Equine herpesvirus-1 (EHV-1) can cause acute upper respiratory tract disease, abortion, neonatal death and neurological disease in horses. Rapid, accurate and timely diagnosis of EHV-1 infection in horses is important to curtail the spread of this pathogen. It has been reported that the neuropathogenic phenotype of EHV-1 can result from a single non-synonymous nucleotide substitution at position 2254 (A→G2254) in open reading frame 30 (ORF30). This was the basis for the development of an allelic discrimination, real-time PCR assay to distinguish between potential neuropathogenic and non-neuropathogenic EHV-1 strains. However, PCR analysis of a panel of EHV-1 abortion isolates revealed that other point mutations within ORF30 could produce false negative results with this previously described assay. Therefore, one of the objectives of this dissertation project was to develop a more sensitive and specific allelic discrimination real-time PCR assay for the detection of EHV-1. This was achieved by redesigning the primers and probes targeting ORF30. The new assay was ten times more sensitive than the original assay, with a lower detection limit of 10 infectious virus particles. While mutations within EHV-1’s genome can hinder diagnosis, they can also impact the virulence of the virus. Objective two, therefore, was to determine if sequential cell passage of T953 would induce sufficient attenuation of the EHV-1 genome to produce a low virulence phenotype. Two separate groups of 28 BALB/c mice were inoculated with either the parental strain or passage 135 (T953 P135) of EHV-1 strain T953. The animals were observed for fourteen days, euthanized and their tissues analyzed for the presence of EHV-1. At the conclusion of the fourteen day observation period, all of the mice infected with T953 P135 survived and regained their pre-inoculation body condition. Furthermore, there were significant differences in virus titer and viral DNA concentrations between T953 P135 and the parental strain, further confirming the attenuated phenotype of the virus. Taken together, data from this study clearly demonstrates that sequential cell culture passage of the neuropathogenic T953 strain of EHV-1 results in attenuation for young adult BALB/C mice.

Equine herpesvirus type-1(EHV-1) is a complex virus known for inducing various forms of disease in horses. In recent years, the number of cases of neurological disease caused by this virus has increased. While there are a number of possible sources for this recent surge, this project set out to determine if a genotypic shift in the latent population of the virus in favor of the neuropathogenic form of EHV-1 is the basis for the recent increase in frequency of EHV-1 neurologic disease. To ascertain if such a shift has in fact occurred, 450 EHV-1 isolates were obtained from fetal tissues resulting from single, sporadic cases of abortion in Thoroughbred broodmares in central Kentucky. Furthermore, the isolates utilized were from different decades (1951-2006) to determine if the genotypic shift was time-related. The isolates were propagated in cell culture, purified and the viral DNA isolated. Real-time allelic discrimination PCR analysis was performed on the DNA samples to identify the genotype of EHV-1. Statistical analysis of the PCR data indicates that the latent mutant population does appear to be increasing. Therefore, the recent increase in the number of outbreaks of EHV-1 neurological disease will most likely continue unless measures are devised to curtail further spread of the pathogen.