was detected in both matrixes studied. These results point out that green leafy vegetables are contaminated with RV and iEV and that the irrigation water would be a source of contamination. The presence of viral genomes and infective particles in food that in general suffer minimal treatment before consumption underlines that green crops can act as potential sources of enteric virus transmission.


Epidemiological, clinical and virological aspects of Rotavirus diarrhea in Yaounde-Cameroon (2014-2016)

Ngoya Roger, Boula Angeline, Kemajou Grace, Nkolo Eric, Ngoh Rose, Koki Paul

Centre Mère et Enfant de la Fondation Chantal BIYA, Cameroon

INTRODUCTION: Rotavirus is the major cause for more than 80% of childhood gastroenteritis. It is a major public health problem in developing countries. This study was to assess the epidemiology, clinical and virological characteristics of community-acquired rotavirus acute gastroenteritis in infants hospitalized in Cameroon.METHODS: It is a retrospective, descriptive and cross-sectional study conducted during the period from January 1, 2014 to December 31, 2016. The registers, the investigation files and the children’s files enabled us to collect the epidemiological and clinical parameters (age, sex, seasonality, clinical signs) and virology of children admitted for acute diarrhea.RESULTS: From 1 January 2014 to 31 December 2016, 1244 cases of acute diarrhea were collected and analyzed, of which 259 were infected with rotavirus (20.82%). Children aged 6-23 months were more affected (50.20%). 176 (67.95%) cases of diarrhea were observed during the dry season. Fever, vomiting, severe dehydration were observed in 259 (100%), 240 (92.66%), 228 (88.03%) cases, respectively. 19 genotypes were highlighted including the most dominant G1P [8] 68 (26.25%) and G3P [6] 66 (25.48%).CONCLUSION: Rotavirus diarrhea is a major health problem in developing countries and affects children under 5years of age. Appropriate and early management and rotavirus vaccination are the most effective preventive measures against rotavirus recommended in our community.


Development of a novel, visual Polymerase Spiral Reaction for detection of Rotavirus C

Kuldeep Sharma, Nitish S Parihar, Arti Shrivas, Vaibhav K Tamrakar, Jyothi Bhat

National Institute for Research in Tribal Health, Jabalpur,Madhya Pradesh, India

Introduction: Rotavirus C is important human enteric and zoonotic pathogen, but their ecology remains to be elucidated. A sensitive, specific, simple, and rapid nucleic acid based diagnostic method; polymerase spiral reaction assay was developed to detect the RVC.Methods: A sum of 137stool samples was collected from the hospitalized children below 5 years of age, having diarrhea for more than five days. The samples were processed for RNA extraction through Trizol method. The PSR reactions were carried out post cDNA synthesis in a 25μl reaction mixture with 1.0μl Bst DNA polymerase, 2.5μl 10X ThermoPol reaction buffer, 0.8M Betaine, 6 mM MgSO4, 1.4mM each dNTPs, 1.6μM RVCVP6 PSR FP and RP primers with 5μl cDNA and incubated for 60 minutes at 59°C, and SYBR Green-I dye was used for result determination. RT-PCR for RVA, RVB and RVC were also applied to the test samples. Seven samples were sequenced.Results: The results of PSR revealed the presence of RVC in 23 samples. All positive samples were from children < 3 years. None of them were positive in RVA and RVB RT-PCR. The obtained sequenced showed 99% identity with porcine rotavirus C and submitted to NCBI from MG676132 to MG676139.Conclusions: The PSR assay established in this study was specific for detection of RVC and no cross-reaction with other RVA and RVB. The detection limit of PSR was 10 times more than RT-PCR. The results showed the potential clinical feasibility of PSR as a useful assay for RVC detection with high sensitivity.


GENETIC CHARACTERIZATION OF STRUCTURAL AND NON-STRUCTURAL GENES OF UNUSUAL ROTAVIRUS STRAINS ASSOCIATED WITH ACUTE GASTROENTERITIS

Vaishali Tatte, Deepthy Maran, Gopalkrishna Varanasi

ICMR-National Institute of Virology, Pune, India, India

Group A rotavirus (RVA), a member of the family Reoviridae remains the leading cause of gastroenteritis in children. Although vaccines are available for the control of rotavirus infections, the global spread of unusual RV strains may present challenges to future vaccine evaluation programmes. In view of this, it is necessary to understand the genetic diversity of the predominant unusual strains associated with the disease.Rotavirus surveillance studies carried out earlier showed increase in the prevalence of unusual RVA strains (7.9-9.1%) in acute gastroenteritis cases (AGE) in Pune, Western India. Of these, three representative unusual RVA strains (G9P[4], G9P[6] and G1P[6]) were selected for the study. Sequencing and phylogenetic analysis of VP7, VP4, VP6, NSP1, NSP2 and NSP3 genes of these strains classified them to the G9- IIId / G9-II and G1-I genotypes, P[4]-5 and P[6]-Ia genotypes, I1 and I2 genotypes, A1 and A2 genotypes, N1and N2 genotypes and T1 and T2 genotypes respectively. Genotype constellation of G9-IIId-P[4]-5-I2-A2-N2-T2, G9-II-P[6]-1a-I1-A1-N1-T1 and G1-I-P[6]-1a-I1-A1-N1-T1 was observed. Nucleotide/amino acid identity of 84-100 %/82.3-100% for VP7, 83.5-99.3%/74.3-99.6% for VP4, 78.7-99%/91-100% for VP6, 78.9-97.9%/67.4-97.1% for NSP1, 82.2-97.5%/59.7-97.1% for NSP2 and 80.2-98.9% /61.7-97% for NSP3 genes with reference strains was noted. Within the Wa-like strains nucleotide/ amino acid divergence of 0.7-12.2% / 0.3-14.1% was noted in all the RV genes studied.The present study highlighted intra-genotypic variations in the RV genes studied. Further emphasize the need for RV surveillance and whole genome classification of unusual RV strains in AGE cases from other regions of the country to understand the diversity.


Rotadial: the first RVA diagnostic kit based on patented VHH antibodies

Celina Guadalupe Vega (1), Marina Bok (1), Juan Ignacio Degiuseppe (2), Alejandra Antonella Rivolta (1), Ana Paula Piantanida (1), Matías Adúriz Guerrero (1), Gustavo Asenzo (1), Andrés Wigdorovitz (1), Juan Stupka (2), Lorena Laura Garaicoechea (1), Viv

1: INTA, Argentine Republic; 2: INEI-Anlis Dr. Carlos Malbrán

Diarrhea is a major cause of infantile morbidity and mortality, especially in developing countries. Annually, more than 1.7 billion episodes of diarrhea occur among children <5 years old, resulting in 525.000 deaths. Rotavirus (RVA) infections are a leading cause of severe, dehydrating gastroenteritis in this group of children. RVA infections still result in >200,000 deaths annually, mostly in low-income countries, even when two oral live vaccines were introduced in 2006. RVA diagnosis is critical not only for medical decisions but also for epidemiological surveillance, outbreak control and to estimate RVA burden and its impact on national health systems. We developed a fast and simple ELISA using patented VHH recombinant antibodies to detect RVA in stools from pediatric patients. This kit was named Rotadial. It provides all the reagents needed and can be performed at room temperature in low-tech laboratories. The VHH antibodies are directed to VP6 protein and can detect all human and animal RVA strains tested so far. Results are obtained in less than 2 h and showed 99% diagnostic specificity and sensitivity. Furthermore, it showed 100% analytical specificity since no cross reactivity with other common infectious agents involved in diarrhea was observed. This kit is being distributed in the National RVA Surveillance Network in Argentina, where a national fully-funded RVA universal mass vaccination programme works since 2015 and it will be commercially available at a competitive price for human and animal RVA detection by next year. To our knowledge, this is the first RVA detection kit based on VHH antibodies.


Detection of rotavirus and distribution of genotypes before and after the introduction of Rotarix in Ireland

Zoe Yandle, Suzie Coughlan, Cillian De Gascun

UCD National Virus Reference Laboratory, Dublin, Ireland

Introduction: Ireland introduced Rotarix into the national immunisation schedule in December 2016. We describe the detection of rotavirus before and after the introduction of the vaccine and the distribution of rotavirus genotypes.Methods: Faecal samples from symptomatic patients were extracted by Roche MagNAPure96 and tested by real-time RT-PCR for rotavirus. Rotavirus wild-type and Rotarix differentiation was introduced in December 2017. A proportion of samples were genotyped by nPCR and electrophoresis.Results: Prior to the introduction of the vaccine from December 2015 to April 2016, 5403 samples were tested for rotavirus. Of these, 1626 patients were ≤ 5 years old and 216 (13.3%) were positive. In addition 3777 patients were >5 years and 35 (0.9%) were positive.In December 2017 to April 2018, 3452 samples were tested. Of these 577 patients were ≤5 years and 24 (4.2%) were wild-type rotavirus positive and a further 14 (2.4%) had detectable Rotarix. There were 2875 patients >5 years and 9 (0.3%) were rotavirus positive.One hundred samples from the pre-vaccine era were genotyped. G1P[8] was the predominant genotype (53%), followed by G9P[8] (30%), G2P[4] (7%), G3P[8] (2%), G4P[8] (4%), G12P[8] (2%), mixed/untypable (2%).In the post-vaccine era 79 samples were genotyped. G1P [8] was the predominant genotype (56%), followed by G2P[4] (20%), G9P[8] (8%), G4P[8] (5%), G3P[8] (4%), G12P[8] (6%), mixed/untypable (1%).Conclusion: Following the introduction of the vaccine rotavirus detection decreased by 89% in patients aged ≤5 years. The G1P[8] genotype is the most common genotype in the pre and post-vaccination era.


WORKSHOP 3: MOLECULAR AND CELLULAR VIROLOGY

Analysis of ribonucleoprotein complexes of IBDV

Carolina Allende-Ballestero, Carlos P. Mata, José R. Castón

Department of Structure of Macromolecules, Centro Nacional de Biotecnología/CSIC, 28049 Madrid, Spain

Infectious bursal disease virus (IBDV) is an avian dsRNA virus of the Birnaviridae family. Birnaviruses have a single ~70-nm-diameter T=13l capsid that encapsidates a polyploid bipartite dsRNA genome (segments A and B), organized as ribonucleoprotein complexes (RNP). Segment A has two partially overlapping open reading frames; the first codes for the nonessential VP5 protein, and the second encodes a polyprotein. The polyprotein is co-translationally self-cleaved by the viral protease VP4. This yields the capsid precursor protein pVP2 (precursor capsid protein), VP3, and VP4. In addition to its RNA-binding activity, VP3 interacts with itself, with the viral polymerase, and/or with pVP2 acting as a scaffolding protein. Segment B is monocistronic and encode the RNA-dependent RNA polymerase, which is packaged within the virion as a free protein (VP1) or is covalently linked to the 5’ ends of the dsRNA molecules (VPg).IBDV virions dialyzed against a low ionic strength basic buffer rendered structurally preserved RNP (comprised of dsRNA, VP3 and VP1/VPg), which were functionally competent for capsid-independent RNA synthesis, and also effectively propagated the virus in transfection experiments. We purified VPg-dsRNA complexes in non-denaturing conditions. Titration of VPg-dsRNA with VP3 (monitored in electrophoretic mobility shift assays) allowed us to determine the VP3 ratio at which RNP properties are restored. Negative stain EM and cryo-EM analysis indicated that RNP are dsRNA molecules wrapped with VP3 monomers. Hybrid methods (EM combined with high resolution X-ray structures) were used to analyze the RNP ends, in which one or two copies of VP1/VPg were found.


Determining the dynamics of birnavirus replication organelle movement in vitro using a split-GFP tagged infectious bursal disease virus (IBDV).

Alice Gray, Andrew James Broadbent

The Pirbright Institute, UK

Birnaviruses are economically important veterinary viruses with a double stranded RNA genome that infect fish, birds, and insects, causing production losses to aquaculture and the poultry industry. However, despite their importance, little is understood of how they replicate within cells. Using reverse genetics, we have made the first reporter birnavirus by tagging a small sub-unit of Green Fluorescent Protein, GFP11, to an infectious bursal disease virus (IBDV) protein to make a split GFP virus (IBDV-GFP11). When DF-1 cells were transiently transfected with the rest of the molecule (GFP1-10) and subsequently infected with IBDV-GFP11, infected cells had multiple green foci in the cytoplasm. When cells were fixed and stained with antibodies against dsRNA and the IBDV VP3 protein, which coats the genome and binds the polymerase, there was a high degree of co-localisation with the GFP signal, suggesting these foci represent replication organelles (ROs). When imaged from 12 hours to 24 hours post-infection, the average diameter of the foci increased while the average number of foci per cell decreased, consistent with the coalescence of multiple ROs over time. When movies were taken of the infected cells at 18 hours post-infection for a period of 2 hours 30 minutes, small foci moved in the cytoplasm and some coalesced, whereas larger foci remained more static. We are now defining the molecular determinants of birnavirus RO dynamics by studying the effect of inhibitors of the cell cytoskeleton. Taken together, the IBDV-GFP11 virus will be a useful tool to study birnavirus-host cell interactions.

217Poster presentations Workshop 3: Molecular and Cellular Virology

A novel rotavirus inhibitor impairs the structural integrity of rotavirus particles and viroplasms

Oscar R. Burrone (1), Catherine Eichwald (2), Giuditta De Lorenzo (1), Elisabeth Schraner (2), Guido Papa (1), Michela Bollati (3), Paolo Swec (4), Matteo de Rosa (3), Mario Milani (3), Eloise Mastrangelo (3), Mathias Ackermann (2), Francesca Arnoldi (1)

1: ICGEB, Italy; 2: Institute of Virology, University of Zürich, Switzerland; 3: Biophysics Institute of the CNR-IBF, University of Milan, Italy; 4: Pediatric Clinical Research Center, University of Milan, Italy; 5: Department of Medicine, Surgery and Health Sciences, University of Trieste, Italy

Since their introduction in 2006, the two rotavirus (RV) vaccines used worldwide reduced significantly the estimated rotavirus-related mortality rate (~215,000 in 2013), but showed very low efficacy in low income countries, where RV remains one of the main causes of death in children. Specific antiviral therapies could thus impact on the still high morbidity and mortality due to RV infections.In this study, we describe the anti-RV activity of ML-60218, a small molecule known as an inhibitor of mammalian RNA polymerase III. In cell-based assays, ML-60218 was found to disrupt already assembled viroplasms, and to hamper the formation of new ones, without the need for de novotranscription of cellular RNAs.This phenotype correlated with reduction in accumulated viral proteins and viral genome segments, disappearance of the hyperphosphorylated isoforms of the viroplasm-resident protein NSP5 and with inhibition of infectious progeny virus production. Invitro, ML-60218caused dose-dependent damage of the structural integrity of purified DLPs, whose transcriptional activity was also compromised in a dose-related manner. The higher order structures formed by the DLP outer layer protein VP6were found severely damaged by ML-60218, whereas VP6 trimerization was not compromised. ML-60218is the first chemical compound found able to i) disrupt viroplasms and ii) damage the stability of DLPs by targeting the viral protein VP6. This molecule thus represents a starting point towards the design of a new class of potent and selective anti-RV compounds.


Cytoplasmic re-localization and colocalization with viroplasms of host nuclear and cytoplasmic proteins, and their role in rotavirus infection

Durga Rao Chilakalapudi, Varsha Tandra, Poonam Dhillon

Indian Institute of Science, India

Rotavirus replicates in the cytoplasm of infected cells in unique virus-induced cytoplasmic inclusion bodies called viroplasms (VM), which are nucleated by two essential viral non-structural proteins NSP2 and NSP5. However, the precise composition of the VM, intracellular localization of host proteins during virus infection, their association with VM or role in rotavirus growth remained largely unexplored. By mass-spectrometry analyses, we identified several host heterogeneous nuclear ribonucleoproteins (hnRNPs) and AU-rich element-binding proteins (ARE-BPs) and cytoplasmic proteins from uninfected MA104 cell extracts in the pull-down (PD) complexes of purified viroplasmic proteins NSP2 and NSP5. The majority of the host proteins interacted with the viroplasmic proteins in an RNA-independent manner. Time-course immunoblot analysis of the nuclear and cytoplasmic fractions from rotavirus-infected and mock-infected cells and immunofluorescence confocal microscopy analyses of virus-infected cells surprisingly revealed sequestration of the majority of the re-localized host proteins in the viroplasms. Ectopic over-expression and siRNA-mediated down-regulation of expression analyses revealed that the host proteins either promote or inhibit viral protein expression and progeny virus production in virus-infected cells. This study demonstrates that rotavirus induces cytoplasmic re-localization and sequestration of a large number of nuclear and cytoplasmic proteins in viroplasms, subverting essential cellular processes in both the compartments to promote rapid virus growth, and reveals that the composition of rotavirus viroplasms is much more complex than that is currently understood, laying the foundation for a new direction of research on host-virus interactions in rotavirus biology.


A New Form of Human Rotavirus Produced from Human Intestinal Enteroids

Sue E. Crawford, Sarah E. Blutt, Sasirekha Ramani, Daniel R. Laucirica, Winnie Zou, Xi-Lei Zeng, Umesh Karandikar, James Broughman, Mary K. Estes

Baylor College of Medicine, USA

Human stem cell-derived intestinal enteroid (HIE) cultures are a new model to study host-pathogen interactions. Our previous studies in 3-dimensional HIEs provided new insight into human rotavirus (HRV) host restriction, pathophysiology and innate epithelial responses. A surprising finding was the lack of infectivity of HRV produced from MA104 cells on HIEs grown as monolayers on 96-well plates, unless the monolayer was wounded prior to infection. The requirement for wounding led us to ask whether HRVs infect HIEs from the apical or basolateral surface. Duodenal, jejunal, ileal and colonic HIEs are susceptible to infection from the basolateral, but not the apical, epithelial surface. We next evaluated whether virus is released apically or basolaterally and whether released virus is infectious on HIE or MA104 cell monolayers. Basolateral infection of HIEs resulted in apical release of newly synthesized virus that is infectious on both HIE and MA104 cell monolayers grown on 96-well plates. Treatment with detergent greatly reduced the infectivity of apically released virus from HIE monolayers on HIEs but not MA104 cells. The ESCRT protein TSG101 was detected on apically released virus from HIEs but not on the detergent-treated virus by Western blot. Our results suggest a new form of HRV is released apically from HIEs that is associated with membranes and may use ESCRT vesicular trafficking for apical release.


