Location: Arthropod-borne Animal Diseases Research
2022 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
This is the final report for this project which terminated December 2021. See the report for the replacement project, 3020-32000-020-000D, “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.
Accomplishments
