Overview of the Arthropod-Borne Animal

Diseases Unit – OCT 2015 USAHA

David Scott McVey, D.V.M.,Ph.D., D.A.C.V.M.

Research Leader, Supervisory Veterinary Medical

Officer

USDA, ARS, Arthropod-Borne Animal Diseases

Research Unit (ABADRU)

Center for Grain and Animal Health Research

(CGAHR) Manhattan, KS

ABADRU’s Current Program

To solve major endemic, emerging, and exotic arthropod-borne disease problems in U.S. livestock

BSL-2 Research Domestic bluetongue (BTV) Domestic vesicular stomatitis virus (VSV) Epizootic hemorrhagic disease virus (EHDV) MP-12 Rift Valley fever virus (RVFV) Flaviviridae

BSL-3 Research Exotic bluetongue (BTV) Vesicular stomatitis virus in animals (VSV) Wild type Rift Valley fever virus (RVFV) Schmallenberg virus (SBV) and African Swine Fever Virus (ASFV) Flaviviridae

Current ABADRU Staff

Research Leader

Dr. D. Scott McVey, VMO

Scientists

Dr. Barbara Drolet-Research Microbiologist

Dr. William Wilson-Research Microbiologist

Dr. Lee Cohnstaedt-Research Entomologist

Dr. Dana Nayduch – Research Entomologist

Dr. Robert Pfannenstiel – Field Entomologist

Virologist, Computational Biologist, VMO

Multidisciplinary Problems:

Multidisciplinary Research Team

MAMMALIAN HOST

INSECT VECTOR

INFECTIOUS AGENTS

Entomologists

Veterinarians Virologists

Current Status of ABADRU

Currently 4 CRIS projects:

1. Orbivirus

2. Rift Valley fever

3. Veterinary Entomology – Vector Biology

4. Predictive Biology – Arboviruses

Viral Genetics/Computational

Biology Scientists

Summary - Whole Genotyping Studies BTV-2 in California BTV-11 from Canine Abortions EHDV-7 Israeli isolate pathogenic in cattle

Introduced BTV-3 to

USA Isolates AK, FL, MS 1999-2012

Caribbean and Central American isolates Barbados, Costa Rica, El Salvador, Honduras, Panama 1988-1991

EHDV North American strains Strains of type 1 and 2 from AL, CA, LA, TX, Alberta Strains of EHDV2 from 2012 cattle outbreak

Gaudreautl et al., J Vet Diagn Invest 26: 553, 2014 Gaudreautl et al., J Vet Diagn Invest In Press, 2015 Wilson et al., J Gen Virology In Press, 2015

Genome Segment

(Gene)

No. of strains

analyzed Related BTV strain Accession No. Gene Identity

1 (VP1) 69 BT6 USA2006/01

BT11 MTQ2010/MQ

GQ506536

JQ972861

97.6%

97.6%

2 (VP2) 171 BT2 USA2006/FL NA 94.5%

3 (VP3) 125 BT2 USA2003/FL NA 96.4%

4 (VP4) 81 BT2 USA1982/OnaB AY855272 99.9%

5 (NS1) 92 BT2 USA2006/FL

BT2 USA1982/OnaB

NA

M97680

98.0%

98.0%

6 (VP5) 113 BT2 USA1982/OnaB AY855278 99.9%

7 (VP7) 145 BT2 USA1982/OnaB AF188660 99.8%

8 (NS2) 105 BT2 USA1982/OnaB AY855287 99.5%

9 (VP6/NS4) 148 BT2 USA1982/OnaB AF403421 99.5%

10 (NS3) 301 BT2 USA1982/OnaB L08628 99.8%

BTV- 2 in California

MacLachlan et al., Emerg. Inf. Diseases 19:66, 2013

Gaudreault et al., JVDI, in press.

What Happens in the Host During Culicoides Midge feeding??

What physiological changes occur in the host in response to the bite wound and the insect saliva deposited during feeding?

What types of immune responses are elicited?

What role, if any, do these host responses play in the ability of virus in the insect’s saliva to infect?

Drolet Lab

Culicoides Blood Feeding: Murine Model

30’

Drolet Lab

hemorrhage

vasodilation

Midge feeding results in hemorrhaging, dilation of blood vessels and swelling (edema) due to wounds caused by mouthparts as well as the host’s immune responses to the insect saliva

Effect of Culicoides Bites on Arbovirus Transmission

Drolet Lab

The host’s reaction to feeding damage and saliva results in recruitment of specific immune cells to the bite site.

These cells are known targets for infection by bluetongue and epizootic hemorrhagic disease viruses

Infected cells drain to nearby lymph nodes

Once in the nodes, more immune cells are made in response to the infection (hyperplasia) and viruses establish centers of replication and efficiently spread throughout the host via the lymphatic system

Hyperplasia

Cells in the walls of blood vessels (endothelium) are also known targets for BTV and EHDV

These cells are exposed to virus because midge mouthparts breach vessels and midge saliva causes dilation and permeabilization of the vessel walls

Both the bite damage and the reaction to the saliva make it very easy for viruses to get into vessels, infect the endothelial cells and spread throughout the host via the circulatory system

Effect of Culicoides Bites on Arbovirus Transmission

Drolet Lab

Culicoides feeding results in a very favorable environment for arboviral infection by recruiting susceptible cell types to the bite site and facilitating dissemination throughout the host via blood and lymph circulatory systems.

