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ARS Home » Plains Area » Manhattan, Kansas » Center for Grain and Animal Health Research » ABADRU » Research » Research Project #432252

Research Project: Orbivirus Pathogenesis, Epidemiology, and Control Measures

Location: Arthropod-borne Animal Diseases Research

2018 Annual Report

1a. 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.

1b. 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.

3. Progress Report:
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.

4. Accomplishments
1. 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.

Review 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.