The Role of DGAT1 in Rotavirus Viroplasm/Lipid Droplet Formation

Sue Ellen Crawford (1), Zheng Liu (1,2), Jeanette Criglar (1), Mary K. Estes (1)

1: Baylor College of Medicine, USA; 2: Rice University, USA

Lipid droplets (LDs) are essential for the replication of rotavirus (RV) and multiple other viruses and intracellular bacteria. LDs are multi-functional organelles that consist of a core of neutral lipids surrounded by a phospholipid monolayer coated by LD-associated proteins. The current model of LD formation posits that increased accumulation of neutral lipids between the two leaflets of the endoplasmic reticulum (ER) triggers budding of LDs from the ER. However, the molecular mechanisms of LD formation remain elusive. Because replication and assembly of RV occurs in viroplasms that interact with LDs physically and functionally, we sought to elucidate how RV induces LD biogenesis. A proteomic analysis of NSP2-interacting cellular proteins identified DGAT1, an ER-localized neutral lipid synthase. Our studies revealed that infectious virus production from DGAT1-silenced cells or DGAT1-/- mouse embryo fibroblasts is increased 5-fold compared to controls; viroplasm/LDs form earlier in DGAT1-silenced cells; and DGAT1 interacts with ubiquitinated NSP2 and is degraded early during infection. These results extend previous observations that inhibiting lipogenesis prior to RV infection blocks viral replication and viroplasm/LD formation; neutral lipid in the viroplasm/LD accumulates in the ER prior to infection; and lipidomics of RV-infected cells showed an increase in phospholipids. Altogether, these results suggest a new hypothesis that RV infection mediates the degradation of DGAT1 for viroplasm/LD biogenesis by redirecting precursor lipids into an alternative pathway that generates phospholipids for the formation of viroplasm/LDs. Delineating how RV exploits LD biogenesis is expected to reveal mechanisms of LD biogenesis that may be relevant to multiple pathogens.


Rotavirus replication is correlated with S/G2 interphase arrest of the host cell cycle.

Selene Glück (1), Antonino Buttafuoco (1), Anita F Meier (1), Francesca Arnoldi (2), Vogt Bernd (1), Elisabeth M Schraner (1), Mathias Ackermann (1), Catherine C Eichwald (1)

1: Institute of Virology, University of Zurich, Switzerland; 2: Dept. of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy

In infected cells, rotavirus (RV) replicates in viroplasms, cytosolic structures that require a stabilized microtubule (MT) network for their assembly, maintenance of the structure and perinuclear localization. Therefore, we hypothesized that RV could interfere with the MT-breakdown that takes place in mitosis during cell division. Using synchronized RV-permissive cells, we show here that RV infection arrests the cell cycle in S/G2 phase, thus favoring replication by improving viroplasms formation, viral protein translation, and viral assembly. The arrest in S/G2 phase is independent of the host or viral strain and relies on active RV replication. RV infection causes cyclin B1 down-regulation, consistent with blocking entry into mitosis. With the aid of chemical inhibitors, the cytoskeleton network was linked to specific signaling pathways of the RV-induced cell cycle arrest. We found that upon RV infection Eg5 kinesin was delocalized from the pericentriolar region to the viroplasms. We used an MA104-Fucci system to identify three RV proteins (NSP3, NSP5, and VP2) involved in cell cycle arrest in the S-phase. Our data indicate that there is a strong correlation between the cell cycle arrest and RV replication.


The dynamics of both filamentous and globular mammalian reovirus viral factories rely on the microtubule network.

Catherine C Eichwald (1), Mathias Ackermann (1), Max L Nibert (2)

1: Institute of Virology, University of Zurich, Switzerland; 2: Dept. of Molecular Genetics and Immunobiology, Harvard Medical School, Harvard University, Boston MA, USA

Mammalian reovirus viral factories (VFs) form filamentous or globular structures depending on the viral strain. In this study, we attempt to characterize the dynamics of both filamentous and globular VFs. We present evidence demonstrating that globular VFs are dynamic entities coalescing between them, thereby gaining in size and concomitantly decreasing in numbers during the course of the infection. Additionally, both kinds of VFs condense into a perinuclear position. Our results show that globular VFs rely on an intact MT-network for dynamic motion, structural assembly, maintenance and perinuclear condensation. Interestingly, dynein localizes in both kinds of VFs, having a role at least in large globular VFs formation. To study filamentous VF dynamics, we used different transfection ratios of µNS with filamentous µ2. We found an MT-network dependency for VF-like structures perinuclear condensation. Also, µNS promotes VFLSs perinuclear positioning as well as an increase in acetylated tubulin levels


Adaptive mutations occurring during serial passages of an avian/mammalian rotavirus reassortant in cell culture

Michael Burwinkel, Alexander Falkenhagen, Eva Trojnar, Reimar Johne

Federal Institute for Risk Assessment, Germany

The rotavirus genome consists of 11 segments of dsRNA. After infection of a cell with two different parent rotaviruses, reassortant progeny viruses can be created, which contain a mixture of genome segments from both parent viruses. By the use of reverse genetics systems (RGS), distinct reassortants can be created, which may have different replication efficiency. Knowledge about genome regions critical for efficiently replicating reassortants may therefore be useful for targeted generation of viable recombinant rotaviruses for basic and applied science. Using a helpervirus-dependent RGS, an artificial rotavirus reassortant containing the VP4 gene of chicken rotavirus strain Ch2G3 in the backbone of simian rotavirus strain SA11 was generated. The reassortant virus could be grown and passaged on MA104 cells, but it replicated only to titers of 104 TCID50/ml, which was consistently lower than that of both parent viruses (106 TCID50/ml for Ch2G3 and 108 TCID50/ml for SA11). After 10 consecutive passages on MA104 cells, the reassortant virus replicated to titers of 106 TCID50/ml. Sequencing of the VP4 gene of the multiple passaged virus indicated the presence of point mutations, in some cases leading to amino acid exchanges as compared to the initial reassortant.The results indicate that rotavirus reassortants with low replication capacity can acquire adaptive mutations conferring efficient growth within a relatively short time periode. However, the identified mutations in the VP4 gene have to be tested for their contribution to the replicative phenotype. In addition, possible mutations in other genome segments have to be analyzed in the future.


Unravelling the complexity of AHSV NS3: a targeted investigation of its functional domains.

Linda Ferreira-Venter (1), Eudri Venter (2), Vida van Staden (1)

1: Dept. of Biochemistry, Genetics and Microbiology, University of Pretoria, South Africa; 2: Laboratory for Microscopy and Microanalysis, University of Pretoria, South Africa

African horse sickness (AHS) is a highly infectious, often fatal, insectborne disease of equids endemic to sub-Saharan Africa and caused by AHS virus (AHSV). This study focusses on the AHSV non-structural protein 3 (NS3), an integral membrane protein involved in virus trafficking and release, also contributing to cellular pathogenesis. To gain insight into the role of the different NS3 domains in mediating these various functions, six reverse genetics-derived mutant viruses, each expressing a modified version of NS3, were generated and compared to the wild-type virus in mammalian cells. This comparison included cytotoxicity assays, virus quantification techniques, transmission electron microscopy, immunogold labelling and laser scanning microscopy.Results showed that both transmembrane domains were essential for NS3’s proper processing and intracellular transport. Their disruption caused altered localisation of NS3 to the nucleus, either as foci or thin fibres, the latter formed through putative association with NS1. The cytoplasmic late-domain motifs and C-terminal region did not influence NS3’s distribution, as even when modified, NS3 still localised to perinuclear vesicles and the plasma membrane. Phenotypically, all mutant viruses exhibited varying degrees of delayed cell death, including significantly reduced release with either the abnormal cytoplasmic aggregation of virus particles, or tethering of virions to the plasma membrane. Infection with the mutants also resulted in the formation of larger virus inclusion bodies. Irrespective of virion distribution, all mutants showed varying degrees of altered localisation of capsid protein VP5 to the plasma membrane. Cumulatively these results hint at a complexity of domain functions for facilitating NS3 pleiotropy.


Polybasic motif within non-structural protein 3 of Bluetongue virus is important for cell release

Thomas Labadie, Sophie Jegouic, Polly Roy

Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, WC1E 7HT, UK

Bluetongue virus (BTV) is an insect-borne virus infecting sheep and cattle. In the course of the infection, a non-lytic release of viral particles is observed, leading ultimately to the lysis of infected cells. The non-structural protein 3 (NS3), which is the only glycosylated membrane protein of BTV, is involved in the virus non-lytic release to the plasma membrane transport network, by using the endoplasmic reticulum and Golgi apparatus. The mechanism by which the NS3 protein hijack this transport network is not yet understood.In this project, multiple sequences alignment of the NS3 protein from several members of the orbivirus genus allowed us to identify novel and conserved polybasic motifs (PBMs) as potential Golgi export signals. Using site directed-mutagenesis and reverse genetics in addition to molecular virology and immunofluorescence technics, we demonstrated that the PBMs present in the NS3 protein are important for efficient cell-to-cell spread. Substitution of amino acids in the PBMs of the NS3 protein decrease the viral fitness in both mammalian and insect cells, with significant delay in the viral particles release. Our data also revealed that these mutations induce a segregation of the NS3 proteins in the Golgi apparatus, with a lower NS3 surface expression level in the infected cells. Finally, these mutations inhibit the secretion of high-density viral particles associated with lipid membranes.Altogether, our data reinforced the role of the NS3 protein in the non-lytic release of BTV particles, mediated by a PBM-dependent mechanism.


Combining classical and reverse genetics to study the multiple proteins of mammalian orthoreoviruses affecting induction of and sensitivity to the interferon response

Delphine Lanoie, Emmanuelle Degeorges, Stéphanie Côté, Véronique Sandekian, Guy Lemay

Université de Montréal, Canada

In our efforts to get a more complete picture of the reovirus determinants affecting the interferon response, various strategies were used, resulting in viruses differing in either induction and/or sensitivity to the response. Initially, a virus was selected for interferon sensitivity following chemical mutagenesis and the use of reverse genetics allowed us to identify a single amino acid substitution in λ2 that is responsible for increased sensitivity. In the course of this work, it was also observed that the wild-type virus laboratory stock is more resistant and induces less interferon than the commonly used virus obtained by reverse genetics. The μ2 and λ2 proteins were found to be responsible for the difference in sensitivity while μ2 and λ1 are involved in the difference of interferon secretion. In addition to the well-known implication of μ2, this further established the importance of λ2 while revealing a role of λ1 for the first time. In our most recent work, a Vero-cell-adapted virus turned out to be also more sensitive to interferon compared to the wild-type laboratory stock. Surprisingly, this was mapped to a single mutation overlapping the σ1 and σ1s coding regions. The absence of selective pressure in interferon-defective Vero cells has likely allowed selection of this new virus; recent data suggest that σ1s is the determinant involved. Further characterization of the different proteins of these different viruses (stability, subcellular localization, …) will be presented. Altogether this work supports the idea that multiple reovirus proteins are involved in the control of interferon response.


Modifications to the cellular transcriptome and alternative RNA splicing after mammalian orthoreovirus infection

Simon Boudreault (1), Martin Bisaillon (1), Guy Lemay (2)

1: Université de Sherbrooke, Canada; 2: Université de Montréal, Canada

In recent works, the use of RNA-seq has been introduced in order to study the modifications that could occur to both the cellular transcriptome and alternative RNA splicing landscape following infection by different viruses. In our laboratories, this has been originally applied to examine the situation during infection of cultured cells with mammalian orthoreovirus. As expected, the transcriptome was deeply modified, especially by increased transcription of various cellular genes involved in the host-cell antiviral response. These data have been used to establish a panel of a dozen genes that are now examined by quantitative RT-PCR following infection with different viruses recently obtained in the laboratory; these viruses differ in either their sensitivity or induction of the interferon response. Somewhat more surprisingly for a cytoplasmic RNA virus, the alternative splicing of various cellular transcripts was also significantly affected, especially for genes involved in RNA metabolism including splicing itself. Interestingly, the expression level of certain known splicing factor was also strongly affected. Using a “bystander” assay, in which infected cells were grown on a semipermeable membrane, we recently observed that changes in alternative splicing of some genes require the presence of the virus. These changes were not observed in bystander cells, despite increased transcription of antiviral genes and splicing factors. Current work examines the role of different viral proteins in the modifications observed in the cellular transcriptome and alternative RNA splicing during mammalian orthoreovirus infection and this will be further discussed.


Mal de Río Cuarto virus minor viroplasm component P6 interacts with an atypical wheat thioredoxin

Gabriela Llauger (1), Demián Esteban Monti (1), Carlos María Figueroa (2), Alberto Álvaro Iglesias (2), Mariana del Vas (1)

1: Instituto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires, Argentina; 2: Instituto de Agrobiotecnología del Litoral (IAL) - Centro Científico Tecnológico Santa Fe (CCT), Santa Fe, Argentina

Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) causes the most devastating maize disease in Argentina. The virus infects planthopper vectors and several grass species. Wheat is currently the best model system to study MRCV interaction with plants. Like other members of the family, MRCV replicates and assembles within cytoplasmic viral inclusion bodies called viroplasms, which are composed by the non-structural viral proteins P9-1 and P6. Both proteins are able to self-interact and to interact between them, and contain PEST sequences that serve as proteolytic degradation signals by the UPS. Despite the relevance of MRCV viroplasm structural organization and dynamics for the design of effective antiviral strategies, the details of this process are still unknown. In the current study, we sought to identify host viroplasm components by screening a wheat cDNA Y2H library, employing P6 as bait. P6 has 788 residues coding for a 90 kDa protein. We demonstrated that P6 can interact with a predicted thioredoxin (TRX) containing an atypical WCRKC active site. Using P6 truncated mutants, we established that the region spanning residues 106 to 788 is required for such interaction. Thioredoxin activity was confirmed by performing insulin reduction assays, and was completely abolished in TRX mutants lacking the cysteine residues within the active site. Additionally, this atypical TRX displayed a mild glutaredoxin activity. P6 has 14 cysteines mostly distributed throughout its C-terminal moiety and therefore is a putative TRX target. Implications of this interaction and its possible role in viroplasm formation within MRCV infection are discussed.


Pseudomonas Phage Phi6 Envelope Formation and Phi6-Specific Vesicle Production in Escherichia coli

Outi L Lyytinen, Daria A Starkova, Minna M Poranen

Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland

Pseudomonas phage phi6 is unique among bacteriophages since it has a lipid-protein envelope as an outermost layer. Phi6 infects plant pathogenic Pseudomonas bacteria and it is a well-known model organism for dsRNA virus assembly. The assembly pathway for its core and the viral genome packaging and replication processes have been described in molecular detail. However, the mechanisms of the envelope assembly of the phi6 are largely uncharacterized. According to the current knowledge, the major envelope protein P9 and the non-structural protein P12 are the minimal protein components needed for the phi6 envelope assembly. In addition to these minimal requirements, the phi6 membrane includes four integral membrane proteins: the fusogenic protein P6, the spike protein P3, the putative holin protein P10 and the minor membrane protein P13. Due to the relatively simple structure of the phi6 envelope, it has a potential to become a model for the virus envelope assembly. In 2012, Sarin et al. [1] found out that phi6-specific vesicles can be produced also in Escherichia coli. In our study, we expressed the phi6 membrane proteins P9, P6 and P3 and the non-structural protein P12 in different combinations in E. coli to study the viral envelope assembly and to produce phi6-specific vesicles for potential biotechnological applications.[1] Sarin, LP, Hirvonen, JJ, Laurinmäki, P, Butcher, SJ, Bamford, DH, Poranen, MM (2012) Bacteriophage Phi6 Nucleocapsid Surface Protein 8 Interacts with Virus-Specific Membrane Vesicles Containing Major Envelope Protein 9. Journal of Virology. 86:9, 5376-5379.


Further analysis of Ibaraki virus VP6 to produce fluorescence-labeled orbiviruses

Eiko Matsuo (1), Marina Hamaji (1), Hiroko Omori (2), Hideyuki Tsuji (1), Akari Saito (1), Polly Roy (3), Keiichi Saeki (1), Takeshi Kobayashi (2), Junichi Kawano (1)

1: Kobe Univesity, Japan; 2: Osaka University, Japan; 3: London School of Hygiene and Tropical Medicine

Recently, live cell imaging techniques have been developed in several viruses, including dsRNA virus. However, the inside-label of the dsRNA virus with fluorescence probes was never reported. In Reoviridae, only orbiviruses bear a unique protein, VP6, which is believed to be essential for orbivirus replication as it is likely to recruit the virus genome into the particle. In our recent studies, two specific regions of VP6, the loop region and VP3 binding region, were identified. In this study, based on these findings, we generated fluorescence-labeled Ibaraki virus (IBAV) to visualize the early step of IBAV infection. For generation of the VP6-labeled IBAV, we used newly developed plasmid-based reverse genetics system for IBAV as well as the reassortant method. As helper cells, BHK cells constantly expressing functional tagged-VP6, in which either Flag-tag or EGFP was inserted into a part of the loop region of VP6 in frame, were used to rescue the replication-deficient IBAV. EGFP gene was also inserted into S9 gene to produce viable VP6-labeled IBAV. In addition, using one of the helper cells, BHK-EGFPVP6 cells, and VP3/6-binding defective IBAV, “Single-cycle” VP6-labeled IBAV was generated. Binding of the virus to BHK cells was observed using fluorescence microscopy. In addition, neutralization antibodies against IBAV prevented binding of these viruses to cells. Moreover, using flow cytometry analysis, the binding abilities of IBAV in various conditions were quantified. Further study using VP6-labeld viruses will be discussed.


Whole genome analysis of African G12P[6] and G12P[8] rotavirus strains with evidence of porcine-human reassortment at NSP3 and NSP4 genes

Fortunate Mokoena (1), Mapaseka Seheri (1), Martin Nyaga (2), Nonkululeko Magagula (1), Arnold Mukaratirwa (3), Augustine Mulindwa (4), Almaz Abebe (5), Angelina Boula (6), Tsolenyanu Enyonam (7), Kebareng Rakau (1), Ina Peenze (1), Jason Mwenda (8), Jef

1: Sefako Makgatho Health Science University, South Africa; 2: University of Free State, Bloemfontein,South Africa; 3: University of Zimbabwe-College of Health Sciences; 4: Mulago National Referral Hospital; 5: Ethiopian Public Health Institute; 6: Chantal Biya Foundation, Yaoundé, Cameroon; 7: Tokoin teaching hospital of Lome, Togo, West Africa; 8: WHO African Regional Office, Brazzaville, Congo

Group A rotaviruses (RVA) represent the most important etiologic agent of acute gastroenteritis (AGE) in infants and young children worldwide. There has been an increase in global detection and reported cases AGE caused by RVA genotype G12 strains, especially on the African continent. We aimed to investigate the possible origin and genomic relationship between African G12 strains and globally circulating rotaviruses. Therefore, the whole genome sequences of 36 RVA G12 strains in combination with P[6] and P[8] genotypes detected from the Southern (South Africa, Zimbabwe), Eastern (Ethiopia and Uganda), Central (Cameroon) and Western (Togo)s Africa were sequenced using the Sanger method and IonTorrent PGM. Genomic analyses of the 11-segments of the study strain MRC-DPRU861 (Ethiopia) displayed a DS-1-like genetic constellation of G12-P6-I2-R2-C2-M2-A2-N2-T2-E2-H2. The remaining G12 strains had a Wa-like genetic backbone of G12-P[6]/P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, with two Ethiopian strains (MRC-DPRU850 and MRC-DPRU5010) and South African strain (MRC-DPRU309) displaying DS-1-like intergenogroup reassortment. Overall, 9 gene segments (VP1-VP3, VP6, NSP1-NSP5) of African G12 strains were determined to be genetically related to globally circulating human G12, G9 and G1 with nucleotide identities of 98.7%-99.9%, 87.5%-98.5% and 82.4%-97.0% respectively. Phylogenetic analysis revealed that strain MRC-DPRU861 (Ethiopia) clustered closely with DS-1 like Malawian strain (MAL88), as well as other globally circulating G2P[4] strains. Interestingly, the NSP4 and NSP3 genes of six African G12 strains were closely related to porcine strains and porcine-human reassortant strains detected in South Africa and Kenya. These findings provide evidence of interspecies transmission and occurrence reassortment events between human and porcine strains.


