Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. This is the final report for this project which terminated December 2021. See the report for the replacement project, 3020-32000-020-00D, ⿿Ecology of Hemorrhagic Orbiviruses in North America for additional information. Objective 1. Progress was made towards orbivirus transmission zones and serosurveillance as described below. In collaboration with Kansas State University (KSU), serological surveys of cattle herds in Kansas and Nebraska were conducted in 2016. High transmission rates of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) were detected, yet low incidence of clinical disease was reported. In the presence of infected midges, wildlife and livestock are continually at risk for BTV or EHDV infection and within-farm transmission rates can be high. This endemic boosting may contribute to population immunity and low incidences of disease. A bead-based diagnostic test was developed to determine whether a cow has been infected with BTV, EHDV, or both, simultaneously in a single serum sample. The assay showed increased performance compared to the antibody test currently in use for orbiviruses in veterinary diagnostic laboratories. Rapid, accurate identification of these pathogens can help reduce time in providing supportive therapies for sick animals, identify the need for and extent of quarantine/animal restriction policies, and inform predictive risk assessments. Sheep are the primary susceptible livestock species for BTV. Experimental sera were used to develop and lab-validate a bead-based diagnostic assay for BTV. Field sera from Wyoming were used to field-validate the assay. Comparisons with western blots and available antibody tests were used to determine sensitivity and specificity. The sheep assay can detect antibody to all endemic BTV serotypes simultaneously in a single serum sample. Deer serve as sentinel species for both BTV and EHDV and provide critical information for epidemiology and livestock risk assessments. Additionally, deer farming generates an estimated $8 billion for the U.S. economy. The development of a test to detect both BTV and EHDV in deer sera was initiated with serum samples from collaborators in Florida. Comparisons with western blots and currently availalble antibody assays with field samples from Kansas, Georgia, and Florida to determine sensitivity and specificity are ongoing. Having a rapid, sensitive serological assay to detect all endemic BTV and EHDV serotypes in this key wildlife species will inform virus circulation rates and livestock disease risk modeling. Objective 2. Progress was made towards understanding virus-vector-host interactions of the orbiviruses BTV and EHDV as described below. BTV stocks grown in Culicoides midge cells and containing cellular proteins were 28 times more lethal in mice than stocks grown in the hamster cells typical used to grow virus. Thus far, whole genome sequencing of the stocks has not identified significant differences that would have led to amino acid changes such as viral protein folding, charge, or hydrophobicity. This suggests insect proteins in the stock may have caused the increased virulence. In a follow-up study to determine if increased virulence could be elicited from just midge salivary proteins, mice were inoculated with BTV with or without salivary proteins. Analysis of daily blood samples and necropsy tissues for infectious virus and viral RNA suggests the salivary proteins caused higher virus titers and longer persistence in tissues and caused slightly more severe clinical disease in mice but did not cause significant increases in mortality. In collaboration with KSU, the immunogenicity of Culicoides cell culture proteins were evaluated as potential vaccine adjuvants. Cellular proteins were fractionated and tested in mice with a known antigen. None of the protein fractions significantly enhanced immune responses to the known antigen. In collaboration with Mississippi State University, four midge salivary genes were cloned and expressed to evaluate their ability to elicit innate and adaptive immune responses in sheep in the presence of a killed BTV vaccine and commercial adjuvant. Sera was tested for anti-BTV antibody levels and cytokines are being analyzed. Sheep receiving salivary proteins had a slightly faster antibody response compared to sheep receiving the BTV vaccine alone. In collaboration with KSU, the effect of BTV infection on insect gene expression in female C. sonorensis midges was performed on midges that were fed with blood spiked with virus in media, blood with media only, or a sugar meal. RNA was sequenced and compared across treatments to identify differentially expressed genes, gene families, and gene networks. This research provides insight into how midges respond genetically to orbivirus infection, may elucidate