Location: Arthropod-borne Animal Diseases Research2013 Annual Report
1a. Objectives (from AD-416):
Objective 1: Determine the molecular and biological characteristics of emergent, re-emergent, and previously uncharacterized bluetongue virus isolates. Sub-objective A. Develop means to detect and characterize emergent viruses. Objective 2: Assess the risk of North American ruminants to emergent, re-emergent, and previously uncharacterized bluetongue virus isolates. Sub-objective A. Develop BTV “vector-transmitted” infectious models in target ruminant species to facilitate disease pathogenesis, disease transmission and vaccine efficacy studies. Sub-objective B. Identify mammalian host innate and adaptive responses to insect transmitted BTV.
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 ever-increasing 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:
Relative to Objective 1 (Determine the molecular and biological characteristics of emergent and previously uncharacterized bluetongue virus isolates) the following progress was made: The bluetongue virus type 17 (BTV-17) core protein (VP7) genome sequence has been used to design X-TAG primers for polymerase chain reaction (PCR) amplification and oligonucleotide capture for an orbivirus fluorescent X-TAG microsphere assay. The BTV-17 and epizootic hemorrhagic disease virus type 1 (EHDV-1) non-structural protein 1 (NS1) genome sequences have been used to design X-TAG primers for PCR amplification and oligonucleotide capture for a BTV/EHDV differential fluorescent X-TAG microsphere assay. The outer capsid (VP2) gene sequences of BTV types 2, 10, 11, 13, and 17 have been used to design X-TAG primers for PCR amplification and oligo capture for a BTV serotype specific fluorescent X-TAG microsphere assay. The VP7 and NS1 genes of BTV-17 were cloned into a cloning vector (pCR2) and an expression vector (pET). The VP2 genes of BTV-2, 10, 11, 13, and 17 were cloned into a cloning vector (pCR2) and an expression vector (pET). The VP7 and NS1 genes of EHDV were cloned into a cloning vector (pCR2). Relative to Objective 2 (Develop BTV “vector-transmitted” infectious models in target ruminant species to facilitate disease pathogenesis, disease transmission and vaccine efficacy studies) the following progress was made: A needle-free inoculation system was tested on sheep carcasses and live sheep with various settings to achieve optimal transdermal inoculum delivery. Injections of blue dye in the carcasses were used to evaluate depth and consistency of delivery. Biting midge (Culicoides sonorensis) salivary proteins were collected on membranes, purified and concentrated. Proteins were identified by mass spectrometry and compared to known salivary proteins of other arthropods. Mice were injected intradermally with salivary proteins. At one, two and three days post inoculation, mice were euthanized. Blood, lymph nodes and injection site skin were harvested for analysis by flow cytometry, CBC chemistry panel, and histology. Specific mouse cell populations related to immune responses were up-regulated by the salivary proteins.
1. Infection of North American sheep by the European Bluetongue serotype 8 (BTV-8) virus. To determine the risk of US sheep to BTV-8, ARS researchers at Manhattan, KS inoculated sheep with BTV-8 obtained from The Netherlands. The disease level observed was similar to domestic BTV virus types and less severe than what has been reported for European sheep species. Virus was detected by real time PCR in blood and distributed widely in tissues. Our results indicate that if BTV-8 were to be introduced into the U.S., our sheep populations would be highly susceptible to infection, but clinical disease would not be more severe than outbreaks resulting from many of our domestic BTV types.
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