Development of a novel reverse genetics system for Rotavirus

Guido Papa (1), Francesca Arnoldi (2), Giuditta De Lorenzo (1), Gianluca Petris (3), Marco Bestagno (1), Oscar R. Burrone (1)

1: Molecular Immunology Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy; 2: Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy.;3: Centre for Integrative Biology (CIBIO), University of Trento, Laboratory of Molecular Virology, Trento, Italy.

Despite recent new developments on RV reverse genetics, most systems based on the use of a helper-virus require a selective pressure for the genome segment (gs) of interest. Our approach is based on the use of a selective marker (i.e. EGFP) that could in principle be used with any gs. A T7-driven viral-like RNA encoding EGFP fused to the viral protein of interest separated by the Tobacco Etch Virus protease (TEVp) cleavage site is expressed in virus infected cells.In vivoprocessing by TEVp (in an MA104-TEVp stable cell line) yields the fully functional viral protein and EGFP. Cells infected with a RV harbouring the recombinant segment and sustaining viral replication become EGFP positive and can be selected by FACS-sorting for EGFP expression and further cultured with fresh cells. Infection-sorting cycles were repeated several times to isolate reassortant RVs with only partial results and some difficulties in rescuing the recombinant viruses. As one possible limitation may be due to the low accessibility of the viroplasm to the cytosolic exogenous virus-like mRNA we developed a system to force its recruitment into these viral compartments, with the aim of enhancing availability for packaging.Our reverse genetics system can be applied to any genome segment, and eventually any virus, as long as the size of the recombinant gene is compatible with packaging into infective particles.


Impaired growth phenotype of recombinant rotaviruses expressing VP3 with a defective 2’,5’-phosphodiesterase (PDE)

Jin Dai (1), ASHA ANN Philip (1), Chantal A Agbemabiese (2), Guido Papa (3), John Patton (1)

1: Indiana University, Indiana, USA; 2: University of Ghana, Ghana; 3: International Center for Genetic Engineering and Biotechnology, Italy

Activation of the 2’, 5’-oligoadenylate synthetase (OAS) - ribonuclease (RNase) L pathway during viral infection results in the degradation of viral and cellular RNAs, creating an environment unfavorable for virus replication. Small RNAs generated by the OAS/RNase L pathway can further inhibit virus replication by upregulating interferon expression and triggering apoptosis. RNase L activity is induced by short 2’-5-oligoadenylate (2-5A) oligomers that are synthesized by OAS. Group A rotaviruses can counter the OAS-RNase L pathway through the action of a 2’,5’phosphodisterase (PDE) domain located at the C-terminus of the viral capping enzyme, VP3. The VP3 PDE active site includes two catalytic histidine residues: H718 and H797. To study the importance of the VP3 PDE in context of infection, we used a reverse genetics system to generate recombinant SA11 rotaviruses expressing VP3 with inactive PDE domains. This was accomplished by mutating H797 to alanine (SA11 PDE/H797A) or arginine (SA11 PDE/H797R). The fact that we could recover and grow the SA11 PDE/H797A and PDE/H797R mutant viruses demonstrates that the activity of the VP3 PDE domain is not essential for virus replication. However, because the growth of both mutant viruses was significantly impaired in comparison to wildtype virus (based on virus titer and plaque size), implies an important role for the VP3 PDE in suppressing antiviral innate immune responses. Interestingly, the mutant viruses retain the capacity to express NSP1 -the viral interferon antagonist - suggesting that the VP3 PDE suppresses an antiviral immune activity that is not dependent on interferon expression.


Modifications of the Rotavirus Plasmid-Based Reverse Genetics System

Asha A Philip (1), Jin Dai (1), Chantal A Agbemabiese (2), Guido Papa (3), Promisree Choudhury (1), Sarah Katen (1), John Patton (1)

1: Indiana University, Indiana, USA; 2: University of Ghana, Ghana; 3: International Center for Genetic Engineering and Biotechnology, Italy

In adapting the plasmid-based rotavirus reverse genetics (RG) system, we explored modifications that could simplify or improve recovery of recombinant viruses. Out of this effort evolved two RG protocols, one with support plasmids (RG+), the other without (RG-). Both protocols were developed using SA11 pT7 plasmids provided by Dr. Takeshi Kobayashi (Osaka Univ) and BHK-T7 cells provided by Dr. Ulla Buchholz (NIH). The protocol essentials are as follows. BHK-T7 cells, grown in GMEM containing 5% FBS, 10% tryptose-phosphate broth (TPB), 2% NEAA, 2% Geneticin, and 1% penicillin-streptomycin, are seeded into 12-well plates. One hour prior to transfection, media on the BHK-T7 cells is replaced with SMEM containing TPB and NEAA. Transfection mixtures are prepared with OptiMEM and Mirus TransLT-LT1 reagent and contain, for the RG+ protocol, 0.8 mcg each of the 11 SA11 pT7 plasmids, 0.8 mcg of pCMV-NP868R (ASFV capping enzyme), and 20 ng of pCAG-p10FAST (reovirus fusion protein), or, for the RG- protocol, 0.8 mcg of each SA11 pT7 plasmid, except pT7 (NSP2) and pT7 (NSP5), which are 2.4 mcg each. At 2 days posttransfection, the BHK-T7 cells are overseeded with MA104 cells in serum-free M199 media containing trypsin. At 5 days posttransfection, lysates prepared from the transfected cells are used to infect MA104 monolayers in 6-well plates. The MA104 cells are harvested 3-7 days later, depending on the extent of CPE, and analyzed by PAGE for viral dsRNA. These protocols have allowed routine recovery of recombinant viruses in our laboratory, including viruses with impaired growth phenotypes.


ROTAVIRUS INFECTION INDUCES ALTERNATIVE SPLICING OF THE STRESS-REGULATED TRANSCRIPTION FACTOR XBP1.

Mariela Duarte (1,2), Patrice Vende (1), Annie Charpilienne (1), Cécile Laroche (1), Didier Poncet (1)

1: I2BC CNRS INRA, France; 2: Université d’Evry, France

The Unfolded Protein Response is a final common pathway in the response of a broad variety of stresses disturbing the Endoplasmic Reticulum (ER). The transcription factor XBP1 is a major regulator of UPR. In the course of studying stress induced by rotavirus infection, we discovered a new XBP1 mRNA. This polyadenylated mRNA (XBP1-es) corresponds to a canonical nuclear splicing of XBP1 pre-RNA, that skips exon 4. XBP1-es encodes an Xbp1 protein that contains complete DNA-binding and activating domains. The XBP1-es was detected upon infection with several cell culture-adapted rotavirus strains (“ES+ strains”), but not with other -although cell culture-adapted and commonly used- strains (“ES- strains”). Identification of the viral gene that triggers this exon skipping by classical viral genetic using monoreassortants between ES+ and ES- strains demonstrate the involvement of gene 7 encoding NSP3. Swapping eIF4G- and RNA-binding domains of NSP3 from ES+ and ES- strain in recombinant rotaviruses shows the involvement of the eIF4G binding domain. ES+ and ES- phenotypes were also correlated with the efficency of nuclear relocalization of the cytoplasmic PABP during infection.Our results demonstrate that rotavirus infection perturbs the nuclear processing of XBP1 pre-RNA. This finding unravels a possible novel way to activate the transcription factor XBP1. As XBP1 is also involved in the innate immune response, our results raise the question of the role of this alternative splicing in the innate immune response.


Molecular characterization of rotavirus infections in children attending Dr George Mukhari Academic Hospital and Oukasie Primary Healthcare and their association with histo-blood group antigen profiles

Kebareng Rakau, Maemu Gededzha, Mapaseka Seheri

Department of Virology, Diarrhoeal Pathogens Research Unit, Sefako Makgatho Health Sciences University, South Africa

BACKGROUND: Recent studies have reported that human rotaviruses recognise histo-blood group antigen (HBGA) as host receptors. HBGA are oligosaccharides encoded by ABO, H, Secretor and Lewis genes. Rotavirus P[4] and P[8] bind the H antigen and Leb HBGA, while P[6] binds the H antigen only and are Lea and Leb negative. The predominance of P[6] in Africa leads to the speculation that the greater population of Africans are Lea and Leb negative. We investigated the association of rotavirus with HBGA in children attending Dr George Mukhari Academic Hospital and Oukasie Primary Healthcare.METHODS: In this cross-sectional study, 99 paired diarrheal stool and saliva samples were collected from children. Stool samples were tested for group A rotavirus using ProSpecTTM EIA. A cocktail of primers was used to characterize rotavirus into G and P types. The saliva samples were tested for Lea, Leb, H antigen and ABO antigens using specific monoclonal antibodies. RESULTS: Rotavirus was detected in 22.2% of stool samples. Rotavirus strains detected were G1P[8], G9P[8], G9P[6] and GxP[8]. The children were characterised as secretor 20.2%, nonsecretor 44.4% (Lea Leb negative) and partial 35.3% HBGA’s. Rotavirus P[6] infected children that were Lea Leb HBGA negative. Contradictory to literature rotavirus P[8] infected children possesing different HBGA types not only the Leb positive. CONCLUSION: The predominance of Lea and Leb negative HBGA phenotype in this study could explain the prevalence of P[6] in African children. The type of HBGA available on the cell during rotavirus entry influences the type of strain causing infection.


Molecular Characterization of Unusual Rotaviruses G12P[11] Strain Identified in Neonates Admitted at Neonatal Intensive Care Units (NICUs) : A study from Pune, Western India

Sujata Ranshing (1), Ashish Bavdekar (2), Umesh Vaidya (3), Manas Behera (4), Gopalkrishna Varanasi (1)

1: National Institute of Virology,Pune-411001, India; 2: Pediatric Research Unit, KEM Hospital Research Centre, Pune-411011, India; 3: Neonatal Intensive Care Unit, KEM Hospital Research Centre, Pune-411011, India; 4: Neonatal Intensive Care Unit and Pediatric Unit, Smt. Kashibai Navale Medical College and General Hospital, Pune 411041, India

Nosocomial infections represent a significant epidemiologic and economic problem worldwide. Rotaviruses (RV) cause infections in neonates that are in general nosocomial and differ both clinically and epidemiologically than pediatric group. These viral infections in neonates are predominantly asymptomatic and are often associated with unusual rotavirus strains which are different from those under circulation. So far very limited data is available on RVs in neonates admitted at NICU. The knowledge of RV strains circulating in neonates is important since these might affect the efficacies of rotavirus vaccines. The aim of the present study is to determine the prevalence of RV infections among neonates in NICUs and to characterize RV strains to understand their genotype distribution. Stool specimens (n=701) were collected from April 2016-March 2018 from 624 neonates admitted mainly for prematurity, low birth weight, associated respiratory distress syndrome sepsis, asphyxia, etc from two tertiary care Hospitals, Pune, western India. Stool specimens were tested for Group A rotavirus by ELISA and positive samples were subjected to multiplex RT-PCR for VP7 (G) and VP4 (P) genotyping. Representative strains of PCR amplicons were sequenced. Rotavirus positivity was detected in 24.92% of the clinical specimens analyzed. Majority of the neonates were asymptomatic (99%) for any diarrheal symptoms. Nearly 91.55% and 77.92% of the strains were typed for G and P genotypes respectively. The study highlights high frequency of unusual G12P[11] (Human-Bovine Reassortant) rotavirus strains among neonates for the first time in western India. Phylogenetic analysis of the partial VP7 coding gene revealed G12 strains in lineage III.


Detection of Rotavirus NSP3 interactions during infection

Carlos Sandoval-Jaime, Carlos Arias, Susana López

Instituto de Biotecnología UNAM, Mexico

Viruses are obligate intracellular parasites, and therefore are dependent on the translational machinery of infected cells for the production of their own viral proteins and their viral genome. The success of these processes needs on first instances of the viral mRNA and the cellular translational machinery interaction, which at the end results in the shutdown of host cell translation. In the case of rotavirus, the protein responsible for taking over the control of protein synthesis is NSP3. Several methodologies have revealed that NSP3 is capable of interacting with various cellular proteins (eIF4G, RoXaN, Hsp90). However, the strategies used in those experiments were not been designed to detect the possible interactions of NSP3 during infection, so those patterns of interaction of NSP3 with viral proteins are unknown. Identify these interactions will allow us understanding the different protein expression phenotype observed in the presence of NSP3. We perform bio-identification assays (BioID) using the NSP3 as bait fused to a promiscuous biotin ligase (BirA) in infected cells. With this technique, biotinylation takes place only in proteins that are at close proximity of the NSP3-BirA fusion. Our technique has allowed the identification of differential biotinylation patterns on infected and uninfected cells transfected with our construction. Further experiments will be performed to determine the nature of those differences.


Investigating the interaction of the viral inclusion bodies formed during African horse sickness virus infection with the host protein synthesis apparatus

Litia Yssel (1), Eudri Venter (2), Vida van Staden (1)

1: Department of Genetics, University of Pretoria, Pretoria, South Africa; 2: Laboratory for Microscopy and Microanalysis, University of Pretoria, Pretoria, South Africa

African horse sickness virus (AHSV) is transmitted by arthropods and causes fatal disease in horses, with major economic implications in Africa. Shortly after infection, AHSV induces the formation of cytoplasmic viral inclusion bodies (VIBs) by aggregation of non-structural protein NS2. The VIBs form the sites of viral genome replication and assembly of progeny virions. It remains to be elucidated which strategy AHSV employs to promote viral protein synthesis whilst suppressing host translation. This study investigated whether components of the host translation apparatus are sequestered to the VIBs, thereby becoming the sites of viral protein production. Immunolabelling and confocal laser scanning microscopy were used to characterize the VIBs both in AHSV-infected BSR cells and in Sf9 insect cells expressing NS2 from a recombinant baculovirus. This was followed by a ribopuromycylation assay to investigate the sites of active protein synthesis. We showed that in AHSV-infected cells a component of the 60S large ribosomal subunit, the ribosomal protein L11, shifted from a diffuse cytoplasmic distribution at 8 hours post infection (h pi) to colocalize with NS2 in the VIBs from 24 h pi onwards. L11 also localized to the VIB-like structures in the Sf9 cells. The puromycin labelling also colocalized extensively with the NS2 signal in AHSV-infected cells, indicating the presence of actively translating ribosomes within the VIBs. These results provide the first evidence that orbiviruses like AHSV might actively sequester ribosomes to the VIBs to translate viral proteins, thereby subverting host translation mechanisms to confer a replicative advantage to the virus.


WORKSHOP 4: IMMUNITY AND PATHOGENESIS

Asymmetric recirculation of rotavirus-specific memory B cells induced by intra-rectal immunization with 2/6-virus-like particles (2/6-VLPs) throughout the gut-associated lymphoid tissues.

Davide Agnello, Damien Denimal, Amandine Lavaux, Cecile Pitoiset, Pierre Pothier, Alexis de Rougemont

Laboratoire de Virologie et Centre National de Référence des Virus Entériques, Centre Hospitalier Universitaire de Dijon, Dijon, France

We previously showed that intra-rectal immunization with rotavirus 2/6-virus-like particles (2/6-VLPs) protects adult mice against rotavirus infection and induces a higher rotavirus-specific IgA production in the small intestine, in comparison with immunization by the intra-nasal route. Indeed rotavirus-specific IgA antibody-secreting cells (ASCs) induced by intra-rectal immunization with 2/6-VLPs express the integrin α4β7, which enables them to bind to the addressin MAdCAM-1 and migrate to the gut.Here we investigated if also rotavirus-specific memory B cells (MBCs) express different homing receptors and localize into different tissues, when generated by immunization with 2/6-VLPs administered through different routes. Whereas only ~40% of 2/6-VLP-specific MBCs induced by intra-nasal immunization express α4β7, virtually all MBCs induced by intra-rectal immunization are α4β7+. Conversely, MBCs induced by intra-rectal immunization lack the lymph-node-homing receptor L-selectin, which is instead expressed by MBCs induced by intra-nasal immunization. Therefore, rotavirus-specific MBCs induced by intra-rectal immunization preferentially recirculate among the gut-associated lymphoid tissues (GALTs), such as Peyer’s patches (PPs) and mesenteric lymph-nodes (MLNs), which express MAdCAM-1, but are instead largely excluded from extra-intestinal lymph-nodes, whereas intra-nasal immunization induces a significant number of 2/6-VLP-specific MBCs also in peripheral lymph-nodes. Consistently, adoptive transfer of antigen-specific MBCs from β7-sufficient mice, previously immunized with 2/6-VLPs by the intra-rectal route, generates rotavirus-specific ASCs in MLNs, PPs and spleen of recipient mice, after oral challenge with rotavirus. On the contrary, rotavirus-specific ASCs are detectable only in the spleen of recipient mice transferred with β7-deficient MBCs, underscoring the importance of this asymmetric recirculation of α4β7-expressing MBCs throughout the GALTs.


Intracellular neutralisation of rotavirus by antibodies

Sarah Caddy, Leo James

MRC-Laboratory of Molecular Biology

Antibodies targeting the rotavirus protein VP6 are highly prevalent in polyclonal antibody responses to rotavirus infection. However, as VP6 is only exposed once rotavirus particles enter cells and uncoat the outer capsid to become double-layered particles (DLPs), the mechanisms by which anti-VP6 antibodies work is uncertain. Despite this, anti-VP6 antibodies are protective in mice, indicative of a functional role. This project therefore sought to examine how anti-VP6 antibodies prevent rotavirus infection.To confirm that anti-VP6 antibodies are able to neutralise intracellularly, a novel assay was developed whereby anti-VP6 antibodies were introduced into the cytoplasm of cells using NeonTM electroporation. This enabled colocalisation of anti-VP6 antibodies with DLPs. Antibody electroporation resulted in neutralisation of several rotavirus strains in multiple cell lines.Results showed that intracellular neutralisation was independent of the intracellular antibody receptor TRIM21, the proteasome and the autophagosome. This suggested that a cell-free assay could be a valuable means of testing VP6-targeted neutralisation. This was verified using an in-vitro transcription (IVT) assay; purified DLPs were incubated with antibodies, then mRNA production measured. Results for both the electroporation and IVT assays were highly comparable for a panel of monoclonal and polyclonal antibodies.Future work aims to determine whether the presence of antibodies that neutralize intracellularly can be correlated with susceptibility to rotaviral disease. This is to be investigated firstly using a mouse model of rotavirus infection, and secondly by testing serum from an ongoing Rotarix vaccine trial. It is anticipated that intracellular neutralisation could be a novel correlate of protection.

243Poster presentations Workshop 4: Immunity and Pathogenesis

Identification of Interferon Stimulated Genes That Restrict Reovirus Infection

Tanner Gill, Geoffrey Holm

Colgate University, USA

Mammalian reovirus is a common non-pathogenic model utilized to study the important interactions between viral infection and the cellular immune response. The innate immune response to reovirus involves the production of type 1 interferons (IFNs), which ultimately results in the activation of specific subsets of interferon stimulated genes (ISGs) with unique antiviral effects. While some antiviral ISGs have been extensively characterized for reovirus, many yet remain unidentified. Therefore, we used an overexpression flow cytometry screen to identify ISGs that restrict reovirus infection. Overall, 22 candidate ISGs were selected based on microarray expression data from reovirus-infected cells and prior identification as having an antiviral effect on other RNA viruses. These candidates, including two positive controls (IFITM3 and CH25H), were expressed via GFP-fusion plasmids, and their effect on reovirus infection was analyzed by flow cytometry. From this screen, five ISGs, including both positive controls, were found to significantly decrease reovirus T3D infection: IFITM3, CH25, XAF1, ADAP2, and C19orf66. Ongoing studies include fluorescence microscopy analysis of the newly identified anti-reoviral ISGs to determine their cellular localization pattern, and functional analyses to determine the stages of the reovirus replication cycle that are affected by ISG activity.


