Location: Arthropod-borne Animal Diseases Research
2021 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. 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.
Accomplishments
1. 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.
2. 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.
3. 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).
Review 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.