anti-viral defense mechanisms, and may determine the effect of BTV infection on midge neurosensory function. Progress was made in determining whether orbivirus infection alters host- seeking feeding behavior of biting midges. Studies to determine if midges have a bloodmeal temperature preference showed an increased preference for higher temperatures for the midge⿿s first meal, suggesting they may target infected, febrile animals for feeding. This favors virus transmission to the insect vector. Lower temperature bloodmeals were then preferred for subsequent feedings, suggesting that after midges feed on infected febrile hosts, they may then target healthy, non-febrile animals. This increases the likelihood of virus being transmitted from infected to healthy animals. Results inform transmission dynamics and overall BTV epidemiology, critical for outbreak control strategies. With collaborators at the University of Georgia, increases in the amounts of EHDV in deer were shown to correlate with increases in midges successfully acquiring and transmitting virus. It was also determined that midges can become infected after feeding on deer that no longer has a detectable viremia. In collaboration with Texas A&M, Wolbachia, a bacteria that naturally lives in midge midguts, was shown to affect the ability of both BTV and EHDV to infect midge cells. In collaboration with Clemson University and University of South Carolina, Aiken, changes were identified in midge sensory and neural tissue gene expression after EHDV infection. Downregulated genes for sensory functions (especially vision), behavior, learning and memory are important in pathways related to cell/tissue structure and integrity, eye structure and neural development. Genes for immune processes, odor and light detection were upregulated. EHDV infection of midges may affect sensory and neural tissues which may change the light wavelength needed to optimally trap infected insects. In collaboration with researchers at Wageningen University, The Netherlands, small changes in specific proteins of BTV significantly affected its ability to multiply in and be transmitted by midges. This suggests that strains of BTV with small genetic differences, may have significantly different abilities to infect, disseminate within, and be transmitted by midges. In collaboration with researchers at Wageningen University, The Netherlands, bacteria populations in the gut of Culicoides midges changed when midges were exposed to antibiotics. This change reduced the ability of gut bacteria to dampen virus replication and subsequent transmission. The use of antimicrobial compounds at livestock farms might therefore increase the risk of midge-transmitted viruses. In collaboration with KSU, the dynamics of EHDV infection in midges was characterized. Virus infected the insect⿿s midgut, escaped the midgut, amplified, and was disseminated to other organs. Virus reached the salivary glands for bite transmission 5 days after feeding on an infectious blood meal. Rates of infection, dissemination, and viral titers were determined. Eleven new serotypes of BTV have been isolated in the southeastern U.S. since 1999. Serotype 3 has been isolated multiple times and spread as far as Mississippi. Whole genome sequence analysis revealed these serotypes reassorted with endemic U.S. virus types. Previously we showed that Culicoides midges deposit saliva containing allergens, proteases, and anti-hemostatic factors into the dermis when biting to facilitate blood-feeding. If infected, virus is also deposited. To understand the extreme efficiency with which midges transmit orbiviruses, the physiological and immune responses to saliva and the bite were examined in a mouse/midge feeding model. Skin, blood, and lymph node cells and cytokines were analyzed. Midge feeding resulted in mast cell degranulation triggering a potent pro-inflammatory Th-mediated cellular response. Hematomas, edema, vasodilation, and leukocyte infiltration to the bite sites were observed and were highly favorable for midges to feed completely. Virus can rapidly establish infection in infiltrating lymph cells and exposed endothelium and then disseminate in the animal through the lymph and circulatory systems. Culicoides midges collected during an EHDV outbreak at a Minneapolis zoo were received, taxonomically identified, and tested for EHDV to better understand which species contributed to the outbreak. Two midge species, C. sonorensis and C. variipennis, were likely transmitting EHDV to the zoo animals to cause the outbreak. An embryonated chicken egg infection/transmission model was developed and is being optimized for midge-transmitted orbivirus investigations. This provides an in vivo transmission model without use of animals and provides a key capacity for all midge transmission competence research for ARS.