Characterization of molecular targets for differential regulation of the type I and III interferon induction and signalling pathways by rotavirus NSP1

Gennaro Iaconis (1,2), Miren Iturriza-Gomara (2), Mark Boyce (3), Steve Goodbourn (4), Neil Blake (2), Ben Jackson (1), Julian Seago (1)

1: The Pirbright Institute; 2: University of Liverpool; 3: Oxford University; 4: St Gerorge’s, University of London

Rotavirus (RV) is the leading cause of severe dehydrating diarrhoea in infants infecting almost every child by 3-5 years of age and causing approximately 590,000 gastroenteritis-associated deaths worldwide. The virus is ubiquitous in all mammalian species and cross-species infections have been reported. However, RV is host restricted during heterologous infection for reasons that are not fully understood.Type I and type III interferons (IFNs) constitute the first line of defence against viral zoonotic infection. They are expressed when specific viral components, such as dsRNA, are detected within the cells. Once secreted, IFNs bind their receptors and induce the expression of interferon-stimulated genes, establishing an antiviral state.RV NSP1 is able to subvert IFN induction and signalling in a strain-specific manner, which may be linked to the observed host-range restriction. To establish if host-range restriction is related to the function of NSP1, the yeast-2-hybrid system was used to investigate the interaction of different components of the host IFN induction pathway with NSP1 proteins from different RV strains. In addition, the ability of NSP1 to (a) block induction of type I and III IFN and (b) to antagonise downstream IFN signalling has been investigated using a series of luciferase reporter assays. I present data showing NSP1 does exhibit species-specific interactions, down regulation of NF-κB activity and inhibition of IFN production.


Investigating the role of akirin, a conserved transcription factor of arthropods, in the pathogenesis of C. sonorensis derived KC cells caused by African Horse Sickness Virus infection

Fidan Karatas, Wilma Fick

University of Pretoria, South Africa

Culicoides midges act as transmission vectors of economically important arboviruses, including African horse sickness virus (AHSV), a double-stranded RNA virus causing one of the most serious diseases of equids. Virus infection causes severe cytolytic effects in mammalian cells, however, infection is persistent in midges with cells showing no cytopathic effects. The ability of the virus to replicate and disseminate from the midge during blood-feeding stands central to the vector capacity of the midge. Vector-based control strategies targeting genes with vital roles in the midge life-cycle and limits virus transmission requires an understanding of the virus-vector interphase and identification of suitable candidate genes. One such candidate is akirin, aconserved transcription factor involved in blood meal digestion, reproduction and development of arthropods. Studies in ticks and mosquitoes have indicated that akirin also plays a role in the innate immune response to viral infections. Based on the hypothesis that akirin performs similar functions in Culicoides, our study aims to investigate the role of akirin in midges and its effect on AHSV replication using an RNAi-based knockdown approach. Akirin-specific knockdown was performed in C. sonorensis-derived KC cells, a cell line previously reported to mount an anti-viral RNAi response that inhibited bluetongue virus infection. We present our results on dsRNA-triggered inhibition of the akirin gene in the absence or presence of AHSV. Fluorescence and confocal microscopy was used to confirm delivery of dsRNA molecules and tracking its cellular location. Expression levels of cellular genes were measured using qRT-PCR and viral proteins by western blotting.


Bat MRV isolate SI-MRV01 dissemination after oral inoculation of neonatal BALB/c mice

Tina Mikuletič (1), Tanja Švara (2), Mitja Gombač (2), Marko Kolenc (1), Andrej Steyer (1)

1: University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia; 2: University of Ljubljana, Veterinary Faculty, Ljubljana, Slovenia

Recently, mammalian orthoreoviruses (MRVs) were detected for the first time in European bats, and the closely related strain SI-MRV01 was isolated from a child with severe diarrhoea in Slovenia. The aim of our study was to elucidate the pathogenetic potential of this strain in mouse model and to test whether virus spreads to organs from the oral entry site.The 3-4 days old suckling mice BALB/c were orally inoculated with 105 TCID50 of bat MRV isolate SI-MRV01. Mice were weighed daily and observed twice a day for clinical signs of disease. Upon the onset of critical symptoms the mice were euthanized with CO2. Organs (spleen, small intestine, thymus, lung, liver, brain, heart and kidney) were collected and prepared for (i) molecular analysis using specific bat MRV real-time RT-PCR, (ii) histopathological studies, (iii) ultrathin sections for electron microscopy and (iv) virus titration.The onset of symptoms (weight loss, coordination disturbance) appeared in average after 8 days post infection. The molecular analysis revealed virus RNA in all tested organs with brain, heart and lung showing the lowest ct value. The relative increase in virus concentration was up to 4 log10 in brain and up to 3 log10 in lungs and heart. The electron microscopy of ultrathin sections showed disseminated viral particles in brain, heart and lungs.Our results confirmed that strain SI-MRV01 disseminates from gastrointestinal tract to organs. The most affected organ eight days post infection was brain, which indicates viral neurotropism and the potential of its dissemination.


CHOICE OF VACCINE AND STUDY OF THE CAREFUL USE OF PHARMACY DURING THE INTRODUCTION OF ROTAVIRUS VACCINE IN DRC

Médard NGOY KILUBA

Pediatric Kalembelembe hospital, Democratic Republic of the Congo

INTRODUCTION: A feasibility study has been initiated in August 2009 by the extended program of vaccination in the Democratic Republic of Congo in collaboration with some partners to evaluate the scale of the Rotavirus diarrhoea in the children under five years. METHODOLOGY: We carried out a retrospective study of 6 last years on the cases of intestinal invagination taken into care by the Pediatric Hospital of Kalembe Lembe RESULTS: From 2012 to 2017, it comes to a total of 1,240 cases treated in surgery, 115 cases of acute intestinal invagination that is 9 % made known, 107 surgical operations, 46 cases of death i.e. 40%.Although these vaccines are not scheduled for vaccinal dates, but they are marketed in DRC. The noticeable causes of intestinal invaginations are: cleaning cleaning of the intestines from local products 41 cases with 13 deaths; congenital malformation 16 cases with 8 deaths; 34 cases with 11 deaths cused by food; typhoid fevers 11 cases with 3 deaths; other cases remain unknown without any cause 13 cases with 11 deaths. No case made known after using Rotavirus vaccine. CONCLUSION: The intestinal invagination turned out to be rare adverse reactions, but significant, anti rotavirus vaccine of the first generation. New Rotarix and Rota Teq vaccines were each one another the subject of study of great scope designed to exclude a risk of intestinal invagination similar to that observed with the RotaShield.


Evaluation of recombinant Newcastle Disease Viruses (NDV) as candidate vaccine delivery vectors for rotavirus VP7 and NSP4 in mice

Larise Oberholster (1), Jasmin Aschenbrenner (1,2), Christiaan Potgieter (3,4), Hester Gertruida O’Neill (1)

1: Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa; 2: School of Life Sciences, Technical University of Munich, Germany; 3: Delatmune (Pty.) Ltd., Lyttelton, Centurion, South Africa; 4: Department of Biochemistry, North-West University, Potchefstroom, South Africa

There is mounting evidence for interspecies transmission of rotavirus (RV) from animals to humans which contribute to RV strain diversity and emphasises the need for a One Health approach in RV control. The development of the Newcastle disease virus (NDV) reverse-genetic system has made it possible to use attenuated NDV (La Sota) as a vaccine vector in non-avian species. Because of natural host-range restrictions, there is no virus spread rendering the use of attenuated NDV in mammals inherently safe. NDV is antigenically distinct from common animal and human pathogens and will thus not be affected by a pre-existing immunity. In this study, the ability of RV outer capsid protein, VP7, and the enterotoxin, NSP4, to induce immune responses in mice upon delivery with recombinant NDVs expressing these proteins, was evaluated. Using the genome sequence of South African group A bovine RV, recombinant NDVs were engineerd to express the bovine RV VP7 and NSP4 proteins. Protein expression was confirmed using immunofluorescent monolayer assay (IFMA) and western blot analysis. The ability of the recombinant NDVs to elicit an immune response was evaluated in laboratory-bred adult mice. Mice were vaccinated twice, and blood samples were collected 3 weeks after each immunization. Seroconversion was determined using virus neutralizing assays, enzyme-linked immunosorbent assays (ELISAs) and immunofluorescent monolayer assays (IFMAs). Results obtained following the evaluation of the recombinant NDVs in mice will be presented.


CD8 T CELL RESPONSES TO AN IMMUNODOMINANT EPITOPE WITHIN THE NON-STRUCTURAL PROTEIN NS1 PROVIDES WIDE IMMUNOPROTECTION AGAINST BLUETONGUE VIRUS IN IFNAR(-/-) MICE.

Alejandro Marin-Lopez (1), Eva Calvo-Pinilla (1), Diego Barriales (2), Gema Lorenzo (1), Sergio Utrilla-Trigo (1), Ruyman Alonso-Ravelo (1), Alejandro Brun (1), Juan Anguita (2), Javier Ortego (1)

1: INIA-CISA, Spain; 2: CIC bioGUNE, Spain

The development of vaccines against Bluetongue, a prevalent livestock disease, has been focused on surface antigens that induce strong neutralizing antibody responses. Because their antigenic variability, these vaccines are usually serotype restricted. We now show that a single highly conserved non-structural protein, NS1, expressed in a modified vaccinia Ankara virus (MVA) vector can provide multiserotype protection in IFNAR(-/-) 129 mice against Bluetongue virus that is largely dependent on CD8 T cell responses. We found that the protective antigenic capacity of NS1 resides within the N-terminus of the protein and is provided in the absence of neutralizing antibodies. The protective CD8 T cell response requires the presence of a specific peptide within the N-terminus of NS1, since its deletion ablates the efficacy of the vaccine formulation. These data reveal the importance of the non-structural protein NS1 in CD8 T cell-mediated protection against multiple BTV serotypes when vectorized as a recombinant MVA vaccine.


CROSS-PROTECTIVE IMMUNE RESPONSES AGAINST ASHV AFTER VACCINATION WITH AVIAN REOVIRUS muNS MICROSPHERES AND MODIFIED VACCINIA VIRUS ANKARA

Eva Calvo-Pinilla (1), Alejandro Marín-López (1), Natalia Barreiro-Piñeiro (2), Diego Barriales (3), Sergio Utrilla-Trigo (1), Ruyman Alonso-Ravelo (1), Javier Benavente (2), Alejandro Brun (1), José Manuel Martínez-Costas (2), Javier Ortego (1)

1: INIA-CISA, Spain; 2: CIQUS-Universidade de Santiago de Compostela, Spain.; 3: CIC bioGUNE, Spain

African horse sickness virus (AHSV) is an insect-borne pathogen that cause acute disease in horses. In an effort to improve the safety and cross-protective immunity of current available vaccine, new generation vaccines have being developed. In this work we have generated and tested novel immunization approaches comprised of reovirus muNS protein microspheres containing proteins VP2 or NS1 of AHSV-4 (MS-VP2/NS1) and modified vaccinia virus Ankara (MVA) as delivery antigen vector expressing the same antigens (MVA-VP2/NS1). IFNAR (-/-) mice immunized with MS-VP2/NS1 or prime-boost MS-MVA-VP2/NS1 vaccination were protected against AHSV-4 infection. Immune responses triggered by MS-MVA-VP2/NS1 regime were higher than the homologous vaccination, with a significant difference in the level of neutralizing antibodies (PRNT50 = 1.75 and 1.05 respectively). Moreover the amount of NS1 specific cytotoxic CD8+T cells was also significant higher after heterologous vaccination.A polyvalent vaccination against AHSV would need to induce protective immunity across all nine serotypes. Homologous vaccination with MS-NS1 or MVA-NS1 reduced clinical signs and viraemia with a partial protection against AHSV-4. The average of viral load was ten times lower in vaccinated than in non-vaccinated animals. Interestingly, prime boost vaccination based on MS-NS1 and MVA-NS1 prevented AHSV infection after challenge with homologous (AHSV-4) and heterologous (AHSV-9) viruses. No viraemia or clinical disease were observed in immunized animals. These results suggest that MS-NS1/MVA-NS1 is a promising vaccine candidate against multiple serotypes of AHSV.


A complement factor NcTEP3 transcriptional regulated by Toll pathway inhibits the RDV infection of leafhopper by inducing antimicrobial peptides

Rui Qiao, Yu Wang

Peking university, China, People's Republic of China

The leafhooper (Nephotettix cincticeps) is a rice insect that transmits plant viruses, and causes more serious enconomic losses. Rice dwarf virus (RDV), which belongs to the genus Phytoreovirus in the family Reoviridae, is one of the most widespread and disastrous rice-infecting virus causing great yield losses in Aisa. Up to now, our knowledge about leafhopper transmiting viruses still remains little.Insects lack an immunoglobulin-based adaptive immune response. Thus, the innate immune system of insects plays a central role in the antiviral processes. Under the condition of viral infection, the innate immune system in leafhopper is regulated to against RDV infection. The complement system of insect is much conserved and similar to the mammal, acts as a key factor in connecting of humoral immunity and cell-mediated immunity and functions during the early phase of infection and directly mediates pathogen elimination. However, the component of complement system is not clear and poorly understood in insect. Based on the transcriptomics sequencing, three complement factors, TEP1, TEP2 and TEP3, are annotated in leafhopper. DsRNA silencings of NcTEP1, 2 and 3 indicate that only NcTEP3 shows a role of antiviral defense. NcTEP3 is regulated by a Toll signaling pathway factor NcDrosal, which is induced to a higher expression upon RDV infection. Furthermore, NcTEP3 directly interacts with the outer capsid protein P2 of RDV, resulting in an induction of Toll pathway produced antimicrobial peptides NcDenfein1 and NcDefensin3, which were known for inactivating and restraining the viral transmission.


Modulation of Neonatal Rotavirus Infection by Interplay between Human Milk Oligosaccharides, Milk Microbiome and Infant Gut Microbiome

Sasirekha Ramani (1), Christopher Stewart (1), Daniel Laucirica (1), Nadim Ajami (1), Liya Hu (1), B. V. Venkataram Prasad (1), Lars Bode (2), Gagandeep Kang (3), Mary Estes (1)

1: Baylor College of Medicine, USA; 2: University of California, San Diego, USA; 3: Christian Medical College, India

Bioactive molecules in breast milk, such as human milk oligosaccharides (HMOs), are key players for protection of the newborn infant from infectious diseases and for healthy intestinal and immunological development. HMOs are known to act as decoy receptors for several enteric pathogens. We used multidisciplinary approaches including infectivity assays, nuclear magnetic resonance, HMO profiling and 16S rRNA gene sequencing of samples from 167 mother-infant pairs to evaluate whether HMOs are decoy receptors for rotavirus strain G10P[11] that shows an almost exclusive tropism for neonates, and to determine whether differences in HMO profiles explain differences in clinical outcomes between neonates. Infectivity assays showed that, contrary to the effect observed for many enteric pathogens, HMOs were not decoy receptors for G10P[11] rotavirus in vitro. Population studies showed significantly higher levels of HMOs Lacto-N-tetraose (LNT), 2’-fucosyllactose (2’FL), and 6’-siallylactose (6’SL) in milk from mothers of rotavirus-positive neonates presenting with gastrointestinal symptoms, compared to mothers of asymptomatically infected or rotavirus-uninfected neonates. The levels of these HMOs also correlated with distinct differences in maternal milk and infant stool microbiome of the symptomatic group, characterized primarily by the increased abundance of Enterobacter/Klebsiella. LNT and 2’FL also improved the infectivity of Rotavac, an asymptomatic neonatal G9P[11] vaccine in vitro, offering new insights into maternal factors that could promote the performance of live, attenuated vaccines. Molecular and translational insight into the interplay between HMOs, milk microbiome, and infant gut microbiome provided a new perspective on host-microbial interactions and pathogenesis of neonatal infections, with potential implications for rotavirus vaccines.


Determination of rotavirus binding bacteria by fluorescence-activated cell sorting and next generation sequencing.

Jesús Rodríguez-Díaz (1), Antonio Rubio-del-Campo (1), Javier Buesa (1), Roberto Gozalbo-Rovira (1), Susana Vila-Vicent (1), Cristina Santiso-Bellón (1), Delgado Susana (2), Natalia Molinero (2), Abelardo Margolles (2), Vicente Monedero (3), Maria Carmen

1: Department of Microbiology, Faculty of Medicine, University of Valencia, Spain; 2: Institute of Dairy Products of Asturias (IPLA-CSIC); 3: Department of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC),

Gut microbiota has emerged as a key factor in the infection of intestinal viruses including enteroviruses, noroviruses and rotaviruses. In the present work we aimed to determine which members of the gut microbiota physically interact with rotavirus in children.To achieve our objective five stool samples from kids suffering diarrhea produced by G1P[8] rotavirus were selected and their microbiotas were filtered and purified using differential centrifugation. The resulting bacteria were incubated with FITC-labelled anti rotavirus antibody and separated by fluorescence-activated cell sorting (FACS). After sorting, fluorescent and non-fluorescent microbial sorted-populations were determined by sequencing the V3-V4 region of the 16S rDNA gene using Illumina technology.Ruminococcus genus were the best rotavirus binders with a ratio of fluorescent to non-fluorescent bacteria >6. Other genera including Oxalobacter, Pseudomonas and Pediococcus presented a ratio >2.We further characterized the Ruminococcus-human rotavirus interaction using two Ruminococcus gauvreauii strains and the G1P[8] rotavirus Wa strain. Binding of this rotavirus to the two R. gauvreauii strains was confirmed by fluorescent microscopy and transmission electron microscopy.These data, together with our previous findings showing a negative correlation between Ruminococcus and antibody titers to rotavirus and norovirus in healthy individuals, make us to propose a pivotal physical interaction between this bacterial group and human rotaviruses. Furthermore, they reveal a likely mechanism on how Ruminococcus could negatively affect rotavirus infection and open new possibilities for anti-rotaviral strategies.


