Location: Arthropod-borne Animal Diseases Research
2019 Annual Report
Objectives
Objective 1. Determine vector biology and environmental maintenance of orbiviruses to inform future surveillance programs.
Sub-Objective A. Using historical data available from veterinary diagnostic laboratories, identify areas of active orbivirus transmission and subsequently identify ecological characteristics within these distinct transmission areas.
Objective 2. Identify determinants of orbiviral replication in vertebrate and invertebrate hosts.
Sub-Objective A.Identify factors in virus-vector-host interactions to inform the development of improved, vector-enhanced experimental animal infection models.
Sub-Objective B. Identify the factors modulating adaptive mammalian immune responses to orbiviruses to inform the development of vaccines.
Sub-Objective C. Determine the effect of EHDV replication mechanisms on vector competence and transmission.
Approach
Bluetongue virus (BTV) is transmitted by Culicoides midges to wild and domestic
ruminants, especially sheep, and results in significant economic losses from
decreased animal production and non-tariff trade restrictions on animals and animal products. Of the 26 BTV serotypes, only five are considered domestic to the U.S., although 10 exotic types have been introduced since 1999. There is an everincreasing need for veterinary diagnostic laboratories to reliably detect multiple serotypes in submitted samples. We propose to develop rapid, sensitive, specific diagnostic assays to detect and differentiate multiple serotypes of BTV and anti-BTV antibodies in cattle and sheep from a single blood or serum sample. There are major gaps in understanding underlying mechanisms of disease and transmission of different serotypes, not only at the level of virus-vector-host interaction, but also at the herd and animal population levels. One major issue is our inability to experimentally demonstrate clinical bluetongue disease in sheep and cattle, critical for understanding pathogenesis and vaccine development and evaluation. Traditional injection infection models completely remove the insect from the equation and expose cell types and elicit immune responses atypical of natural infections. These dissimilarities may play a significant role in the clinical disease differences seen in natural versus laboratory infections. We will evaluate the role of virus delivery
routes (subcutaneous versus intradermal) and the role insect salivary proteins play in virus infection, pathogenesis and immune responses to BTV. The long term goal is to develop a robust BTV infection and disease animal model; a critical need for bluetongue infection, pathogenesis and vaccine research.
Progress Report
Progress related to Objective 1: Collaborators at Kansas State University (KSU) have identified 500 serum samples from cattle across 9 regions of Kansas which represent diverse geographic regions and varied cattle management operation systems. Importation permits have been obtained for enzyme-linked immunosorbent assay (ELISA) kits from overseas, the kits have been ordered and sent to KSU-Veterinary Diagnostic Laboratory (KSU-VDL). ELISA testing of the cattle sera by KSU-VDL is ongoing to determine the presence of antibodies to the hemorrhagic orbiviruses, bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). Subsequent analysis of testing results will provide an estimate of serological prevalence in distinct regions of Kansas and transmission zones will be determined. Additionally, preliminary syndromic serological testing of VDL submissions for BTV and EHDV have been discussed and may be initiated after the surveillance data are available.
Progress related to Objective 2: Interferon receptor knock-out (IFNAR) mice and the parental strain (C57 Black) mice were inoculated with bluetongue virus that was mixed with Culicoides salivary proteins or media alone. Blood samples were taken daily to monitor viremias and multiple tissue samples were taken during necropsies. Blood and tissues samples have been processed and tested by viral plaque assay and real time polymerase chain reaction (PCR) for viral titers and systemic distribution. Analysis of results is in progress. Preliminary data suggests mice receiving virus that had been mixed with Culicoides salivary proteins showed slightly more severe clinical disease scores. Additionally, these mice had higher titers of virus in tissues and longer persistence of ribonucleic acid (RNA) in tissues.
Culicoides salivary proteins are being collected and purified to use in a sheep study to determine their effect on the protective innate and adaptive immune responses elicited by a bluetongue killed vaccine.
With collaborators in The Netherlands, we showed that genome segment 10 (S10, which encodes nonessential NS3/NS3a protein, is required for virus release from cultured Culicoides cells and virus propagation in the competent vector insects, Culicoides sonorensis. Additionally, we showed that this same protein is important for bluetongue viruses to escape the midgut barrier in Culicoides sonorensis biting midges.
EHDV and BTV proteins were cloned and expressed for use in developing a multiplex fluorescent microsphere immunoassay to detect antibodies to these viruses in deer sera. Field sample test validation is ongoing with field collected white-tailed deer sera.