Impacts
(N/A)
Publications
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Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. Objective 1. Progress on Orbivirus transmission zones and serosurveillance continued as follows: Serological surveys of cattle herds in Kansas and Nebraska conducted in 2016 showed high transmission rates of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), yet low incidence of clinical disease. Disease incidence in white- tail deer and other susceptible wild ungulates also provided evidence of virus transmission in these regions. To date, factors which contribute to emergence of more significant clinical disease in livestock and wildlife populations in any given vector season remain undefined and require more in-depth studies of population-level serology (with technically improved assays), more thorough disease investigation and diagnostic studies, and more thorough field ecology studies (climatology, entomology, vector habitat, and disease epidemiology). Development of a bead-based assay to determine exposure of cattle to BTV, EHDV, or both in a single serum sample was completed and validated with both experimental and field- collected sera. The assay provides a rapid, sensitive, specific assay for orbivirus serosurveillance in cattle. Development of a bead-based assay to determine exposure of white-tailed deer to BTV, EHDV, or both in a single serum sample was initiated with field-collected deer sera. The completed assay will provide a rapid, sensitive, specific serosurveillance assay for the captive cervid industry. Objective 2. Progress on Orbivirus (BTV and EHDV) investigations of virus- vector-host interactions continued as follows: BTV grown in Culicoides midge insect cells was shown to be more virulent to mice than virus grown in mammalian cells that are traditionally used for experimental animal studies. Sequencing efforts have begun in order to determine whether specific differences in viral genomes is associated with the observed increased virulence. Protein analyses and mouse studies have begun for identification and evaluation of immunogenic proteins from midge cell culture as potential vaccine adjuvants for killed orbiviral vaccines. Two Culicoides midge salivary genes thought to have immunogenic properties have been cloned and expressed to evaluate their ability to elicit innate and adaptive immune responses in sheep. Culicoides midge species collected during an EHDV outbreak at a Minneapolis zoo were received from zoo officials, taxonomically identified, and are being tested for EHDV to better understand which species contributed to the outbreak. Development of an embryonated chicken egg infection model was initiated for midge- transmitted arbovirus investigations. When complete, this will provide an in vivo transmission model without use of animals and will provide a key capacity for all midge transmission competence research for ARS researchers in Manhattan, Kansas. Research on the effect of BTV infection on insect gene expression in female Culicoides sonorensis midges was performed. Female midges were fed either blood spiked with virus in cell culture media, blood with culture media only, or a sugar meal. RNA was sequenced and is being compared across treatments to identify genes, gene families, and gene networks that are differentially expressed. This collaborative research will provide insight into how midges respond genetically to orbivirus infection, elucidate anti-viral defense mechanisms, and determine the effect of BTV infection on midge neurosensory function. Future behavioral studies will determine whether altered gene expression results in altered neurosensory behavior. Record of Any Impact of Maximized Teleworking Requirement: Due to maximum teleworking and a full-stop mandate by the collaborator⿿s university on large animal studies from the start of pandemic to date for the sake of animal welfare, the sheep studies were not initiated in FY20- 21. It is unclear whether they will be rescheduled. ACCOMPLISHMENTS 01 Rapid, cost-effective orbivirus diagnostics for cattle. Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are transboundary, midge-transmitted orbiviruses that cause moderate to severe disease in domestic and wild ruminants. Economic losses to the cattle industry are due to decreased animal production, but most significantly from trade restrictions on movement of animals and animal products. ARS researchers, in Manhattan, Kansas, developed a bead-based diagnostic test that can simultaneously determine whether a cow was infected with BTV, or EHDV, or both, in a single serum sample. When compared to antibody tests currently in use for orbiviruses in veterinary diagnostic laboratories, the bead-based assay showed increased performance. For effective disease surveillance and accurate trade regulation implementation, rapid, sensitive, specific assays such as this allows veterinarians, animal health inspectors, and livestock owners to determine if cattle have been exposed to one or both of these orbiviruses. 02 Bacteria in the gut of Culicoides midges affect their ability to transmit viruses. Culicoides midges transmit several viruses to domestic and wild ruminants in the United States and in Europe. ARS researchers, in Manhattan, Kansas, in collaboration with researchers at Wageningen University, the Netherlands, showed some midge species had higher rates of virus infection after being exposed to antibiotics due to changes in the bacterial communities in their guts. Resident gut bacteria may dampen virus transmission in biting midges and the use of antimicrobial compounds at livestock farms might therefore have an unexpected contradictory effect on the health of animals by increasing the risk of Culicoides-transmitted arbovirus transmission. Understanding how gut bacteria influence the ability of insect vectors to transmit viruses contributes to the search for new and effective disease control strategies for vector-borne diseases. 03 High rates of Orbivirus transmission yet low rates of clinical disease in Kansas and Nebraska cattle in 2016. Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) are orbiviruses that infect both domestic and wild ruminants and are transmitted but Culicoides biting midges. BTV has the greatest economic impact with losses attributed to effects on animal health and productivity, as well as non- tariff trade restrictions that affect the sale and movement of animals and germplasm. ARS researchers, in Manhattan, Kansas, in collaboration with researchers at Kansas State University and the University of Nebraska-Lincoln showed the serological and case diagnostic evidence of BTV and EHDV transmission, with limited disease incidence. It is not clear what factors allow high transmission rates with little clinical disease, nor what factors contribute to emergence of more significant clinical disease in livestock and wildlife in certain years and regions. Disease in white-tailed deer and other susceptible ungulates also provided evidence of virus transmission, although again the incidence of disease was low. A more complete understanding of the BTV and EHDV ecologies will require more in-depth study of population-level serology (with technically improved assays), more thorough disease investigation and diagnostic studies, and more thorough field ecology studies (climatology, entomology, and disease epidemiology).
