Location: Arthropod-borne Animal Diseases Research
2017 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
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
1. 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.
2. 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.