EHDV BTV

BTV EHDV

EHDV BTV

Microspheres with differing fluorescent signatures

Each microsphere is labeled with either EHDV or BTV specific antigens

Beads are mixed together and incubated with serum from animals containing antibodies to EHDV, BTV, both, or neither

A species-specific secondary antibody conjugated with a red protein-pigment (Phycoerythrin, PE) is added to detect serum antibody binding

The microspheres are “read” for the presence of the fluorescent signature and PE signal.

Multiplex Fluorescent Microsphere Assays to Detect Viral Antibodies

BTV microsphere

EHDV microsphere *

BTV+ cutoff EHDV+ cutoff

Serum samples with antibodies to BTV, EHDV, both or neither

Multiplex Fluorescent Microsphere Assays to Detect Viral Antibodies

†Cutoff values are 2 standard deviations above negative background

*The ratio of PE intensity to number of beads is reported as the Median Fluorescent Intensity (MFI)

†

Microspheres with differing fluorescent signatures

Each microsphere is labeled with EHDV or BTV specific oligo linker

Beads are mixed together and incubated with the biotinylated cDNA

The microspheres are “read” for the presence of the fluorescent signature and PE signal.

Multiplex Fluorescent Microsphere Assays to Detect Viral RNA

RNA is extracted from blood samples from animals infected with BTV, EHDV, both or neither. Biotinylated viral primers are used in PCR make biotinylated cDNA virus targets

Streptavidin PE complexes are added which bind to the biotin

BTV EHDV

EHDV BTV

EHDV BTV

Current EHDV research at ABADRU

Experimental infection of WTD with EHDV-2

1. Compare infection rates in C. sonorensis after feeding on WTD with variable viremia profiles over the course of infection.

2. Determine if the bite from a single infected C. sonorensis can transmit EHDV to susceptible WTD.

M. Lee

EHDV and BTV

L Cohnstaedt

Host Pathogen

Environment

Disease

Broad research interests in epidemiology, transmission, and pathobiology of EHD and BT in domestic and wild ruminants

Research to incriminate regional vectors of EHDV and BTV are

needed

Culicoides Ecology Research Projects

Ongoing field and laboratory studies aimed at better understanding Culicoides ecology as it relates to the epidemiology of BTV, EHDV, and other arboviruses

Culicoides Ecology Research Projects

Primary objectives

1. Determine Culicoides spp. distribution and abundance in areas of Orbivirus outbreaks in cattle and deer

2. Determine the impact of habitat characteristics (biotic and abiotic) on breeding site quality for Culicoides spp.

3. Understand the ruminant host associations of Culicoides spp. and evaluate suspect vector species for susceptibility to Orbivirus infection

Culicoides control and surveillance

• Insecticidial sugar trap – Photo attraction of sugar feeding

midges

– Insecticidal sugar bait effectiveness studies • Toxic substances

• Non-toxic substances (natural products)

• EHD virus detection from single infected midge feeding

Dana Nayduch Research Molecular Biologist

Culicoides sonorensis - Biting midge

Molecular and microbiological studies of vector competence:

• Comparative transcriptomics

• Molecular and cellular biology of midge, gene function

• Establishing RNAi as tool for midge functional analyses

• Current work: Microbe-midge and virus-midge interactions

Arthropod-Borne Animal Diseases Research Unit

Nayduch Lab

1st reference transcriptome for Culicoides • Will guide genome project • 1st report of expression

profiles associated with diet • Described molecular

components of anautogeny/vitellogenesis

Nayduch Lab

First description of innate immune

pathways in midges

Inducing RNA interference in the arbovirus vector, Culicoides sonorensis

1st demonstration of RNAi in Culicoides • Identified and annotated the core

machinery of the siRNA and apoptosis pathways in C. sonorensis.

• Developed successful dsRNA injection technique for inducing knockdown

• Showed functional RNAi mechanism by IAP1 depletion (phenotype, mRNA level)

M.K. Mills, D. Nayduch, K. Michel

Conclusion

Research Recommendations

Report Published

Orbivirus Gap Analysis

Vector-Borne and Zoonotic Diseases / June 2015, 15(6)

http://online.liebertpub.com/toc/vbz/15/6#utm_source=ETOC&utm_medi

um=email&utm_campaign=vbz

Gap Analysis Summary Virology: Elucidating key aspects of virus-vector-host interactions,

pathogenesis, epidemiology, and control strategies.

Surveillance: Understanding the ecosystems supporting the arthropod transmission of BTV and EHDV in different climatic and geographic zones.

Diagnostics: Improving the distribution of diagnostic capabilities nationally, with the availability of validated tests and sample processing capacity.

Vector Control: Understanding midge-host attack behavior to develop better vector control measures.

Vaccines: Addressing the gaps in the limited number of available vaccines for BTV, and EHDV for which there are no vaccines.

Research Goals from an ABADRU

Perspective

Model surveillance approaches – regional

Serological, virus, molecular, regional, inter-epidemic ??

Collaborative test improvement/development

Availability

QA/QC

Epidemiology/Interpretation

Pathogenesis

Innate, inflammatory, vector

Immunity

Inform vaccine research and development

D. Scott McVey, DVM, PhD

Research Leader – ABADRU

scott.mcvey@ars.usda.gov

785-537-5561

Contact