Impacts
(N/A)
Publications
- McGregor, B.L., Erram, D., Alto, B.W., Lednicky, J., Wisely, S.M., Burkett- Cadena, N.D. 2021. Vector competence of Florida Culicoides insignis (Diptera: Ceratopogonidae) for epizootic hemorrhagic disease virus serotype 2. Viruses. 13(3):410. https://doi.org/10.3390/v13030410.
- Drolet, B.S., Reister-Hendricks, L.M. 2021. A duplex fluorescent microsphere immunoassay for detection of bluetongue and epizootic hemorrhagic disease virus antibodies in cattle sera. Viruses. 13(4):682. https://doi.org/10.3390/v13040682.
- Kopanke, J., Lee, J., Stenglein, M., Carpenter, M., Cohnstaedt, L.W., Wilson, W.C., Mayo, C. 2021. Exposure of Culicoides sonorensis to enzootic strains of bluetongue virus demonstrates temperature- and virus-specific effects on virogenesis. Viruses. 13(6):1016. https://doi.org/10.3390/ v13061016.
- Mohlmann, T., Vogels, C., Goertz, G., Pijlman, G., Ter Braak, C., Te Beest, D., Hendriks, M., Nijhuis, E., Warris, S., Drolet, B.S., Van Overbeek, L., Koenraadt, C. 2020. Impact of gut bacteria on the infection and transmission of pathogenic arboviruses by biting midges and mosquitoes. Microbial Ecology. 80:703-717. https://doi.org/10.1007/s00248-020-01517-6.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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.
Impacts
(N/A)
Publications
- 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.
- 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.
- 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.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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 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 01 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. 02 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.
Impacts
(N/A)
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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Objective 1: We have identified collaborators at the Kansas State University (KSU) Veterinary Diagnostic Laboratory (VDL) who have previously collected sets of sera from cattle from multiple regions of the state of Kansas. These collections of sera represent diverse geographic regions and varied cattle management operation systems. As a means of passive, retrospective surveillance, these sera will be tested for the presence of antibodies to the closely related hemorrhagic orbiviruses, bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), by enzyme-linked immunosorbent assay (ELISA). Subsequent analysis of testing results will provide an estimate of serological prevalence in distinct regions of Kansas. We anticipate testing to begin in Fall of 2018. Further testing of these sera will depend on the results. 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. Objective 2: Interferon receptor knock-out (IFNAR) mice and the parental strain (C57 Black) mice were inoculated with bluetongue virus that had either been propagated in mammalian or insect cell cultures. Blood samples were taken daily to monitor viremias and multiple tissue samples were taken during necropsies. Blood and tissues samples are being processed and tested by viral plaque assay and real time PCR for viral titers and systemic distribution. Mice receiving insect-sourced virus had a death rate 28 times higher than mice receiving the same dose of virus that was propagated in mammalian cells. Culicoides salivary proteins were collected and purified and IFNAR mice were bred for use in an animal study to examine the effects of Culicoides salivary proteins on the virulence of bluetongue virus. 