Location: Arthropod-borne Animal Diseases Research
2020 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
Objective 1: Toward better understanding the maintenance of orbiviruses in the environment and help design future surveillance programs, significant progress was made on developing serology tests to detect antibody to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in cattle, sheep, and deer. Serology tests inform livestock owners as to where and how often orbiviruses are present in their animal herds, and the overall level virus circulation in specific regions in the U.S. This, in turn, informs their level of outbreak risk for these hemorrhagic diseases. Currently there are five serotypes, or strains, of BTV recognized as endemic in the U.S. and three strains of EHDV. Diagnostic assays must detect antibody to all 8 of these viruses to accurately diagnose whether an animal has been exposed. Fluorescent bead-based serological tests for BTV and EHDV are in various stages of development. These tests can detect antibodies to all the U.S. BTV and EHDV from a single serum sample, are cost-effective, accurate, and faster than the currently available tests.
Cattle can become infected with either BTV, or EHDV, or both simultaneously. The need for a rapid, cost effective diagnostic assay to detect both BTV and EHDV antibodies in cattle was identified by livestock production stakeholders. Sera from experimentally infected cattle was used to develop, optimize, and lab-validate the assay. Field samples from South Dakota and Nebraska were then used to field-validate the assay for both viruses. Comparisons with other diagnostics such as western blots and enzyme-linked immunosorbent assays (ELISAs) were conducted to compare sensitivity and specificity. The cattle assay is capable of detecting antibody to all five endemic BTV strains and all three endemic EHDV strains and can discern BTV from EHDV antibodies in a single serum sample.
Sheep are highly susceptible to BTV with mortality rates as high as 50%. Sera from experimentally infected sheep was used to develop, optimize and lab-validate the test for BTV. Field sera from Wyoming was then used to field-validate the test. Sensitivity and specificity levels of the test were determined by comparing results with other available tests. The sheep assay is capable of detecting antibody to all five endemic BTV strains in a single serum sample.
Deer farming is one of the fastest growing industries in rural America, generating nearly $8 billion for the U.S. economy and supporting tens of thousands of jobs. Additionally, deer serve as the sentinel species for both BTV and EHDV outbreaks and are key to understanding the epidemiology of these diseases and livestock disease risk assessments. The need for a rapid, cost effective diagnostic assay to detect both BTV and EHDV antibodies in deer was identified by deer farmer, wildlife management, and animal diagnostic laboratory stakeholders. Assay development was initiated with archived sera from collaborators in Florida. Sensitivity and specificity levels will be determined by comparing testing results with other available diagnostic assays. Additional samples from recent outbreaks in Kansas, Florida, and Georgia were recently acquired for subsequent field-validation of the assay.
Objective 2: Toward understanding of how orbiviruses replicate themselves in both animals and insects, progress was made on several virus-vector-host investigations. Identifying genetic and protein factors involved in these interactions help to develop improved, vector-enhanced experimental animal infection models, find targets within the insect vector that could be used to block virus transmission, and potentially identify other organisms within the insect that may be exploited to block transmission. Progress was made on the analysis of the effects of Culicoides salivary proteins on mammalian immune responses and the subsequent effects on BTV infections. Experimental infection of cattle and sheep often lack the clinical disease of natural, midge-transmitted infection seen in outbreaks. This makes it very difficult to study pathogenesis and evaluate vaccine candidates. We found mammalian immune responses to the saliva of the biting midge results in an inflammatory reaction that most likely contributes to the clinical disease seen in natural infections. This may lead to a more natural, vector-enhanced, clinical disease animal infection model for future investigations.
Blood feeding is a critical component of BTV and EHDV transmission by insects. Fevers in infected sheep, cattle, and deer often correlate to high amounts of virus in the blood. ARS researchers are investigating whether midges have a blood meal temperature preference and if this preference changes when insects are infected with BTV or EHDV. Preliminary results show an increased preference for higher bloodmeal temperatures for the midge’s first meal. This suggests midges may target infected, feverish animals for bloodmeals. This is an advantage to the virus as it increases the likelihood of being picked up by the insect. Results also showed an increased preference for lower temperature bloodmeals on subsequent feedings, suggesting that infected midges may then target healthy, non-febrile animals. Again, this is an advantage to the virus as it increases the likelihood of virus being transmitted to healthy animals. These results will inform transmission dynamics and overall BTV and EHDV epidemiology, critical for outbreak control strategies.
Wolbachia is a bacterium that lives in the gut of many insect species without deleterious effect to the insect. This gut microbe has been shown to alter infection rates of several viruses for those insect species. Recently, low levels of Wolbachia were detected in Culicoides midges. In collaboration with Texas A&M researchers, the effects of Wolbachia on BTV and EHDV infections of Wolbachia infected Culicoides cells was initiated. Preliminary results show Wolbachia is able to inhibit BTV infection in cells. Results of these in vitro cell culture studies will inform whether in vivo insect infections should be pursued and whether increasing and stabilizing Wolbachia infections in midge populations could be an effective strategy for controlling midge transmission of orbiviruses.
Accomplishments
1. Very minor genetic differences in viruses can result in major differences in their ability to be transmitted by midges. Transmission of viruses by insects is a complex mechanism consisting of many different processes in both the insect and in the animals on which they feed. Insects must obtain virus from an infected animal during blood feeding. The virus must then replicate itself and disseminate within the insect so that it reaches the salivary glands to be transmitted to another animal when the insect takes its next blood meal. Bluetongue virus (BTV) is transmitted by biting midges (Culicoides) and causes severe, economically devastating disease in sheep, cattle and deer. Researchers at Manhattan, Kansas, in collaboration with researchers at Wageningen University, The Netherlands, used genetic approaches to determine that small changes in specific proteins of the virus significantly affect the virus’ ability to multiply in these insects and therefore dictate whether virus could be transmitted or not. To date there are 28 different types of BTV that cause varying degrees of disease and are transmitted by various Culicoides spp. midges. These results suggest that types of BTV differing only slightly in their genetic make-up, may have significantly different abilities to infect, disseminate within, and be transmitted by midges. Understanding these specific virus-vector interactions is crucial for determining vector competence, assessing bluetongue disease risk, and predictive modeling.
Review Publications
Van Gennip, R., Drolet, B.S., Rozo Lopez, P., Roost, A., Boonstra, J., Van Rijn, P. 2019. Vector competence is strongly affected by a small deletion or point mutations in bluetongue virus. Parasites & Vectors. 12:470. https://doi.org/10.1186/s13071-019-3722-2.
Schirtzinger, E.E., Jasperson, D.C., Ostlund, E.N., Johnson, D.J., Wilson, W.C. 2018. Some recent US Bluetongue virus serotype 3 isolates found outside of Florida indicate evidence of reassortment with endemic co-circulating serotypes. Virus Genes. 99:157-168.
Sunwoo, S., Noronha, L.E., Morozov, I., Trujillo, J.D., Kim, I., Schirtzinger, E., Faburay, B., Drolet, B.S., Urbaniak, K., McVey, D.S., Meekins, D.A., Palmer, M.V., Balaraman, V., Wilson, W.C., Richt, J.A. 2020. Evaluation of a baculovirus-expressed VP2 subunit vaccine for the protection of white-tailed deer (Odocoileus virginianus) from epizootic hemorrhagic disease. Vaccines. 8(1):59. https://doi.org/10.3390/vaccines8010059.