Protein deficiency reduces the efficacy of an oral attenuated human rotavirus vaccine in a human infant fecal microbiota transplanted gnotobiotic pig model

Linda J. Saif (1), Ayako Miyazaki (1,3), Sukumar Kandasaamy (1), Husheem Michael (1), Stephanie Langel (1), Francine Paim (1), Juliet Chepngeno (1), Moyasar Alhamo (1), David Fischer (2), Huang-Chi Huang (1), Vishal Srivastava (1), Dipak Kathayat (1), Lo

1: The Ohio State University, Food Animal Health Research Program, VPM Dept, OARDC, CFAES, Wooster, Ohio USA; 2: University of Detroit Mercy, School of Denistry, Detroit, Michigan, USA; 3: National Institute of Animal Health, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan

The low efficacy of rotavirus (RV) vaccines in developing countries, where malnutrition is prevalent, remains a major concern for global health. To understand the effects of protein malnutrition on RV vaccine efficacy, we elucidated the innate, T cell and cytokine immune responses to attenuated human RV (AttHRV) vaccine and virulent human RV (VirHRV) challenge in germ-free (GF) pigs or human infant fecal microbiota (HIFM) transplanted gnotobiotic (Gn) pigs. All pigs were fed protein-deficient or -sufficient bovine milk diets. We analyzed serum levels of tryptophan (TRP) and its metabolite, kynurenine (KYN) that are important in immune regulation. In the protein deficient, compared with sufficient pigs, we observed altered serum IFN-α, TNF-α, IL-12 and IFN-γ responses to the oral AttHRV vaccine. Post- VirHRV challenge, the protein-deficient vaccinated pigs had: 1) Lower protection rates against diarrhea and significantly increased virus shedding titers (HIFM transplanted, but not GF pigs); 2) Suppression of multiple innate immune parameters and HRV-specific IFN-γ producing T cells; and 3) Decreased serum KYN levels, suggesting that altered TRP metabolism negatively affected immune responses. Collectively, our findings confirm the negative effects of protein deficiency (exacerbated in HIFM transplanted pigs with decreased Firmicutes-to-Bacteroides ratios), on innate, T cell and cytokine immune responses to HRV and on vaccine efficacy, on TRP-KYN metabolism and on gut microbiota. Our novel insights into the immunological impairment and reduced efficacy of a HRV vaccine in a protein deficient, HIFM pig model may be applicable to the reduced effectiveness of oral vaccines in malnourished children in developing countries.


The effect of supplementation of fatty acids, with varying degrees of saturation, on rotavirus yield and replication in MA104 cells

Wico Sander, Trudi G O’Neill, Carlien H Pohl

Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa

Rotavirus (RV) replication is dependent on the formation of viroplasms, which associate with lipid droplets (LDs). It was shown previously that this association is essential for RV replication. LD functions include triglyceride storage, protein transport and eicosanoid synthesis. Changes in fatty acid (FA) composition of LDs can affect these functions. Therefore, the effect of supplementation of FAs, with varying degrees of saturation, on RV infection was evaluated.MA104 cells were grown for 24h in DMEM, supplemented with 50 μM of FAs. The effect of supplementation on RV SA11 was monitored by determining the rate of viral replication with growth studies, followed by TCID50 titrations; the effect on viral yield using TCID50 titrations 16h post infection and the effect on viral RNA yield was monitored by qRT-PCR 16h post infection. The production of the pro-inflammatory eicosanoid, prostaglandin E2 (PGE2), was determined by ELISA.Results showed that the unsaturated FAs [oleic acid (18:2), γ-linolenic acid (18:3), sciadonic acid (SCA) (20:3)] increased the rate of viral replication during the first 24h. However, the saturated FA [stearic acid (18:0)] did not have an influence on the rate of replication. PGE2 results showed that RV infection of unsupplemented cells caused an increase in PGE2 production. In addition, supplementation with 18:3, a precursor for arachidonic acid (substrate for PGE2 production) showed a further increase in PGE2 production during RV infection. Data will be discussed in context with the metabolised FA profiles as determined by gas chromatography.


Rotavirus regulates the STAT1 antiviral response in infected and uninfected bystander cells by two discrete mechanisms.

Adrish Sen (1,2), Harry Greenberg (1,2)

1: Depts of Medicine, Molecular Microbiology & Immunology, School of Medicine, Stanford University, California, USA; 2: VA Palo Alto, Palo Alto, California, USA

Several interferon (IFN) types, by activating the Signal Transducer and Activator of Transcription 1 (STAT1) protein, substantially restrict the replication and pathogenicity of heterologous RV strains (i.e. RVs isolated from a non-native host species). We previously demonstrated that RVs potently inhibit host STAT1 responses to various IFNs in both RV-infected (i.e. RV antigen-positive, RV+) and uninfected “bystander” (i.e. RV antigen-negative, RV-) cells. We also showed that during RV infection, endogenous type I, II, and III IFN receptors (IFNRs) are efficiently degraded exclusively in RV+ infected cells, coincident with STAT1 inhibition. Our new findings demonstrate that RV NSP1 is responsible for degrading IFNRs and delineate a novel domain involved in NSP1 stability and the degradation of IFNRs.Interestingly, RV also inhibits IFN-directed STAT1-Y701 phosphorylation in bystander cells. In contrast to RV+ cells, bystanders do not undergo IFNR degradation during infection indicating the existence of a second RV strategy to inhibit STAT1 signaling in uninfected cells. We now demonstrate that RV infection induces STAT3 phosphorylation in vitro, which is required for the RV ability to spread to adjacent uninfected cells. In suckling mice, differences in intestinal STAT3-pY705 activation and STAT3-induced canonical transcriptional responses correlate with contrasting STAT1-dependent replication phenotypes of the homologous murine EW versus the heterologous simian RRV RV strains. These findings reveal a novel role for STAT3 in the replication and intestinal restriction of RVs that is likely related to RV inhibition of STAT1 in bystander cells.


A novel vaccine approach for insect borne diseases; Disabled Infectious Single Animal (DISA) vaccine for Bluetongue

Piet A. van Rijn (1,2), Femke Feenstra (1), Rene G.P. van Gennip (1)

1: Wageningen BioVeterinary Research, the Netherlands; 2: North-West University, Potchefstroom, South Africa

Insect borne bluetongue virus (BTV) (Orbivirus, Reoviridae) is circulating in large parts of the world corresponding to transmission by specific species of Culicoides biting midges. Currently, >28 serotypes have been identified. Today, BTV is a serious threat for formerly BT-free countries in moderate climate conditions. Conventionally live-attenuated vaccines (LAVs) can have shortcomings associated to safety issues and DIVA incompatibility. We have developed BTV deficient for NS3/NS3a protein which is grown on standard cell lines guaranteeing cost-competitive vaccine production. BTV without NS3/NS3a is completely safe (not virulent, no viremia, and consequently no uptake by midges). Safety is further augmented as virus propagation in midges is abolished (no spread by midges), and is named BT Disabled Infectious Single Animal (DISA) vaccine. DISA vaccination results in rapid, complete, serotype specific protection, and enables differentiating infected from vaccinated animals (DIVA) based on PCR diagnostics and NS3 based ELISAs. The immune reaction is robust as shown for smaller vaccine dose and different vaccination routes. The BT DISA vaccine platform has been applied for many serotypes by exchange of serotype immunodominant outer shell proteins. BT DISA vaccines can be used as mono-serotype vaccine but also allows safely combined cocktails to achieve broad protection or in a tailor-made fashion to control specific multi-serotype field situations. BT DISA vaccines will be tested in cattle as well as for cocktail formulations within the EU-funded project PALE-Blu. BT DISA vaccines are the solution to combat Bluetongue in a cheap, safe, effective and controllable program for different field situations.


Bioinnovo IgY DNT: towards the end of newborn calf diarrhea

Celina Guadalupe Vega (1), Marina Bok (1), Maren Ebinger (2), Lucia Rocha (1), Verónica Pinto (3), Christian Knapp (3), Alejandra Antonella Rivolta (1), Viviana Parreño (1), Andrés Wigdorovitz (1)

1: INTA, Argentine Republic; 2: El Mangrullo Farm; 3: Vetanco S.A.

Neonatal calf diarrhea is one of the main causes of mortality during the first weeks of life. A new product based on IgY antibodies was launched to market as Bioinnovo IgY DNT. It is enriched in IgY antibodies to Rotavirus A –RVA, Coronavirus, Escherichia coli and Salmonella sp. We present the results of the official field trial of IgY DNT performance against natural RVA-associated diarrhea in newborn dairy calves under artificial rearing in a dairy farm named El Mangrullo in Buenos Aires Province, Argentina. Thirty newborn Holsteins calves were randomly assigned to IgY DNT-treated (IgY) or negative control group. Treated animals received daily 20 g of Bioinnovo IgY DNT twice a day together with milk during the first 14 days of life. The IgY group showed 20% reduction in diarrhea morbidity together with a delay in one day in diarrhea onset and a lower severity when compared with control animals. Calves receiving IgY had a significant reduction in diarrhea duration when compared with control group, from 4.64 to 1.53 days. RVA was the only infectious agent detected in this study, with similar incidence in both groups (47%). IgY-treated animals showed a delay in the onset of RVA shedding and the average duration of RVA shedding was significantly shorter and of lower titers than control group, as calves in IgY group shed RVA for only one day and it was associated with diarrhea only in 29% (2/7) of the cases. This demonstrates the beneficial impact of IgY DNT against RVA diarrhea in calves.


Pre-vaccination rotavirus serum IgA and IgG, and stunting are associated with low rates of seroconversion to Rotarix® at 1 year of age among Bolivian infants

Daniel E Velasquez Portocarrero (1), Paulina Rebolledo (2), Umesh Parashar (1), Baoming Jiang (1), Juan Leon (2)

1: Centers for Disease Control and Prevention, Atlanta, GA, USA; 2: Emory University, Atlanta, GA, USA

Induction of persistent levels of serum IgA against rotavirus (RV) is an important goal for childhood immunization.Infants (N=168) recruited in El Alto, Bolivia, received two doses of Rotarix® at 9 [±1] and 19 [±2] (mean age in weeks [SD]). Blood specimens were obtained at 9 [±1] (before dose 1), 29 [±3], and 53 [±3] weeks of age. Serum RV-specific IgA and IgG was measured by enzyme-linked immunosorbent assays. Seroconversion was defined as a ≥ 4 fold rise in RV-IgA titer at the second or third blood draw (at 1 year of age) compared to the first draw.RV-IgA seroconversion was 63% (106/168) at second blood draw and 83% (139/168) at 1 year of age. Infants that seroconverted at the second blood draw had a higher rate of seroconversion at 1 year of age compared with infants who did not [(92% (98/106) vs 66% (41/62), p<0.001]. Infants who had RV-IgA pre-vaccination, compared with infants who did not, had a lower serconversion rate at 1 year [48% (10/21) vs 88% (129/147), p<0.001]. Also, infants who had high levels of RV-IgG at pre vaccination, compared with infants who did not, had a lower serconversion rate at 1 year [76% (72/95) vs 92% (67/73), p=0.007]. Additionally, stunted, compared with non-stunted, infants at the second blood draw had lower seroconversion at 1 year of age [63% (12/19) vs. 87% (126/145), p=0.015]High levels of pre-vaccination IgA and IgG, and stunting appeared to diminish the RV-IgA seroconversion at 1 year of age after Rotarix® immunization


Using gnotobiotic (Gn) pig model for pre-clinical evaluation of candidate rotavirus vaccines

Lijuan Yuan

Virginia Polytechnic Institute and State University, USA

With the two vaccines RotaTeq and Rotarix on the market, placebo-controlled efficacy trials of new vaccination approaches will be ethically unallowable and comparator studies will require enormous sample sizes. Gn pigs present the most ideal animal model for pre-clinical evaluation of candidate rotavirus vaccines. There are four distinctive advantages of using Gn pig models to study the immunogenicity and protective efficacy of rotavirus vaccines: 1) neonatal pigs provide an immunologically naïve background that allows clear identification of the immune responses to a single vaccine; 2) high similarities in gastrointestinal physiology and immune responses make the results observed in pigs highly translatable to humans; 3); Gn pigs can be reconstituted with defined levels of maternal antibodies; and 4) Gn pigs can be transplanted with defined human gut microbiota. Gn pig model not only permits exploration of the efficacy of new vaccination approaches, it also leads to the identification of immune correlates of protection. Questions have been addressed with Gn pig model include: What are the basic parameters of the intestinal and systemic antibody and T cell responses to live vaccines versus non-replicating vaccines in naïve hosts? How do these responses correlate with protection? How does the type of vaccine antigen and route of administration influence the intestinal and systemic immune responses to rotavirus and the degree of protection? How effective different adjuvants in enhancing vaccine efficacy? How do maternal antibodies and gut microbiota influence immunogenicity and protective efficacy of rotavirus vaccine? Lessons learned from Gn pig model guide future vaccine development.


Disabled Infectious Single Animal (DISA) vaccine for African Horse Sickness

Piet A. van Rijn, Sandra G.P. van de Water, Christiaan A. Potgieter, and René G. P. van Gennip

Department of Virology, Wageningen Bioveterinary Research (WBVR), Lelystad, The NetherlandsDeltamune (Pty) Ltd, Lyttelton, Centurion, South AfricaDepartment of Biochemistry, North-West University, Potchefstroom, South Africa

African horse sickness virus (AHSV1-9), (Orbivirus, Reoviridae) is an insect borne virus causing disease with a mortality of >90% for naïve domestic horses. Conventionally live-attenuated vaccines (LAVs) can have shortcomings associated to safety issues, and do not exhibit DIVA. We have regenerated virulent AHSV deficient for NS3/NS3a expression which is grown on standard cell lines and is completely safe (not virulent, no viremia, and consequently no uptake by midges), and shows protective capacity. Vaccine safety is further augmented as orbivirus propagation without functional NS3/NS3a protein is abolished in midges (no spread by midges), and this deletion mutant is explored as Disabled Infectious Single Animal (DISA) vaccine platform by exchange of serotype immunodominant outer shell proteins. Lack of immunogenic NS3/NS3a enables differentiating infected from vaccinated horses (DIVA principle). AHS DISA vaccines sharing the DISA vaccine platform can be used as mono-serotype vaccines and can be safely combined to achieve broad protection or to combat specific multi-serotype field situations in a tailor-made fashion. In conclusion, except for protection, the experimental AHS DISA vaccine platform fulfils all important vaccine criteria. Current results show ample room for improvement of the efficacy (virus backbone, vaccine dose, revaccinations, and possibly the vaccination route. Special attention should be given to onset of immunity in the light of emergency vaccination in AHS-free countries, and to broad protection in countries endemic for several AHSV serotypes. The AHS DISA vaccine platform should be further investigated to combat African Horse Sickness in a safe, effective, cheap and controllable vaccination campaign.


WORKSHOP 5: STRUCTURE

mu1 protein dependent aggregation of reovirus particles promotes reassortment

Deepti Thete, Anthony Snyder, Pranav Danthi

Indiana University, USA

Reassortment following infection with segmented viruses increases viral diversity and promotes viral evolution. Reassortment requires coinfection of cells with more than one virus. Traditionally, infection of host cells by each of these viruses is considered to be an independent, random event. More recently, formation of vesicles containing multiple virions, attachment of multiple virions to surfaces of bacteria, or aggregation of virions has been demonstrated to result in formation of multivirion infectious units. Formation of such collective units could make coinfection by multiple strains anon-random and increase the efficiency of reassortment. We demonstrate that particles of mammalian reovirus can aggregate by incubation at 37°C under physiological conditions, even at low particle concentrations. When particles of two different prototype strains, T1L and T3D, are incubated together at 37°C, they form mixed aggregates. Importantly, when reassortment frequency of progeny viruses produced from cells infected with a mixture of non-aggregated or aggregated T1L and T3D is measured, there are significantly more reassortants recovered when particles were aggregated. We further demonstrate that properties of the mu1 protein outer capsid protein influence the propensity of virus particle aggregation. These data identify mu1-dependent aggregation as a new determinant of reovirus coinfection and reassortment.


Structural and biophysical analysis of human picobirnavirus capsid

Alvaro Ortega-Esteban (1), Carlos P. P. Mata (1), M. Jesús R. Espinosa (1), Pedro J. de Pablo (2), José R. Castón (1)

1: Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), 28049 Madrid, Spain; 2: Departamento de Física de la Materia Condensada, Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain

Picobirnaviruses (PBV) are non-enveloped viruses with two dsRNA genomic segments. PBV have a wide host range, including insects, birds and humans. Their effects on the host organism are mild, related to diarrhea and gastroenteritis. Rabbit PBV is characterized by X-ray crystallography; the ~40 nm-diameter, icosahedral T=1 capsid is based on 60 cuasi-symmetric capsid protein (CP) dimers of a single protein (also referred to as a “T=2”, 120-subunit capsid) [EMBO J. 2: 1655, 2009].We work with the human PBV (HPBV). Starting from a synthetic DNA, we successfully expressed the HPBV CP in Escherichia coli. We prepared three CP variants able to form virus-like particles (VLP): CP, a complete version of the original amino acid sequence; pCP: a processed form that lacks the first 45 N-terminal residues; and HT-CP, the original sequence with an extra 36-residue His tag.We used 3D cryo-electron microscopy (cryo-EM) at near atomc resolution and atomic force microscopy (AFM) to describe the structure and biophysical properties of these HPBV VLP. The HPBV capsid is built of 60 copies of two-fold symmetric CP homodimers, labelled A and B, with an intricate interface. N-terminal region of polypeptide chain A of CP and HT-CP interacts with the heterologous packaged RNA. HPBV pCP-based VLP might contain heterologous proteins, which suggests that differences at the CP N terminus assist packaging of different cargos. We studied with AFM whether encapsulation of heterologous cargos alter the physical properties of the VLP. Finally, we established a reversible disassembly/reassembly system for these HPBV VLP.

265Poster presentations Workshop 5: Structure

WORKSHOP 6: USING AND ABUSING HOST PATHWAYS

NS4 and NS5 of orbiviruses: modulation of pathways in insect and mammalian cells

Fauziah Mohd Jaafar (1), Pierre Olivier Vidalain (2), Damien Vitour (1), Stephan Zientara (1), Peter P.C. Mertens (3), Houssam Attoui (1)

1: UMR1161 Virology, INRA-ANSES-ENVA, France, 94700; 2: UMR8601-LCBTP, université Paris Descartes, France, 75006; 3: The School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Leicestershire UK, LE12 5RD

NS4 and NS5 are additional non-structural proteins recently identified for the orbiviruses [for the tick-borne Kemerovo virus group, bluetongue viruses (BTV) and other insect-borne orbiviruses]. Secondary structure predictions suggest both proteins are organised as coiled-coils. Our studies localise these proteins to the cytoplasm and nuclei of infected cells. Reverse genetics using wild-type BTV-1 and NS4, NS5 or NS4/NS5 deletion mutants have shown both proteins to be non-essential in certain cell cultures, or in infected animal models. However, deletion mutants replicated to lower levels than wild type BTV-1.Our protein-protein interaction studies show NS4 and NS5 to be multitask proteins interfering with multiple cellular pathways, including innate immune pathways in mammalian and arthropod cells. In arthropod cells (midge or tick cells) these proteins suppress RNA silencing by modulating transcription of RNAi pathway components. In mammalian cells, they modulate both the RNAi response and the interferon response. NS4 also interacts with proteins involved in capping/de-capping mRNA and may play a role in transition from cap-dependent to cap-independent translation in mammalian cells. When overexpressed in mammalian cells, NS4s of insect-borne or tick-borne orbiviruses can permit a tick-only orbivirus (St Croix River virus) to initiate replication in usually non-permissive mammalian cells.


