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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
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