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. A manuscript was published describing the dynamics of EHDV-2 infection in Culicoides sonorensis midges. Virus was found to infect the insect�s midgut, escape the midgut, amplify and be disseminated to other organs by way of the hemolymph; a fluid in the insect analogous to blood. Virus reached the salivary glands as soon as 5 days after feeding. This study was the first to examine EHDV-2 infection dissemination, infection prevalence, and viral titer simultaneously within C. sonorensis over the course of infection. Our data suggests that C. sonorensis midges are likely to be able to transmit EHDV-2 5 days after ingesting a blood meal from an infected animal. In addition, this study is the first to identify the physiological basis for the lack of EHDV-2 vertical transmission in its insect vector. A manuscript was published describing the immunological responses of mice to midge feeding, how that relates to the ability of midges to feed to repletion, and why Culicoides midges are such efficient vectors of orbiviruses. EHDV and BTV proteins are being cloned and expressed for use in developing a multiplex fluorescent microsphere immunoassay to detect antibodies to these viruses in deer sera. After development, lab validation and field validation, this assay will be transferred to the University of Florida to be utilized in orbivirus serosurveillance of white-tailed deer. Eleven new serotypes of BTV have been isolated in southeastern United States, predominantly Florida. One, serotype 3, has been isolated multiple times and spread as far as Mississippi. Whole genome sequence analysis revealed that these strains are reassortant virus with U.S. strains; that is, the viruses have some gene segments from an endemic strain. The data also indicated that there are at least two different lineages currently circulating it the U.S. Subsequent to this initial manuscript the characterization of BTV serotype 3 strains isolated from an outbreak in white-tailed deer has been conducted. Accomplishments 01 Physiological and immunological responses to Culicoides sonorensis blood feeding: A murine model. Blood feeding Culicoides spp. biting midges are of great agricultural importance as livestock, equine, and wildlife pests and as vectors of the orbiviruses bluetongue, epizootic hemorrhagic disease, and African horse sickness. To obtain a blood meal, midges deposit saliva containing a myriad of proteins into the skin to facilitate feeding. Infected midges deposit virus along with the salivary proteins during feeding. The extreme efficiency with which midges are able to transmit orbiviruses is not clearly understood, as much is still unknown about the physiological trauma of the bite and immune responses to saliva deposited during feeding. Of particular interest are the first few hours and days after the bite; a critical time period for any midge-transmitted virus to quickly establish a localized infection and disseminate, while avoiding the hosts� immune responses. A mouse/midge feeding model using colonized Culicoides sonorensis midges was used by ARS scientists in Manhattan, Kansas, to characterize innate mammalian immune responses to blood feeding. Analysis of skin and draining lymph nodes show Culicoides midge feeding elicits a potent pro-inflammatory T-Cell helper (Th)-mediated cellular response with significant mast cell activation, subcutaneous hematomas, hypodermal edema and dermal capillary vasodilation, and rapid infiltration of leukocytes to the bite sites. Mast cell degranulation, triggered by bite trauma and specifically by midge saliva, was key to physiological and immunological responses and the ability of midges to feed to repletion. Our study shows the host�s physiological and immunological responses to midge blood feeding is what makes this vector so efficient in transmitting viruses and critical to understanding the epidemiology and control of these diseases.
Impacts
(N/A)
Publications
- Mills, M.K., Ruder, M.G., Nayduch, D., Michel, K., Drolet, B.S. 2017. Dynamics of epizootic hemorrhagic disease virus serotype 2 infection within the vector, Culicoides sonorensis (Diptera: Ceratopogonidae). PLoS One.