Nuclear localisation of African horse sickness virus non-structural protein NS4 differs across strains

Shareen Boughan (1), Christiaan Potgieter (2), Vida van Staden (1)

1: University of Pretoria, South Africa; 2: Deltamune (Pty) Ltd, South Africa, North-West University, South Africa

African horse sickness virus (AHSV) causes the disease African horse sickness in susceptible equids, and is of major economic importance in Southern Africa. While AHSV replicates exclusively in the cytoplasm, the recently discovered non-structural protein NS4 localises to both the nucleus and the cytoplasm of AHSV-infected cells. As little is known about the functional importance of AHSV NS4 in the nucleus, the aim of this study was to compare the characteristics of NS4 and its presence in different nuclear compartments across all AHSV serotypes.We analysed all nine AHSV reference strains plus one field isolate from each serotype, and compared NS4 sequences and protein expression levels. Confocal microscopy was used to visualize the intracellular localisation of NS4. The NS4 gene sequences encoded one of three different NS4 types, i.e. NS4-I, NS4-II or NLS-NS4-II (contains an N-terminal nuclear localisation signal). Each of these had a distinctly different intracellular NS4 distribution profile. NS4-I showed a mainly nuclear distribution, NS4-II was homogenous in the cytoplasm and the nucleus, while NLS-NS4-II displayed distinct punctate nuclear foci. We also investigated if NS4 can localise to specific nuclear compartments (Cajal bodies, promyelocytic leukemia bodies and nuclear speckles), Interestingly, we observed that AHSV infection resulted in relocation of a component of nuclear speckles to the virus inclusion bodies in the cytoplasm.This study shows that differences exist across all serotypes for AHSV NS4. Because NS4 may localise to nuclear compartments involved in immunity, NS4 may act as a virulence factor which is important in vaccine development.

269Poster presentations Workshop 6: Using and Abusing Host Pathways

Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway

Cindy Kundlacz (2), Aurore Fablet (2), Rayane Amaral Da Silva Moraes (3), Marie Pourcelot (3), Cyril Viarouge (2), Corinne Sailleau (2), Axel Gorlier (2), Emmanuel Breard (2), Stephan Zientara (2), Damien Vitour (2), Grégory Caignard (1)

1: INRA, France; 2: ANSES, France; 3: ENVA, France

Bluetongue virus (BTV) is an arbovirus responsible for bluetongue, a non-contagious disease that affects a wide range of wild and domestic ruminants. In our laboratory, we study the effect of BTV on different cell signaling pathways. In addition to its antagonist role on type I interferon pathway (IFN-I), we have revealed a new function carried by the BTV-NS3 protein as a positive modulator of the MAPK/ERK pathway. This signaling pathway responds to growth factors and activates various phosphorylation events leading in particular to the activation of translation initiation factor 4E (eIF4E) and Elk-1 transcription factors involved in cell survival, proliferation and differentiation. The use of U0126, an inhibitor that block the phosphorylation of ERK by MEK proteins, allowed us to refine the action level of BTV-NS3, namely downstream of the EGF receptor and upstream or directly on MEK1/2. Furthermore, inhibition of MAPK/ERK pathway with U0126 significantly reduced viral titers suggesting that BTV manipulates this pathway to increase replication efficiency. We also have some evidence that the localization of BTV-NS3 at the Golgi apparatus is very important for the activation of the MAPK/ERK pathway. Using different NS3 deletion mutants, we did not show any link between the activation of the MAPK/ERK pathway and the inhibition of IFN-I by BTV-NS3. Altogether, our data provide molecular basis to explain the role of BTV-NS3 as a virulence factor and determinant of pathogenesis and demonstrate that BTV has evolved a single virulence factor to block IFN-I and to activate simultaneous cellular response to growth factors.


Reovirus inhibits NF-kappaB signaling in infected cells by at least two different mechanisms

Andrew McNamara, Pranav Danthi

Indiana University, USA

The capacity of reovirus to induce cell death correlates with viral disease. Reovirus infection leads to the blockade of prosurvival signaling driven by transcription factor NF-kappaB. Moreover, the degree to which reovirus inhibits NF-kappaB correlates with the capacity of the virus to induce cell death. While these observations suggest that the blockade of NF-kappaB regulates cell death following reovirus infection, a mechanism by which NF-kappaB is inhibited has not been identified. We have found that reovirus blocks NF-kappaB signaling in two ways. Early stages of reovirus infection which occur prior to viral gene expression induce proteasomal degradation of critical members of the Inhibitor of KappaB Kinase (IKK) complex. The loss of IKK complex components prevents nuclear translocation of NF-kappaB, thereby preventing gene expression. Serotype 3 strains of reovirus, such as T3A, induce a greater level of cell death with faster kinetics than serotype 1 strains, such as T1L. Consistent with this, T3A causes a more efficient reduction in levels of the IKK complex than serotype 1 strain T1L and, concomitantly, a more efficient blockade of NF-kappaB nuclear translocation. Surprisingly, despite not efficiently inhibiting NF-kappaB nuclear translocation, T1L remains capable of blocking NF-kappaB -dependent gene expression. Unlike T3A, however, efficient inhibition of NF-kappaB-dependent transcription by T1L requires viral gene expression. These data indicate that reovirus uses multiple strategies to block survival signaling by NF-kappaB. The targeting of multiple stages of NF-kappaB signaling by different steps in virus replication may allow the virus to efficiently elicit cell death.


Integrative hormone, metabolomic and transcriptomic profiling reveal hormonal imbalance and strong sugar partitioning alteration in Mal de Rio Cuarto virus-infected wheat

Luis Alejandro de Haro (1), Sofía Maité Arellano (1), Ondrej Novák (2), Regina Feil (3), Analía Delina Dumón (4), María Fernanda Mattio (4), Danuše Tarkowská (2), Gabriela Llauger (1), Miroslav Strnad (2), John Lunn (3), Stephen Pearce (5), Carlos María Figueroa (6), Mariana del Vas (1)

1: Instituto de Biotecnología, Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina; 2: Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany, Czech Academy of Sciences, Czech Republi; 3: Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany; 4: Instituto de Patología Vegetal, Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina; 5: Department of Soil and Crop Sciences, Colorado StateUniversity, Fort Collins, CO, USA; 6: Instituto de Agrobiotecnología del Litoral, UNL, CONICET, FBCB, Argentina

Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) infects monocotyledonous plants and causes the most important viral maize disease in Argentina. Wheat (Triticum aestivum) acts as a winter reservoir for the virus and is currently the best model species to study the disease. Infected plants show severe symptoms, including shortening of internodes, a stunted appearance, increased tillering and reduced root length. Leaves are short, coriaceous and have enlarged veins. To better understand the molecular basis of plant-virus interactions that lead to symptoms, an RNA sequencing and metabolomics approach was used in young systemic leaves. More than 3,000 differentiallyaccumulatedtranscripts were detected at 21 days post inoculation (dpi) compared to mock-infected controls. Infected plants exhibited a strong decrease in TaSWEET13 transcripts, which are involved in sucrose loading to the phloem. Glucose, fructose, sucrose, starch, trehalose-6P, and organic and amino acids all accumulated upon MRCV infection. In addition, several transcripts related to plant hormone metabolism, transport and signaling were increased. Transcripts coding for GA20ox, D14, MAX2 and SMAX1-like, which participate in gibberellin biosynthesis and strigolactone signaling, were reduced. Transcripts involved in jasmonic acid (JA), ethylene and brassinosteroid (BR) biosynthesis, perception and signaling and in auxin transport were also altered. Hormone measurements detected that JA, BRs, abscisic acid and indole-3-acetic acid were significantly accumulated in infected leaves. These results indicate that MRCV causes a profound hormonal imbalance that, together with alterations in sugar partitioning, could account for the symptoms observed in MRCV-infected plants.


Loperamide, a mu-opioid receptor agonist, inhibits reovirus infection

Angela K Berger (1,2), Alison W Ashbrook (3), Maximilian H Carter (4), Karla D Passalacqua (5), Kristen M Ogden (4), Charles M Rice (3), Christiane E Wobus (5), Bernardo A Mainou (1,2)

1: Department of Pediatrics, Emory University, Atlanta, GA, USA; 2: Children’s Healthcare of Atlanta, Atlanta, GA, USA; 3: Laboratory of Virology and Infectious Diseases, Rockefeller University, New York, NY, USA; 4: Department of Pediatrics, Vanderbilt University, Nashville, TN, USA; 5: Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA

Reovirus is a nonenveloped, segmented dsRNA virus that infects most humans during childhood but seldom causes disease. During cell entry, reovirus attaches to cell-surface carbohydrate and junction adhesion molecule-A (JAM-A), a tight junction protein. Reovirus enters cells via receptor-mediated endocytosis and undergoes stepwise proteolytic disassembly in acidified endosomes. Inhibiting cell entry steps results in downstream impairment of infection. To identify host factors that affect reovirus infection, we performed a high-throughput screen of small molecule inhibitors to assess compounds that increase or decrease reovirus infectivity. Loperamide, a mu-opioid receptor agonist that is used as an anti-diarrheal, impairs reovirus infectivity without affecting cell number. Treatment of cells with loperamide does not alter levels of cell-surface JAM-A and does not impact reovirus attachment to cells. In contrast, infection with infectious subvirion particles (ISVPs), which use JAM-A to infect cells but do not require endosomal acidification, are not affected by loperamide. Concomitant with a cell entry impairment, viral disassembly is delayed and formation of viral factories is impaired in loperamide-treated cells. Interestingly, treatment of cells with loperamide does not affect norovirus, rotavirus, Sindbis virus, or yellow fever virus infection, but impairs chikungunya and Zika virus infection. These data suggest that loperamide impairs reovirus infection by affecting cell entry and has anti-viral properties against a group of unrelated viruses.


Rotavirus hinders antiviral RNA interference by degradation of Argonaute-2

Urbi Mukhopadhyay (1), Shampa Chanda (2), Mamta Chawla-Sarkar (1)

1: National Institute of Cholera and Enteric Diseases, India;2: GITAM Institute of Science, India

Introduction: The notion of RNA interference (RNAi) as an antiviral defense system is well established among plant and insect viruses. Recent studies have expanded the horizon of RNAi inhibitors from plant and insect viruses to the mammalian viruses. However, viruses have co-evolved various suppressors-of-RNA-silencing (RSS) to antagonize RNAi-mediated host antiviral strategy. In the present study, the possibility of RNAi inhibition and potential antagonizing effects of RSS were explored during infection with Rotavirus (RV), a diarrheagenic double stranded RNA virus of Reoviridae family.Results:RNA interference mediated inhibition of ectopically expressed eGFP was found to be effectively blocked during early hours of RV infection (2-6hpi), but was restored beyond 6hpi. Argonaute-2 (AGO2), a key effector within RNA-induced silencing complex (RISC), was subsequently identified as the protein which is degraded leading to disruption of the RNAi machinery early during RV infection. RV non-structural protein 1 (NSP1) was further found out to be the RSS which degrades AGO2 protein by ubiquitin-proteasome system independent of co-opted E3 ubiquitin ligases Cullin1 and Cullin3. Interestingly, ectopic overexpression of AGO2 resulted in reduced intracellular RV burden as confirmed by reduced viral titers and viral protein expression.Conclusion:Host RNAi machinery can generate small interfering RNAs (siRNAs) when dsRNA genome of RV replicates in the cytoplasm. This could have potential deleterious effect on viral replication. Thus RV has evolved mechanism of evading this RNAi mediated host antiviral mechanism by degrading the RISC effector protein AGO2 during early hours of infection.


Rotavirus infection down-regulates redox-sensitive transcription factor Nrf2 and Nrf2-driven transcription units

Upayan Patra, Urbi Mukhopadhyay, Mamta Chawla-Sarkar

National Institute of Cholera and Enteric Diseases, India

Introduction: Rotavirus (RV), a non-enveloped double stranded RNA virus of Reoviridae family, is the major causative agent of acute infantile gastroenteritis. Avoidance of cellular stress-response pathways is an essential facet of RV-induced obligatory host reprogramming. Here, the status of redox-sensitive transcription factor Nrf2 and Nrf2-driven transcription units containing antioxidant response element (ARE) is studied post RV infection in vitro.Results: Steady-state Nrf2 protein levels in whole cell lysates and nuclear fractions of MA104 and HT29 cell line were found to get induced till 3hpi followed by a sharp decline as RV-SA11 infection progresses. Reduction in Nrf2 levels during RV-SA11 infection resulted in marked attenuation of Nrf2-driven transcription units leading to lowered expressions of stress-responsive Nrf2 target genes HO-1 and NQO1 both in presence and absence of Nrf2-driven transcriptional inducers. Initial induction of Nrf2 was revealed to be sensitive to treatments with anti-oxidants NAC and PTDC. Reduction of Nrf2 levels beyond 3hpi, however, was found to be independent of cellular redox status as treatment with oxidative stressors failed to rescue Nrf2 levels. Moreover, increasing the half-life of Nrf2 through inhibition of canonical Nrf2 turn-over pathway could not restore Nrf2 levels post RV-SA11 infection. Interestingly, chemical activation of Nrf2-ARE pathway significantly reduced RV RNA transcripts, protein expressions and viral titer post RV infection, emphasizing the importance of Nrf2-ARE pathway as a druggable anti-rotaviral determinant.Conclusion: Stress-responsive Nrf2-ARE pathway has been found to be markedly attenuated beyond initial hours of RV infection. Chemical activation of this deterministic pathway revealed promising anti-RV potential therapeutically.


Investigating cytosolic conveyance of newly synthesised mature African horse sickness virus particles

Ané Pieters, Vida van Staden

University of Pretoria, South Africa

African horse sickness virus (AHSV) causes haemorrhagic disease in horses. In mammalian cells AHSV causes severe cytopathic effect, while insect cells sustain asymptomatic infection. Virus egress, the last stage in the AHSV life cycle during which virus particles are trafficked from viral inclusion bodies to the plasma membrane and released, is thought to contribute to the differences in cellular pathogenicity. Given the 70 nm size of AHSV particles, passive diffusion is improbable. Our study focused on providing preliminary insight into usurpation of cellular trafficking pathways for the egress of newly synthesised mature AHSV particles. We used a combination of molecular, microscopic and biochemical assays in mammalian and insect cells to study the influence of cellular components on virus replication and trafficking. Transmission electron microscopy of AHSV-infected BSR and KC cells demonstrated a close association of mature particles with intracellular vesicles at early times after infection. At later times, mature particles were predominantly distributed as cytoplasmic aggregates. Inhibition of multi vesicular body (MVB) resident lipid phosphatidylinositol-3 kinase (PI3K) at early times post infection resulted in decreased total virus titre, suggesting that the observed virus-containing vesicles might be MVBs. Brefeldin A (BFA)-mediated dissociation of Golgi ADP-ribosylation factor (ARF) and coatomer subunit beta (β-COP) at later times of infection resulted in a significant decrease of virus release in BSR but not KC cells. Thus, our study showed that components of MVB are important for virus production in KC and BSR cells and that ER-Golgi transport is crucial for AHSV egress in BSR cells.


WORKSHOP 7: APPLIED DSRNA VIROLOGY

Isolation and identification of dsRNA viruses from Fusarium species

Akhtar Ali, Samira Mokhtari

Department of Biological Science, The University of Tulsa, Oklahoma, 74104, USA

Fusarium is a large genus of filamentous fungi that contains more than 300 species distributed worldwide. Many species cause plant diseases, such as vascular wilts, root, stalk and rots of cob, seedlings, tubers, bulbs and corms. These diseases are a leading cause of economic losses in important agricultural crops worldwide. Recent studies have shown that a number of mycoviruses were isolated from Fusarium species. Some of these mycoviruses have adverse effects on the growth of fungi and could be used as a potential biological control to reduce the effects of fungal diseases. The purpose of this study is to explore unique mycoviruses from Fusarium species, which could have a hypovirluence against Fusarium species that cause wilt disease in important agricultural crops. In our work, we collected spores of four unknown Fusarium species and grew them in the lab. Three of the species showed signs of various dsRNA viruses while one was free of any mycoviruses. The identification and characterization of these dsRNA mycoviruses are in the process and will be discussed.


Non-vaccine prevention of dsRNA virus infection

Meng-Jung Chen

Chi-Mei Medical Center, Taiwan

Background: Rotavirus infections are common infections in children. It is a genus of double-stranded RNA viruses in the family Reoviridae. Nearly every child is infected with rotavirus at least once by the age of 5. Since rotavirus vaccines are not available sometimes, it is important to understand the non- vaccine prevention of rotavirus infection.Method: We did a review to explore the non- vaccine prevention and management of this dsRNA virus infection.Result: The genome of rotavirus consists of 11 segments of dsRNA. Each genome segment codes for 1 protein with the exception of segment 11, which codes for 2 proteins. Among the 12 proteins, 6 are structural and 6 are non-structural proteins. It is a double-stranded RNA non-enveloped virus. The thing is to follow good hygiene like hand washing and keeping clean. People with rotavirus infection should avoid from work until no more symptoms for 2 days. If working as food processor, the exclusion period should be 3 days. In rotavirus infection, hungry patients should be given small amount of usual foods, but avoid high in fat or sugar. The important issue is to keep from dehydration. Oral rehydration solution is recommended for those with dehydration.Conclusion:With appropriate prevention and intervention, we can minimize the risk of dsRNA virus infection if rotavirus vaccine is not available. However the effect of non- vaccine prevention of rotavirus infection is limited, vaccination should be used for better protection.

279Poster presentations Workshop 7: Applied dsRNA Virology

Identification and initial characterization of dsRNA mycoviruses infecting the chestnut pathogen Cryphonectria naterciae

Carolina Cornejo (1), Helena Braganca (2), Daniel Rigling (1)

1: Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Switzerland; 2: Instituto Nacional de Investigação Agrária e Veterinária, Av. da República, 2780-157 Oeiras, Portugal

The ascomycete genus Cryphonectria includes known tree pathogens. The most studied species is C. parasitica – the causal agent of the destructive chestnut blight disease, which was introduced in North America and in Europe in the early 20th century. Some strains of C. parasitica however exhibit reduced levels of virulence due to a viral infection. The so-called Cryphonectria hypovirus (CHV) attenuates the pathogenicity of C. parasitica reducing its parasitic growth and sporulation capacity. CHVs are cytoplasmatic, unencapsidated dsRNA viruses with genome ranges between 9 and 13 kbp. CHVs have no extracellular phase and are transmitted mainly from infected to non-infected fungal strains via hyphal anastomosis. The discovery of CHV sparked off great interest in mycoviruses as natural biocontrol agents. The aim of this study is to screen additional species of Cryphonectria for the presence of CHV or other RNA viruses, (1) to characterize them molecularly and (2) to test their capacity in hypovirulence on chestnuts trees. We used isolates of C. naterciae to extract dsRNA directly from lyophilized mycelium and to generate an Illumina cDNA library by reverse-transcription including specific sequencing adaptors. Currently, a de novo assembly of the reads is being aligned to viral sequences to characterize them molecularly. The potential for using these Cryphonectria viruses as agents for the biological control on chestnuts trees will be presented.


