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Molecular Basis of Attenuation of the Modified Live Virus Vaccine Strain of Equine Arteritis
U. B. R. Balasuriya
Department of Veterinary Sciences
The United States is the only country with a substantial horse breeding industry that does not have any EAV testing requirements for imported stallions or semen. The situation is complicated by widespread lack of awareness among horse owners and breeders about EAV and differences in breed prevalence of EAV infection. In an effort to clarify the risks associated with EAV infection of horses, we propose to define the genetic determinants of virulence of EAV. The purpose of this study is to identify potential virulence determinants of EAV by sequence analysis of virus strains of defined virulence phenotype.These efforts eventually should facilitate the rapid identification of new strains of the virus of enhanced virulence, more especially their ability to cause abortion in mares. It should also help to better delineate the role of the carrier stallion in generating them, and lead to the eventual development of an improved and MLV vaccine for EAV.
2010 Project Description
The primary goal of this research project is to localize the virulence determinants of equine arteritis virus (EAV) to specific nucleotide residues using site specific mutagenesis of the infectious cDNA clones of the virus. This project provided multiple opportunities to train several undergraduate students, graduate students and postdoctoral scholars, as well as a visiting scientist from France. Recent findings from this study have been presented at several national and international meetings (six) and published in peer reviewed journals (eleven). Furthermore, nucleotide sequences are made available to the public through GenBank. An infectious cDNA clone of current modified live virus (MLV) strain of EAV was developed and a patent application has been submitted.
These studies identified the virulence determinants of EAV and determinants of EAV persistent infection. Amino acid changes in either the replicase (non-structural proteins [nsp1, nsp2, nsp7 and/or nsp10]) or structural proteins (GP2, GP4, GP5 and M) resulted in attenuation of the virulent VB strain of EAV during extensive cell culture passage to produce the modified live virus (MLV) vaccine strain of EAV. Reverse genetic studies showed that recombinant virus with multiple substitutions in the structural proteins was more attenuated than the recombinant virus with substitutions only in the replicase proteins.
Reverse genetic studies also clearly showed that substitutions in the structural proteins, but not the replicase, are responsible for the establishment of EAV persistent infection in a cell culture (HeLa cells) model. Furthermore, localization of virulence determinants of EAV to specific nucleotide residues will facilitate the development of genetically marked modified live virus vaccine for equine viral arteritis (EVA).
A genetically engineered vaccine would allow us to establish a stable validated repository of seed virus stock for live virus production, which would ensure a genetically homogenous virus stock. Additionally, data from these studies will eventually facilitate the rapid identification of new strains of EAV with enhanced virulence, better delineate the role of the carrier stallion in generating them, and the eventual development of improved vaccines for EVA and other arteriviruses (e.g. porcine reproductive and respiratory syndrome in pigs). Compared to the existing means of immunization, the new vaccines will provide a) higher efficacy; b) higher safety; and c) specific discrimination between vaccinated and naturally infected animals.
Moreover, the development of several chimeric infectious clones of virulent and avirulent strains of EAV has allowed us to expand our studies to identify the EAV target cell population in equine PBMCs and to carry out a detailed characterization of the interaction between the target cell population and the virus. Using a panel of five recombinant chimeric viruses, we demonstrated that interactions between GP2, GP3, GP4, GP5 and M envelope proteins play a major role in determining the CD14+ monocyte tropism while the tropism of CD3+ T lymphocytes is only determined by GP2, GP4, GP5 and M envelope proteins but not the GP3 protein.
This study showed for the first time that CD3+ T lymphocytes may play an important role in the pathogenesis of equine viral arteritis when horses are infected with the virulent strains of EAV. These studies will help to better characterize the pathogenesis of EAV infection in horses.
Zhang, J., Stein, D.A., Timoney, P.J., and Balasuriya, U.B.R. 2010. Cure of persistent equine arteritis virus infection in HeLa cells by treatment with a peptide-conjugated morpholino oligomer. Virus Res. 150(1-2):138-142.
Go, Y.Y., Zhang, J., Timoney, P.J., Cook, R.F., Horohov, D.W., and Balasuriya U.B.R. 2010. Complex interactions between the major and minor envelope proteins of equine arteritis virus determine its tropism for equine CD3+ T lymphocytes and CD14+ monocytes. Journal of Virology 84(10):4898-4911.
Broaddus, C.C., Balasuriya, U.B.R., White, J., Timoney, P.J., Funk, R. A., and Holyoak, G.R. 2010. Safety consideration of vaccinating mares against equine viral arteritis in mid and late pregnancy and in the immediate postpartum period. J. Am. Vet. Med Assoc. (in press).
Broaddus, C.C., Balasuriya, U.B.R., White, J., Timoney, P.J., Makloski, C., Torrisi, K., and Holyoak, G.R. 2010. Infectivity of embryos following insemination of donor mares with equine arteritis virus infective semen. Theriogenology (in press).
Zhang, J., Timoney, P.J., Shuck, K.M., Seoul, G., Go, Y.Y., Lu, Z., Powell, D.G., Meade, B.J., and Balasuriya, U.B.R. 2010. Molecular epidemiology and genetic characterization of equine arteritis virus isolates associated with the 2006-2007 multi-state disease occurrence in the USA. J. Gen. Virol. 91:2286-2301.
Brault, S.A., Bird, B.H., Balasuriya, U.B.R., and MacLachlan, N.J. 2010. Genetic heterogeneity and variation in viral load during equid herpesvirus-2 infection of foals. Vet. Microbiol. (Epub ahead of print).
Lu, Z., Dubovi, E.J., Zylich, N., Crawford, P.C., Sells, S., Go, Y.Y., Loynachan, A.T., Timoney, P.J., Chambers, T.M., and Balasuriya, U.B.R. 2010. Diagnostic application of H3N8 specific equine influenza real-time RT-PCR assays for the detection of canine influenza virus in clinical specimens. J. Vet. Diagn. Invest. 22(6):942-945.
Pronost, S., Pitel, P.H., Miszczak, F., Legrant, L., Marcillaud-Pitel, C., Hamon, M., Tapprest, J., Balasuriya, U.B.R., Freymuth, F., and Fortier, G. 2010. Description of the first recorded major occurrence of equine viral arteritis in France. Equine Vet. J. (Epub ahead of print).
Pronost, S., Cook, R.F., Fortier, G., Timoney, P.J., and Balasuriya, U.B.R. 2010. Relationship between equine herpesvirus-1 myeloencephalopathy and viral genotypes . Eq. Vet. J. 42(8):672-674.
Go, Y.Y., Snijder, E.J., Timoney, P.J., and Balasuriya, U.B.R. 2010. Characterization of equine humoral antibody response to the nonstructural proteins of equine arteritis virus. Clin. Vaccine Immunol.(2010 Dec 8. [Epub ahead of print]).