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Equine herepsvirus-1 (EHV-1) associated neurologic disease (also known as equine herpesvirus myeloencepalopathy [EHM]) is caused by specific genomic variants of the virus and is associated with a 15-50% case-fatality rate. A single nucleotide polymorphism at position 2254 in the EHV-1 DNA polymerase gene (encoded by open reading frame 30 [ORF30) has been linked with the occurance of outbreaks of EHM frequently characterized by high morbidity and high case-fatality rates. Previous studies have identified that strains possessing adenine at nucleotide position 2254 (A2254) in ORF30 are of a nonneuropathogenic phenotype whereas those with guanine at 2254 (G2254) are of a neuropathogenic phenotype. EHV-1 strains of the G2254 genotype replicate more efficiently in the horse and produce significantly higher viral burdens. This increased replicative capacity enhances the virus' ability to infect capillary endothelial cells, leading to interference with the blood supply to the CNS and the appearance of neurologic signs. Recently, Perkins et al. (2009) performed statistical analysis of ORF30 from a large number of EHV-1 isolates (n= 176) and demonstrated that the odds of having neurologic disease with the ORF30 G2254 genotype is 162 times greater than those with the opposite A2254 genotype. Despite this strong statistical significance they also found that 24% of isolates from horses with neurologic disease had the nonneuropathogenic A2254 genotype. We have found upon comparison of the results from the allelic discrimination assays with clinical histories provided by the attending veterinarians that there were a number of significant discrepancies between the genotype of ORF30 (A2254 or G2254). Specifically, preliminary studies in our laboratory have identified a number of A2254 genotype EHV-1 isolates from cases of neurologic disease, as well as G2254 genotype viruses from numerous horses where there was no evidence of neurological involvement. In addition, we have identified viruses that have substitutions in ORF30 in addition to A->G2254. For example the RACH modified live virus vaccine strain of EHV-1 has the G2254 genotype but also at least one other substitution in ORF30. Taken together these data suggest that additional mutations in ORF30 or changes in other viral genes may contribute to the development EHM following infection with EHV- 1. Therefore, the focus of this proposal is to identify additional nucleotide substitutions in ORF30 or other viral genes that may contribute to the development of EHM. Based on our preliminary data, we hypothesize that the neuropathogenic phenotype in EHV-1 can result from a nucleotide substitution(s) either in addition to or instead of the previously identified A->G2254 substitution in the ORF30 encoding the viral DNA polymerase. To test this hypothesis we propose to identify additional putative neurovirulence determinants of EHV-1 by sequencing genes essential for replication from an extensive panel of archived viral isolates along with obtaining the full-length genomic sequences of a selected number of well-characterized neuropathogenic and non-neuropathogenic strains of EHV-1 from Europe and North America. We believe that the nucleotide substitutions identified in this study along with associated EHM phenotype (clinical signs) will allow us to develop new allelic discrimination and specific nucleotide sequencing assays for the diagnosis of EHM. Ultimately, these associations will be tested using infectious EHV-1 molecular clones under development in this laboratory.
|Effective start/end date||4/1/10 → 3/31/13|
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