Comparative evaluation of sensitivity and specificity of Immunochromatography kit (IC Kit) for the rapid detection of rotavirus in Bangladesh

Shuvra Dey (1), Sadia Farzan Sifat (1), Md Almamun (1), Nadim Sharif (1), Nasir Uddin Nobel (1), Shilpi Sarkar (2), Anowar Khasru Parvez (1), Ali Azam Talukder (1)

1: Department of Microbiology, Jahangirnagar University, Savar, Dhaka, Bangladesh; 2: Department of Zoology, Jahangirnagar University, Savar, Dhaka, Bangladesh

Background: Rotavirus is the most common cause of acute diarrhea in young children worldwide. Our research intended to evaluate the Immunochromatography kits used for rotavirus by comparing the detection results with the reference method RT-PCR. Methods: A total of 118 fecal samples were collected between January 2016 and December 2016 from children below 5 years old with acute gastroenteritis from Mother and Children Hospital (Chittagong, Bangladesh). The IP-Rota/Noro kit (Immuno-Probe, Japan) was used to detect rotavirus. All the samples were also analyzed by reverse transcription polymerase chain reaction (RT-PCR) as the reference method. Results: A total of 118 stool specimens were tested by Immunichromatography Kit (IP-Rota/Noro kit). Results were found within 15 mins. Among 118 stool specimens 68 and 8 specimens were detected as rotaviurus and norovirus positive by IP-Rota/Noro kit. All specimens were subjected to further analysis by RT-PCR method. In compare with RT-PCR method, the sensitivity and specificity of IP-Rota/Adeno kit for the detection of rotavirus is 100% and 92% and for norovirus is 100% and 100% respectively. In addition, phylogenetic analysis revealed that the majority of rotavirus belonged to G1P[8]. Conclusion: Our findings clearly indicate that rotavirus is the most important enteropathogen responsible for viral gastroenteritis in Bangladesh. The immunochromatography kit provide high specificity and sensitivity as well as good agreement with the reference method RT-PCR for the detection of rotavirus. Finally, it must be suggested that IC kit might be used as an alternative method for the rapid diagnosis of rotavirus.


Epidemiology and burden of dsRNA virus in Taiwan

Chia-Jung Hsieh

Chung-Hwa University of Medical Technology, Taiwan

Background: dsRNA virus cause threat both to fishery and human in Taiwan. We explored the epidemiology and burden of dsRNA virus in Taiwan, with focus on fishery and rotavirus infection.Methods: Review articles to assess dsRNA virus impact both on fishery and human in Taiwan with stress on fish and rotavirus infection.Result: Since 1992, red sea bream in certain area in Taiwan was plaqued by a novel disease. Iridovirus was noted on EM exam, later grouper and sea bass were also infected. In 1995, a new viral disease causing 60% mortality threatened cultured grouper in Taiwan, particles were found in moribund fish. The acridine orange staining and IUDR treatment indicates that the viral genome is double-stranded DNA. Healthy groupers were experimentally injected with TGIV with mortality reaching 100%, and no grouper died in control groups. On the other hand dsRNA virus in human like rotavirus is a infectious gastroenteral disease in children. Rotavirus causes more than 100000 hospitalizations per year in children less than 5 years old, leading to a significant cost. The total cost was several times the expense of rotavirus vaccine. Rotavirus vaccination would prevent about 4 deaths, 10000 hospitalizations, and 60,000 outpatient visits due to rotavirus infection every year.Conclusion: dsRNA virus cause significant burden with potential mortality risk both to fish and human in Taiwan. More studies and researches shold be conducted to understand the threat of dsRNA virus.


Metagenomic approach to detect dsRNA viruses and other human enteric viruses in wastewater samples.

Soizick F. Le Guyader (1), My Phan (2), Sofia Strubbia (1), Julien Schaeffer (1), Marion Koopmans (2), Matthew Cotten (2)

1: IFREMER, France; 2: Erasmus, Netherlands

During the winter gastroenteritis season, a large diversity of human enteric viruses co-circulate in the population and are thus potentially excreted in wastewaters. Full-genome next-generation sequencing (NGS) may now be applied to evaluate this diversity and potentially to identify some predominant strains circulating in the population or some emerging viruses. However, the low abundance in the environment of these physically small particles and with short genome compared to bacteria, make sequencing from wastewater samples a challenge. To overcome this issue both sample preparation and sequence analysis using an optimized pipeline are of primary importance.A method based on polyethylene glycol concentration was adapted by adding acidification and additional cleaning steps and pig-mucin capture. Then libraries were prepared from two protocols for three wastewater samples and submitted for NGS. All read were analyzed using the SLIM pipeline.Following NGS, the total abundance of reoviridae reads in the three samples were between 0.02% to 0.92%, higher than caliviridae reads that varied 0,01% and 0,1%. A complete genome sequence, comprising the 11 fragments of a human rotavirus G9[P8] was detected in one of the sample, interestingly after pig mucin capture.In conclusion, this technique is promising to evaluate the virome diversity however more technical improvment are needed to concentrate and purify human enteric viruses. The pipeline analysis is essential to identify sequence of interest.


Assessing the use of picobirnavirus and infective enterovirus as indicators for faecal contamination tracking in freshwater and its potential to report the presence of rotavirus.

Gisela Masachessi (1), Verónica Emilse Prez (1), Laura Cecilia Martínez (1), Miguel Oscar Giordano (1), Patricia Angélica Barril (2), Jorge Vitorio Pavan (1), Silvia Viviana Nates (1)

1: Instituto de Virología Dr. JM. Vanella, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Argentine Republic; 2: Laboratorio de Microbiología de los Alimentos, Centro de Investigación y Asistencia Técnica a la Industria (CIATI A.C.)

An environmental survey was conducted in order to assess picobirnavirus (PBV) and infective enterovirus (iEV) as indicators of faecal contamination in freshwater and its potential to report the presence of rotavirus (RV). The study was conducted in the San Roque Dam, Córdoba, Argentina, during a three-years-long monitoring campaign. Water samples were concentrated by polyethylene glycol precipitation. RT-PCR was carried out to assess the genome presence of PBV and RV. iEV was evaluated by cell culture-immunofluorescence assay. RV genomic concentration was quantified by qPCR and RV infective particles was estimated.The overall frequency was 62.9% for PBV and 70.4% for iEV. No significant difference was observed in the rates of iEV and PBV in the years studied or a seasonal pattern for the viruses studied. The overall RV frequency was 45.8%; 90.9% of the RV positive samples were also positives for iEV and/or PBV genome. Rotavirus infective particles/10 ml of water were detected in a range of 33-7,466; 90% of the samples revealed up to 100 infective particles, which are enough to initiate a viral infection in the exposed host. At least one of the viruses analysed was demonstrated in the 100% of the samples with values under the level guide for faecal coliforms (<200 MNP/100 ml).Therefore, in this setting, PBV and iEV could be promising indicators for faecal contamination tracking in freshwater. The detection of these viruses could inform about the presence of RV. The evidence of iEV could indicate adequate matrix conditions for maintaining the RV infectivity.


Diffuse contamination of surface waters from the north region of Argentina: human rotavirus detection and characterization.

Patricia Angélica Barril (1,2), Hugo Ramiro Poma (2,3), Georgina Gisela Giordano (4), Verónica Emilse Prez (2,4), Silvia Viviana Nates (4), Verónica Beatriz Rajal (2,3,5)

1: Centro de Investigación y Asistencia Técnica a la Industria - Asociación Civil. Centenario, Neuquén, Argentina.; 2: Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.; 3: Instituto de Investigaciones para la Industria Química (INIQUI), CONICET – Universidad Nacional de Salta (UNSa), Salta, Argentina.; 4: Instituto de Virología “Dr. J. M. Vanella”, FCM, Universidad Nacional de Córdoba. Córdoba, Argentina.; 5: Facultad de Ingeniería, UNSa, Salta, Argentina.

Fecal pollution of water is a serious concern because it is associated with the transmission of pathogens. The aim of this study was to analyze theoccurrence of rotavirus in the surface waters of the Arias-Arenales River in Salta, Argentina, and to statistically correlate its presence with physicochemical and microbiological variables of water quality. Physicochemical variablesincluding temperature, pH, turbidity, conductivity, salinity and dissolved oxygen were measured in situ. Total and thermotolerant coliforms were determined by the Most Probable Number method. For viral analysis, water samples were concentrated by ultrafiltration and rotavirus detection and G-type characterization was performed by RT-nested PCR. The nonparametric Spearman test was applied in order to find associations between the evaluated variables.Rotavirus was found in 4/116 (3.4%) water samples, during the summer and autumn seasons. The strains were characterized as G1, G4 and G9, which are common genotypes circulating in the human population. Rotavirus presence did not correlate with the physicochemical variables analyzed, neither with the total and thermotolerant coliforms, which confirms that there is no quantitative relationship between microbiological indicators of fecal contamination and the presence of rotavirus.The monitoring sites located downstream the discharge of sewage water (treated and untreated) did not reveal rotavirus contamination. These results showed that the Arias–Arenales River presents diffuse contamination by rotavirus, originated from a variety of different sources, and also support the essential need of viral indicators for enhanced monitoring of water quality.


Rotavirus-gastroenteritis and molecular characterization of hospitalized children under 5 years of age with acute diarrhea in Nepal

Jeevan Bahadur Sherchand (1), Sony Shrestha (1), Ocean Thakali (1), Laxman Shrestha (2), Ajit Raymajhi (3)

1: 1Public Health Research Laboratory, Institute of Medicine, Tribhuvan University Teaching Hospital, Department of Microbiology,; 2: Tribhuvan University Institute of Medicine, Department of Pediatrics, Maharajgunj Kathmandu Nepal; 3: Kanti Children’s Hospital Maharajgunj Kathmandu Nepal

Background: Rotavirus gastroenteritis is one of the major childhood morbidity and mortality among children under 5 years of age Nepal. In spite of the substantial burden of rotavirus gastroenteritis in Nepal, rotavirus vaccine has not been introduced. Hence, the study identified the magnitude of rotavirus disease burden and molecular characterization among hospitalized children with acute diarrhea in Nepal.Methods: A total of 3619 stool samples of hospitalized children were collected who had acute diarrhea and less than 5 years of age from January 2013 to December 2017. All samples were tested for rotavirus antigen by ELISA (ProSpecT, USA). ELISA positive stool samples were genotyped.Results: Study of five-year period, the proportion of rotavirus infection was 26% among hospitalized children. The majority of children hospitalized with rotavirus gastroenteritis were less than 2 years of age (84.5%). Rotavirus-associated gastroenteritis hospitalizations occur throughout the year in Nepal, but a distinct peak in winter up to 50% was observed. Of 939 ELISA positive samples, 592 were genotyped by RT-PCR. The most prevalent genotype was G12P [6] (46.2%), followed by G2P [4] (11.3%), G1P [8] (10.6%), G9P [4](7.3%), and G9P[8](5.6%). Mixed infection accounted for 3.3% of cases, 6.2% were partially typed and 9.4% of the samples were G and P untypable.Conclusions: The high proportion of rotavirus gastroenteritis and a diversity of rotavirus circulating strains indicated that there is an urgent need of rotavirus vaccine introduction. This will ultimately help to prevent the morbidity and mortality of rotavirus gastroenteritis in children of Nepal.


Combination of Mutations to Produce Reovirus Variants with Improved Oncolytic Potency

Francisca Cristi-Munoz (1,2), Yip Wan Kong (1,2), Hitt Mary (1,3), Maya Shmulevitz (1,2)

1: Medical Microbiology and Immunology Department, University of Alberta, Canada; 2: Li Ka Shing Institute of Virology; 3: Oncology Department, University of Alberta, Canada

Reovirus is a nonpathogenic virus that naturally inhabits the enteric or respiratory tracts of humans. Reovirus can also selectively infect and replicate in tumor cells and is therefore a candidate for cancer therapy. Given that wild-type reovirus is naturally adapted to enteric environments rather than tumors, our laboratory used positive selection to isolate 13 reovirus variants that replicate more efficiently in tumor cells relative to wild-type reovirus (T3wt). All 13 variants retained poor infectivity towards non-transformed cells, indicating that mechanisms for differential specificity towards cancer cells were not overcome. Since these 13 reovirus variants have 1-4 mutations each, we did not know which mutations were important or dispensable. To identify the mutations critical for enhanced oncolysis, 20 of the mutations were introduced into reovirus using site-directed mutagenesis and reverse genetics. We generated viruses with either single mutations, or various combinations of 2 or 3 mutations. Using plaque size to reflect the proficiency of reovirus replication in tumor cells, beneficial mutations were identified. Importantly, when some beneficial mutations were combined, they demonstrated additive effects on oncolytic potency, suggesting the mutations promote distinct features of reovirus replication or accumulate to augment the same features. The oncolysis-promoting mutations predominated in reovirus structural protein l2 and the s1 cell attachment protein, and a majority reduced s1 protein levels; a mechanism we previously described for increasing oncolysis. Interestingly, some mutations in s1 did not affect s1 levels to T3wt, suggesting that new and distinct mechanisms for enhancing replication in cancer cells are possible.


A synthetic biology approach for African horse sickness vaccine platforms

Meredith Stewart (1), Alejandro Marin-Lopez (2), Vanessa Herder (1), Javier Ortego (2), Massimo Palmarini (1)

1: MRC-University of Glasgow Centre for Virus Research, UK; 2: Centro de Investigación en Sanidad Animal, INIA-CISA, Spain

African horse sickness virus (AHSV) is the causative agent of African horse sickness (AHS), a highly fatal disease of equids. Currently, live attenuated vaccines are used to control AHS especially in South Africa, but they are in general subject to restrictions. We took advantage of previously published AHSV structural and antigenic data to simplify AHSV vaccine development using an AHSV-4 reverse genetics approach. We systematically substituted the tip and central domains of AHSV-4 outer capsid protein VP2 with the corresponding region from other serotypes to generate a chimeric protein. AHSV S2 chimeric and monoreassortant viruses for all 9 serotypes were recovered by reverse genetic using AHSV-4 as backbone and used to immunise guinea pigs. Presence of neutralising antibody titres were determined using a fluorescence-based neutralisation assay. The exchange of the tip and central domain of VP2 switched the serotype specificity of the rescued chimeric viruses, however, sera from AHSV-4VP2DTip only neutralised the homologous AHSV-4 reference virus. Monoreassortants, but not recombinant viruses containing chimeric VP2, induced antibodies with low levels of cross-neutralisation between phylogenetically related serotypes. Interestingly, most of the sera raised were able to neutralise AHSV-4, indicating the presence of other neutralising epitopes within the virus. These results raise the possibly of generating a single virus that affords protection against multiple serotypes. Our research highlights the ability to manipulate the AHSV genome to rapidly generate ‘synthetic’ viruses using a single platform approach for vaccine development.


Nanopore sequencing as a revolutionary diagnostic tool for porcine viral enteric disease complexes identifies porcine kobuvirus and picobirnavirus as important enteric viruses

Sebastiaan Theuns (1), Bert Vanmechelen (2), Quinten Bernaert (1), Nádia Conceição Neto (2), Ward Deboutte (2), Marilou Vandenhole (1), Leen Beller (2), Piet Maes (2), Jelle Matthijnssens (2), Hans J Nauwynck (1)

1: Ghent University, Belgium; 2: KU Leuven, Belgium

Enteric diseases in swine are often caused by different pathogens and thus metagenomics are a useful tool for diagnostics. The capacities of nanopore sequencing for diagnostics of rotavirus and other viruses were investigated here.First, cell culture-grown porcine epidemic diarrhea virus and rotavirus A were pooled and sequenced on a MinION. Reads were already detected at 7 seconds after start of sequencing, resulting in high sequencing depths (19.2 to 103.5X) after 3h. Next, diarrheic feces of a one-week-old piglet was analyzed. Almost all reads (99%) belonged to bacteriophages, which may have reshaped the piglet’s microbiome. Contigs matched Bacteroides, Escherichia and Enterococcus phages. Moreover, porcine kobuvirus was discovered in the feces for the first time in Belgium. Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding (106.42-107.01 copies/swab) and diarrheic signs was not observed during a follow-up study. Retrospective analysis showed the widespread (n=25, 56.8% positive) of genetically moderately related kobuviruses among Belgian diarrheic piglets. In another case of mysterious diarrhea in sows, we were able to detect picobirnavirus upon sequencing with MinION. This dsRNA virus was recently also found by our Laboratories in two other cases of diarrhea from fattening pigs using Illumina and requires further in vitro and vivo investigations.MinION enables rapid detection of enteric viruses. Such new methodologies will change diagnostics, but more extensive validations should be conducted. The true enteric pathogenicity of porcine kobuvirus should be questioned, while its subclinical importance cannot be excluded.


First immune nanobody library against Rotavirus derived from a guanaco

Matias Aduriz Guerrero (1), Cecile Vincke (2), Ema Romao (2), Marina Bok (1), Celina Vega (1), Andres Wigdorovitz (1), Serge Muyldermans (2), Viviana Parreño (1)

1: National Institut of Agricultural Technology, Argentine Republic; 2: Vrije Universiteit Brussel

Rotavirus A (RVA) is the main viral agent causing diarrhea in pediatric patients, accounting for more than 200,000 deaths in children < 5 year old. It is of significant importance to find alternative ways of treatment and prevention. Members of the Camelidae family produce antibodies naturally devoid of a light chain, referred to as heavy chain antibodies. Their variable region, also known as Nanobody, is the smallest molecule capable of recognizing antigens with high affinity. Up to date, the species used for the generation of Nanobodies are domestic Lama glama, Vicugna pacos, Camelus bactrianus and Camelus dromedarius; wild South American camelids, Lama guanicoe and Vicugna vicugna are still unexplored. Since Lama guanicoe is a wild camelid species facing various climate conditions and a hostile environment, it will probably have a potent immune system, possibly including different Nanobody genes. These may show several technological advantages, like higher thermo stability, or resistance to enzymatic digestion, which can be beneficial in the search of a treatment for RVA. A wild guanaco was immunized with 3 doses of bovine RVA commercial vaccine. The immune response was monitored by ELISA. Four days after the final boost, peripheral blood mononuclear cells were isolated and their cDNA was extracted. A Nanobody library was constructed with a final size of 3.7x108 transformants. The library was panned by phage display, using captured RVA virus and 25 Nanobodies were obtained. The RVA specific Nanobodies are being recloned and produced for further analysis and comparison with the already generated Nanobodies derived from llama.


Assessment of improved rotavirus vaccine production in gene-edited Vero cells

Houping Wang (1), Jackelyn Murray (2), Les Jones (2), Ralph Tripp (2), Baoming Jiang (1)

1: Division of Viral Diseases, Center for Diseases Control and Prevention, Atlanta, Georgia, USA; 2: Department of Infectious Disease, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

Background: Currently licensed oral rotavirus vaccines have shown to be safe and efficacious against severe rotavirus disease among children in high and middle income countries. These vaccines are less effective in low-income countries where rotavirus remains a major cause of severe gastroenteritis. In addition, vaccine introduction efforts are hampered by their high cost in those resource-poor settings. Thus, developments of low-cost vaccines using improved technology are needed.Methods: We generated stable knockout cell lines using CRISPR-Cas9 gene editing technology. Silence host genes that increase rotavirus replication were identified and validated using small interfering RNAs. Wild-type (WT) and host gene knockout (KO, ∆LRGUK and ∆WDR62) Vero cell lines were tested for enhanced production of rotavirus vaccine strains CDC-9, Rotarix, RotaTeqG1 and 116E in 24-well plates. Infected Vero cell cultures were evaluated for rotavirus antigens VP6 and VP7 by EIA and infectivity by FFA.Results: We found that the ∆WDR62 Vero cell KO line increased VP6 antigen production of all strains tested by 2-10 fold compared to that of WT at day 5 post-infection. Increased production was observed for VP7 of human G1 rotavirus strains by EIA. In addition, increased infectious titers by up to 3 logs were observed in ∆WDR62 knockout cells infected with the four vaccine strains compared to the WT.Conclusion: Our results show increased production of human and animal rotavirus vaccine strains using KO cell lines. Further studies are in progress to support these findings using larger production platforms.