- Lehiy, C.J., Reister-Hendricks, L.M., Ruder, M., McVey, D.S., Drolet, B.S. 2018. Physiological and immunological responses to Culicoides sonorensis blood feeding: A murine model. Parasites & Vectors. 11(1):358-370.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Culicoides populations and their associations with specific breeding habitats were studied at multiple sites in Kansas that differ in their animal use patterns (beef cattle, dairy units, farmed deer, and bison and cattle grazed prairie). The distribution of known and putative disease vectors in several landscapes of Eastern Kansas has been characterized and data is being analyzed to elucidate relationships with habitat type, soil characteristics, animal use patterns, microbial populations, and variation in seasonal rainfall. Site-specific soil chemistries are being analyzed and will be correlated to midge collections and eventually to orbivirus seroprevalence from those sites in the last several years. Initiated planning discussions with Kansas and Nebraska veterinary diagnostic laboratories to initiate passive, random screening of animal diagnostic samples. Orbivirus seroprevalence within distinct transmission zones will inform environmental maintenance of orbiviruses. Related to Objective 2: Three female and one male IFNAR (interferon alpha/ beta receptor knock out) mice were purchased, bred and maintained over several months to obtain numbers needed for an animal infection study. The effect of cell culture origin on bluetongue virus (BTV) challenge inocula production was investigated. Protocols were established to produce representative cell lysate viral inocula containing cellular proteins for testing and comparisons at two infectious doses. Additionally, the parental mouse strain, C57BL/6J were tested for their susceptibility to the Culicoides cell viral lysate preparation. Preliminary results suggest viral inocula containing Culicoides cellular proteins is more virulent than viral inocula containing mammalian cellular proteins. Additionally, the C57BL/6J mice appear to be refractory to the Culicoides viral inocula preparations, regardless of infectious dose. C. sonorensis salivary proteins were collected on membranes and extracted for further analysis and for eventual use in animal infection studies. An immunohistochemical assay was developed to detect epizootic hemorrhagic disease virus antigen in C. sonorensis tissues. The assay was used to examine the progression of infection over time, midgut infection and escape, salivary gland infection and escape. ARS, in collaboration with Kansas State University, has developed next generation vaccines for epizootic hemorrhagic disease virus (EHDV) type 1, 2 and type 6 that an invention disclosure has been submitted. Accomplishments 01 Dynamics of epizootic hemorrhagic disease virus serotype 2 infection within the vector, Culicoides sonorensis. C. sonorensis biting midge is a confirmed vector of epizootic hemorrhagic disease virus (EHDV), which causes mortality in white-tailed deer and more increasingly in cattle. Currently, of the seven EHDV serotypes, only three are present in the USA, and very few studies have focused on the infection time course of these serotypes within the midge. The objective of the research was to characterize EHDV-serotype 2 infection within the midge by measuring vector infection prevalence, virus dissemination, and viral load over the course of infection. Midges were fed a blood meal containing EHDV-2 and sampled at specific time points post feeding. Midges were processed using virus isolation techniques to calculate infectious virus quantities, using molecular techniques to calculate the number of viral RNA genomes, and using immunohistochemistry to determine if and when the virus moved from the gut of the midge to the salivary glands where it could be transmitted the next time the midge fed. Virus was found to infect the insect�s midgut, escape the midgut, amplify and be disseminated to other organs by way of the hemolymph; a fluid in the insect analogous to blood. Virus reached the salivary glands as soon as 5 days after feeding. This study was the first to examine EHDV-2 infection dissemination, infection prevalence, and viral titer simultaneously within C. sonorensis over the course of infection. Our data suggests that C. sonorensis midges are likely to be able to transmit EHDV-2 5 days after ingesting a blood meal from an infected animal. 02 Non-structural protein NS3/NS3a is required for propagation of bluetongue virus in Culicoides sonorensis. ARS, in collaboration with Wageningen University, The Netherlands, examined the role of nonstructural proteins NS3/NS3a in replication and dissemination of BTV in C. sonorensis midges. Virus strains were generated using reverse genetics and their growth was examined in vitro. The ability of the virus to infect and escape the midgut was examined by separate testing of midge heads and bodies. Although the parental NS3/NS3a expressing strain was not able to replicate and disseminate within C. sonorensis after oral feeding, this virus was able to replicate efficiently when the midgut infection barrier was bypassed by intrathoracic injection, whereas the NS3/NS3a knockout mutant was unable to replicate. This demonstrates that NS3/NS3a is required for viral replication within Culicoides. The lack of viremia and the inability to replicate within the vector, clearly demonstrate the inability of NS3/NS3a knockout DISA vaccine strains to be transmitted by midges and therefore they would be safe to use as vaccines without the risk of subsequent transmission by Culicoides midges.
Impacts
(N/A)
Publications
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