With collaborators at the University of Georgia, Southeast Cooperative Wildlife Infectious Disease Study Group we determined that increases in the amounts of epizootic hemorrhagic disease virus serotype 2 (EHDV-2) in deer correlated with increased likelihood that midges would successfully acquire virus and increased proportion of midges that became competent to transmit the virus to another animal. Additionally, midges became infected after feeding on deer that no longer had a detectable viremia.
Accomplishments
1. Epizootic hemorrhagic disease virus (EHDV) infection affects sensory and neural tissues in Culicoides midges. Female Culicoides biting midges are vectors of EHDV, which causes disease in wild and domesticated ruminants. With collaborators at Clemson University and University of South Carolina Aiken, ARS scientists in Manhattan, Kansas, identified key changes in female midge gene expression profiles occurring during early infection with EHDV type 2. Genes that had decreased expression included those for sensory functions (especially vision), behavior, learning, and memory. These genes are important in pathways related to cell/tissue structure and integrity, eye morphogenesis and neural development. Genes that had increased expression included those for immune processes, odor, and light detection. Our results suggest that EHDV-2 infection of midges may have a significant effect on sensory and neural tissues. These results suggest a change in light wavelength needed to optimally trap infected insects during outbreaks and for viral circulation surveillance.
2. Epizootic hemorrhagic disease virus (EHDV) infections of blood-feeding midges correlate to virus levels in deer. The impact of variation in the level and duration of virus in the blood of infected white-tailed deer on midge infection is not well characterized. With collaborators at the University of Georgia, ARS scientists in Manhattan, Kansas, found that increases in deer EHDV blood titers significantly increased both the likelihood that midges would successfully acquire EHDV and the proportion of midges that reached the titer threshold for transmission competence. Unexpectedly, we identified infected midges after feeding on one deer when virus was no longer detectable. These significant results are important in helping to understand the epidemiology of this disease and suggests that deer with undetectable virus can still serve as a virus source for blood-feeding midges.
Review Publications
Maclachlan, N., Zientara, S., Wilson, W.C., Richt, J., Savini, G. 2019. Bluetongue and Epizootic Hemorrhagic Disease Viruses: Recent developments with these globally re-emerging infections of ruminant animals. Popular Publication. 34:56-62. https://doi.org/10.1016/j.coviro.2018.12.005.
Mendiola, S.Y., Mills, M.K., Maki, E.C., Drolet, B.S., Wilson, W.C., Berghaus, R., Stallknecht, D.E., Breitenbach, J.E., Mcvey, D.S., Ruder, M.G. 2019. EHDV-2 infection prevalence varies in Culicoides sonorensis after feeding on infected white-tailed deer over the course of Viremia. Viruses. 11:371-385. https://doi.org/10.3390/v11040371.
Nayduch, D., Shankar, V., Mills, M., Robl, T., Drolet, B.S., Ruder, M., Scully, E.D., Saski, C. 2019. Transcriptome response of female Culicoides sonorensis biting midges (Diptera: Ceratopogonidae) to early infection with epizootic hemorrhagic disease virus (EHDV-2). Viruses. 11:473. https://doi.org/10.3390/v11050473.
Schirtzinger, E.E., Jasperson, D.C., Ostlund, E.N., Johnson, D.J., Wilson, W.C. 2017. Some recent US Bluetongue virus serotype 3 isolates found outside of Florida indicate evidence of reassortment with endemic co-circulating serotypes. Journal of General Virology. https://doi.org/10.1099/jgv.0.000965.
Wang, L., Lanka, S., Cassout, D., Mateus-Pinilla, N., Li, G., Wilson, W.C., Yoo, D., Frederickson, R. 2019. Inter-serotype reassortment among epizootic hemorrhagic disease viruses in the United States. Transboundary and Emerging Diseases. https://doi.org/10.1111/tbed.13257.
Schirtzinger, E.E., Ruder, M.G., Stallknecht, D.E., Wilson, W.C. 2019. Complete genome sequence of a 2016 Bluetongue virus serotype 3 isolate from Louisiana, USA. Microbiology Resource Announcements. 8:e00128-19. https://doi.org/10.1128/MRA.00128-19.
Schirtzinger, E.E., Jasperson, D.C., Ruder, M.G., Stallknecht, D.E., Chase, C.C., Johnson, D.J., Ostlund, E.N., Wilson, W.C. 2019. Evaluation of 2012 US EHDV-2 outbreak isolates for genetic determinants of cattle infection. Journal of General Virology. 100:556-567.