A DS-1 like human rotavirus G9P[6] vaccine strain for improving efficacy in target populations

Yuhuan Wang, Theresa Resch, Mathew D Esona, Sung-Sil Moon, Baoming Jiang

Centers for Disease Control and Prevention, USA

Monovalent human rotavirus vaccine Rotarix® (G1P[8]) has shown broad cross protection against homotypic and heterotypic Wa-like human rotavirus strains among children worldwide. This vaccine, however, appears to induce slightly less or non-consistent protection against DS-1 like rotavirus P[4] strains in some settings. In addition, children who are secretor or Lewis-negative and are vaccinated with Rotarix® have shown to be prone to infection with P[6] strains. In the present study, we describe the isolation of a DS-1 like human rotavirus G9P[6] strain (CDC-6) that grows to high titer (up to 109 ffu/ml) with predominantly triple-layered particles in Vero cells. Full genome sequencing showed nucleotide and amino acid sequence changes in several genes of the virus from stool to adaption in Vero cells. This adapted virus was tested for its attenuation and safety in neonatal rats by oral gavage inoculation and was found to shed in stool at levels similar to that of Rotarix-inoculated rats. In addition, similar body weight gains were observed in CDC-6, Rotarix and placebo-inoculated rats. By contrast, RRV-inoculated animals had much higher levels of viral shedding in stool and significantly lower gains in body weight. This human G9P[6] strain is as a promising new and potential low-cost vaccine candidate (because of high titer) for global use, particularly in targeted population with secretor or Lewis-negative status and high prevalent DS-1 like P[6] strains.


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Structures of dsRNA viruses

Totivirus Birnavirus Cypovirus

Single layered capsids

Triple layered capsids

Double layered capsids

Cystovirus Orthoreovirus

Also similar: Partivirus, Picobirnavirus, Botybirnavirus

No true capsids

Also similar: Mycovirus, Fijivirus

Endornavirus Hypovirus

Images: Swiss Bioinformatics Institute

Rotavirus Orbivirus

Also similar: Phytoreovirus

ClathrinCaveolin

Non-clathrin, non-caveolin macropinocytosis

Attachment

Activationuptake

TrypsinChymotrypsin

disassembly

Cathepsinsother

proteases?

uptake

Conformational transitions,membrane interaction

low pHlipids

Core delivery

mRNA transcription

Pore formation,membranedisruption

Cell entry of dsRNA viruses


Genome Packaging in dsRNA Viruses

What is packaged ? Single stranded RNA or double stranded RNA.

How is the genome recognized among all other RNA molecules in the host cell?

Mature virions are known to contain the double stranded RNA genome, but the molecular recognition events that lead to this point and how they are coordinated spatially and temporally with the replication of the genome are only recently becoming accessible research questions.

Genome segmentation introduces additional complexity in assembling a viable virion, which requires the inclusion of every segment.

or

What are the molecular interaction(s) that discriminate the viral genome from other RNAs?

Secondary structure, with internal stems and loops

Fully base paired


Is the genome packaged into a pre-assembled particle or is the recognition of the genome a key part of the assembly process?

Genome Packaging in dsRNA Viruses

How is a full complement of genome segments recruited ?Genome segmentation can confer selective advantages but what

mechanism(s) ensure that a complete set of genome segments is packaged? Two broad classes of models are possible.

2. Specific recognition of each genome segment by viral proteins

Segmented genomepackaged

Individual segments

• Assembly halted until all segments are packaged

Segment 1 Segment 2 Segment 3

RNA complexIndividual segments

Recognition of completed complex

Segmented genomepackagedInteraction of

segments (ss form)

1. Interaction of the RNA genome segments prior to packaging

+ empty genome

+ genome

or


IFNAR

IRF7 ISGsNF-κB

IFN

MAVS

RIG-I

RIG-I

IRF3IRF7

TLR3

IκB

NF-κB

SCFβ-TrCP

ISGF3

IRF9STAT1

STAT2

IκB UbUbUb

UbUb

1.HidethegenomeandprotectRNAs

2.Promotehostproteindegradation

3.Preventtranscriptionfactorsfromlocalizingtothenucleus

4.InhibittranscriptionoralterRNAprocessing

5.Turnofftranslation

ISGproducts

HowdodsRNAvirusesinhibittheinnateimmuneresponse?


11. ATTENDEES

Agbemabiese, Chantal Ama Noguchi Memorial Institute for Medical Research, Ghana

Agnello, Davide Centre Hospitalier Universitaire de Dijon, France

Ahmad, Irfan SK.University of Agricultural Sciences & Technology of Kashmir, India

Ahmed, Mohamed Lemine Cb. National Institute of Research on Public Health, Mauritania

Alorcon, RodolfoNational Institutes of Health, USA

Ali, Akhtar University of Tulsa, USA

Allende-Ballestero, CarolinaCNB-CSIC, Spain

Alshaikhahmed, Kinda London School of Hygiene and Tropical Medicine, UK

Amadu, Dele Ohinoyi University of Ilorin Teaching Hospital, Nigeria

Apondi, Ernest Wandera Nagasaki University, Kenya

Aravamudhan, Pavithra University of Pittsburgh, USA

Arias, CarlosInstituto de Biotecnologia/UNAM, Mexico

Armah, GeorgeNoguchi Memorial Institute for Medical Research, Ghana

Arnold, Michelle LSU Health Sciences Center - Shreveport, USA

Attoui, Houssam UMR1161 Virologie INRA-ANSES-ENVA, France

Bekker, Shani May University of Pretoria, South Africa

Beller, Leen KU Leuven, Belgium

Ben Hadj Fredj, Mouna University Hospital Sahloul, Tunisia

Benninghoff, BerndGSK, Belgium

Bennour, Haifa University Hospital Sahloul, Tunisia

Bitra, KavitaBayer crop science, USA

Boehme, KarlUniversity of Arkansas for Medical Sciences, USA

Bonifacio, Joseph Manzano Research Institute for Tropical Medicine, Philippines

Bormann, Joerg University Bremen, Germany

Borodavka, Alexander University of Leeds, UK


Boughan, Shareen University of Pretoria, South Africa

Boula, Angeline Yvette Centre Mère et Enfant, Cameroon

Boulant, Steeve Universtiy Hospital Heidelberg and DKFZ, Germany

Bowen, Michael Dominic CDC, USA

Boyce, Mark University of Oxford, UK

Broadbent, Andrew James The Pirbright Institute, UK

Bucardo, Filemón National Autonomous University of León, Nicaragua

Burrone, Oscar R. ICGEB, Italy

Caddy, Sarah University of Cambridge, UK

Caignard, Grégory INRA, France

Cameron Ruiz, NataliaUniversity of Glasgow, UK

Castells, Matias University of the Republic, Uruguay

Castón, José CSIC, Spain

Chagas, Elaine Evandro Chagas Institute, Brazil

Chen, Meng-Jung Chi-Mei Medical Center, Taiwan

Chilakalapudi, Durga Rao Indian Institute of Science, India

Chissaque, AssucenioInstituto Nacional de Saude, Mozambique

Close, LilaKU Leuven, Belgium

Cooke, Lyndsay Sarah The Pirbright Institute, UK

Cornejo, Carolina Swiss Federal Institute for Forest, Snow and Landscape Research WSL, Switzerland

Cottet, Luis Eduardo Universidad de Santiago de Chile, Chile

Coulson, Barbara S University of Melbourne, Australia

Crawford, Sue Ellen Baylor College of Medicine, USA

Criglar, Jeanette Marie Baylor College of Medicine, USA

Damanka, Susan Afua Noguchi Memorial Institute for Medical Research, Ghana

Danthi, Pranav Indiana University, USA

De Wolde, EsméeMerck & Co., Inc.

32711. Attendees

Deboutte, Ward KU Leuven, Belgium

Debrus, SergeGSK Vaccines, Belgium

Degiuseppe, Juan Ignacio INEI - ANLIS “Dr. Carlos G. Malbrán”, Argentina

del Vas, MarianaNational Institute of Agricultural Technology (INTA), Argentina

Dennis, Francis Ekow Noguchi Memorial Institute for Medical Research, Ghana

Dermody, Terence University of Pittsburgh School of Medicine, USA

Desselberger, Ulrich University of Cambridge, UK

Dey, Shuvra Jahangirnagar University, Bangladesh

Diehl, SeanUniversity of Vermont, USA

Ding, Siyuan Stanford University, USA

Donato, Celeste Michelle Monash University, Australia

Dulwich, Katherine The Pirbright Institute, UK

Duncan, RoyDalhousie University, USA

Eichwald, Catherine C University of Zurich, Switzerland

Erickson, Andrea Kaup University of Texas, USA

Esona, Mathew DCDC, USA

Estes, MaryBaylor College of Medicine, USA

Falkenhagen, AlexanderFederal Institute for Risk Assessment, Germany

Ferreira-Venter, LindaUniversity of Pretoria, South Africa, South Africa

Fix, AlanPATH, USA

Fodha Bouzgarrou, Imene University Hospital Sahloul, Tunisia

Garcés Suárez, Yasel Autonomous National University of Mexico, Mexico

Ghosh, Ananta Kumar Indian Institute of Technology Kharagpur, India

Ghosh, Souvik Ross University School Veterinary Medicine, Saint Kitts And Nevis

Gómez Santiago, Fabián Instituto de Diagnóstico y Referencia Epidemiológicos, Mexico

Greenberg, HarryStanford University, USA


Guimera Busquets, Marc The Pirbright Institute, UK

Guo, Lihua Chinese Academy of Agricultural Sciences, China

Gupta, Shipra Jamia Hamdard, India

Hagbom, Marie Linköping University, Sweden

Hardy, Alexandra MRC - University of Glasgow Centre for Virus Research, UK

Harris, Vanessa Amsterdam Institute for Global Health and Development, Netherlands

Hockman, MeganEmory University, USA

Holm, Geoffrey Colgate University, USA

Hoxie, IreneCity University of New York

Hsieh, Chia-Jung Chung-Hwa University of Medical Technology, Taiwan

Hu, Liya Baylor College of Medicine, USA

Hughes, Joseph MRC-University of Glasgow Centre for Virus Research, UK

Huyzers, MarnoNWU Potchefstroom, South Afrika

Hyser, Joseph Baylor College of Medicine, USA

Iaconis, Gennaro The Pirbright Institute, UK

Ilca, Serban Luca University of Oxford, UK

Isa, PavelIBT, UNAM, Mexico

Jere, Khuzwayo University of Malawi, Malawi

Jiang, BaomingCDC, USA

Johne, Reimar Federal Institute for Risk Assessment, Germany

Joshi, Madhuri Shantanu National Institute of Virology, India

Kadoya, Syun-suke Tohoku University, Japan

Kanai, Yuta Osaka University, Japan

Kannimuthu, DhamotharanNorwegian University of Life Sciences (NMBU), Norway

Karatas, Fidan University of Pretoria, South Africa

Kawagishi, Takahiro Research Institute for Microbial Diseases, Osaka university, Japan

32911. Attendees

Kerviel, Adeline London School of Hygiene and Tropical Medicine, UK

Kirkwood, CarlBill and Melinda Gates Foundation, USA

Kobayashi, Takeshi Osaka University, Japan

Kumar, Dilip Baylor College of Medicine, USA

Labadie, Thomas London school of hygiene and tropical medicine, UK

Lartey, Belinda NaaNoguchi Memorial Institute for Medical Research, Ghana

Lazaro, C. Eures Iyar Garcia Research Institute for Tropical Medicine, Philippines

Le Guyader, Soizick F. IFREMER, France

Lemay, Guy Université de Montréal, Canada

Llauger, Gabriela National Institute of Agricultural Technology (INTA), Argentina

Lopez, SusanaInstituto de Biotecnologia, UNAM, Mexico

Luchs, Adriana Adolfo Lutz Institute, Brazil

Lyytinen, Outi Leena University of Helsinki, Finland

Mainou, Bernardo Alfredo Emory University, USA

Malik, Yashpal Indian Veterinary Research Institute, India

Mandile, Marcelo Gastón Universidad Nacional de Quilmes, Argentina

Marthaler, DouglasKansas State University, USA

Martinez, Iara Magaly Health Sciences Research Institute, National University of Asuncion, Paraguay

Masachessi, Gisela Universidad Nacional de Córdoba, Argentina

Mascarenhas, Joana D’Arc Pereira Evandro Chagas Institute, Brazil

Matsuo, Eiko Kobe Univesity, Japan

Matthijnssens, Jelle KU Leuven, Belgium

McDonald, Sarah Wake Forest University, USA

McNeal, Monica Malone Cincinnati Children’s Hospital Medical Center, USA

Mikuletič, Tina University of Ljubljana, Slovenia


Miller, Cathy Iowa State University, USA

Miño, Samuel INTA, Argentina

Moerman, LeentjeGlaxoSmtihKline Vaccines, Belgium

Mogotsi, Milton Tshidiso University of the Free State, South Africa

Mohan, KrishnaBharat Biotech International Ltd., India

Mohanty, SujitCincinnati Children’s Hospital, USA

Mokoena, Fortunate Sefako Makgatho Health Science University, South Africa

Morris, DonTom Baker Cancer Centre, Canada

Mukherjee, Anupam ICMR-National Institute of Cholera and Enteric Diseases, India

Mukhopadhyay, Urbi National Institute of Cholera and Enteric Diseases, India

Munlela, Benilde António Instituto Nacional de Saúde, Mozambique

Ngomane, Thenjiwe Grace Sefako Makgatho health Sciences university, South Africa

Ngoy Kiluba, Médard Pediatric Kalembelembe hospital, Democratic Republic of the Congo

Ngoya Ebiguide, Roger QuantierCentre Mère Et Enfant De La Fondation Chantal Biya, Cameroon

N’guessan-Kouame, Amani Rebecca Teaching Hospital of Yopougon, Côte d’Ivoire

Nkolo Mviena, Gaston Eric Mother and Child Center Chantal BIYA Foundation, Cameroon

Nomikou, Kyriaki MRC-University of Glasgow, UK

Nordgren, Johan Linköping University, Sweden

Nyaga, Martin University of the Free State, South Africa

Oberholster, Larise University of the Free State, South Africa

Ogden, Kristen Marie Vanderbilt University Medical Center, USA

O’Neill, TrudiUniversity of the Free State, South Africa

Oni, Oluwole Oyetunde Federal University Of Agriculture Abeokuta, Nigeria

33111. Attendees

Ortega-Esteban, Alvaro Centro Nacional de Biotecnología, Spain

Ortego, Javier INIA-CISA, Spain

Palmarini, MassimoMRC, University of Glasgow, UK

Papa, Guido International Center for Genetic Engineering and Bioteghnologies, Italy

Parashar, UmeshCDC, USA

Parker, John S. L. Cornell University, USA

Parreno, Viviana Instituto Nacional de Tecnología Agropecuaria (INTA), Argentina

Patra, Upayan National Institute of Cholera and Enteric Diseases, India

Patton, John Indiana University, USA

Pereira, Priya LeenaGSK, Belgium

Pfeiffer, JulieUniversity of Texas Southwestern Medical Center, USA

Philip, Asha Ann Indiana University, USA

Pieters, Ané University of Pretoria, South Africa

Poljšak-Prijatelj, MatejaUniversity of Ljubljana, Slovenia

Poncet, Didier I2BC CNRS INRA, France

Poranen, Minna University of Helsinki, Finland

Pourcelot, MarieEnva, France

Prasad, BidadiBaylor College of Medicine, USA

Prez, Verónica Emilse Instituto de Virologia Dr JM Vanella, Argentina

Qiao, Rui Peking university, China

Racaniello, VincentColumbia University, USA

Rakau, Kebareng Sefako Makgatho Health Sciences University, South Africa

Ramani, Sasirekha Baylor College of Medicine, USA

Ranshing, Sujata Sudhir National Institute of Virology, India

Ray, Pratima Jamia Hamdard University, India

Raychaudhuri, MithuBharat Buitech, India

Rezende da Silva, JuliaEvandro Chagas Institute, Brazil


Rimstad, Espen Norwegian University of Life Science, Norway

Rodríguez-Díaz, Jesús University of Valencia, Spain

Rojas, José Manuel Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Spain

Roossinck, MarilynPenn State University, USA

Ropiak, HonorataPirbright Institute, UK

Roy, PollyLondon School of Hygiene & T, UK

Saif, Linda J. The Ohio State University, USA

Salgado, Eric Children’s Hospital Boston, USA

Sander, Wico University of the Free State, South Africa

Sandoval-Jaime, Carlos Instituto de Biotecnología UNAM, Mexico

Sen, Adrish Stanford University, USA

Sharma, Kuldeep National Institute for Research in Tribal Health, India

Shepherd, Frances Katen University of Minnesota, USA

Sherchand, Jeevan Bahadur Tribhuvan University Institute of Medicine, Nepal

Shi, Chenyan KU Leuven, Belgium

Shirafuji, Hiroaki National Institute of Animal Health, Japan

Shmulevitz, Maya University of Alberta, Canada

Simsek, CerenKU Leuven, Belgium

Singh, AmyCincinnati Children’s Hospital, USA

Soares, LuanaEvandro Chagas Institute, Brazil

Sonza, SecondoUniversity of Melbourne, Australia

Stanifer, Megan Universtiy Hospital Heidelberg, Germany

Steele, DuncanBilll and Melinda Gates Foundation, USA

Steger, Courtney Virginia Tech, USA

Stewart, Meredith MRC-University of Glasgow Centre for Virus Research, UK

Steyer, Andrej University of Ljubljana, Faculty of Medicine, Slovenia

33311. Attendees

Strydom, Amy University of the Free State, South Africa

Sung, Po-yu London School of Hygiene and Tropical Medicine, UK

Suzuki, Nobuhiro Okayama University, Japan

Suzuki, Tohru National Institute of Animal Health, Japan

Svensson, LennartLinköping University, Sweden

Tao, Yizhi Jane Rice University, USA

Tatte, Vaishali ICMR-National Institute of Virology, India

Taylor, Gwen Marie University of Pittsburgh, USA

Taylor JohnUniversity of Auckland, New Zealand

Theart, LuanNorth West University, South Africa

Theuns, Sebastiaan Ghent University, Belgium

Vainio, Eeva Johanna Natural Resources Institute Finland, Finland

van Dijk, Alberdina Aike North-West University, South Africa

Van Dycke, Jana KU Leuven - Rega Institute, Belgium

van Rijn, Piet A. Wageningen BioVeterinary Research, Netherlands

van Staden, Vida University of Pretoria, South Africa

Varanasi, Gopalkrishna National Institute of Virology, India

Vega, Celina Guadalupe INTA, Argentina

Velasquez Portocarrero, Daniel Eduardo Centers for Disease Control and Prevention, USA

Vitour, Damien ANSES, France

Wall, Gayle Victoria University of Pretoria, South Africa

Wang, Houping CDC, USA

Wang, Yuhuan Centers for Disease Control and Prevention, USA

Wessel, ØysteinNorwegian University of Life Sciences, Norway

Yandle, ZoeUCD National Virus Reference Laboratory, Ireland

Yinda, Kwe ClaudeKU Leuven, Belgium


Yssel, Litia University of Pretoria, South Africa

Yuan, Lijuan Virginia Polytechnic Institute and State University, USA

Zhou, HongUniversity of California, USA

33511. Attendees

13th international double-stranded rna · 14 abstract book | 13th international double-stranded rna...

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