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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.
2011 Project Description
The primary goal of this research project was 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, and to identify the host genetic factors associated with the susceptibility to EAV infection. This project provided multiple opportunities to train several undergraduate students, graduate students and visiting scientist from France, Brazil and Argentina. Recent findings from this study has been presented at several national and international meetings (11) and published in peer reviewed journals (08).
These studies identified the virulence determinants of EAV and determinants of EAV persistent infection, as well has host genetic determinants that are associated with the susceptibility to EAV infection. During this year, most of the research has been focused on studying the mechanisms of virus-host interactions. Understanding how host cellular proteins interact with viral RNA and viral proteins, as well as their role in viral infection, will enable better characterization of the pathogenesis of EAV and establishment of persistent infection in stallions.
Accordingly, we hypothesized that both viral factors and host genetically related factors could influence the outcome of EAV infection in horses. To test this hypothesis, we first combined contemporary molecular biology techniques with dual color flow cytometric analysis to characterize the interactions of viral structural proteins and the equine peripheral blood mononuclear cells in vitro. Results from this study demonstrated that interactions between GP2, GP3, GP4, GP5 and M envelope proteins of EAV play a major role in determining the CD14+ monocyte tropism while the tropism of CD3+ T lymphocytes is determined by GP2, GP4, GP5 and M envelope proteins but not the GP3 protein. Secondly, a genome wide association study using SNP genotyping identified a common haplotype associated with the in vitro CD3+ T lymphocyte/resistance to EAV infection among four breeds of horses.
Subsequently, these studies were extended to establish a possible correlation between the in vitro susceptibility of CD3+ T lymphocytes to EAV and establishment of persistent infection in stallions. Interestingly, carrier stallions with susceptible CD3+ T lymphocyte phenotype to EAV may represent those at higher risk of becoming persistently infected.
Finally, the precise effect of EAV on the immune system of horses, innate and humoral immunity, was studied. Horses were shown to mount a strong humoral antibody response to nonstructural proteins (nsps) 2, 4, 5 and 12 of EAV, whereas nsps 1, 2 and 11 suppressed the type I interferon production.
Findings from these studies have opened up new directions for future EAV research using genomic and proteomic approaches to study host cell factors involved in EAV attachment and entry and establishment of persistent infection in the stallions.
Broaddus, C. C., Balasuriya, U. B. R., White, J., Timoney, P. J., Funk, R. A., and Holyoak, G. R. 2011. Evaluation of the safety of vaccinating mares against equine viral arteritis during mid or late gestation or during the immediate postpartum period. J. Am. Vet. Med Assoc. 238(6):741-750.
Go, Y. Y., Snijder, E. J., Timoney, P. J., and Balasuriya, U. B. R. 2011. Characterization of equine humoral antibody response to the nonstructural proteins of equine arteritis virus. Clin. Vaccine Immunol. 18(2):268-279.
Firth, A. E., Zevenhoven-Dobbe, J. C., Wills, N. M., Go, Y. Y., Balasuriya, U. B. R., Atkins, J. F., Snijder, E. J., and Posthuma, C. C. 2011. Discovery of a small arterivirus gene that overlaps the GP5 coding sequence and is important for virus production. J. Gen. Virol. 92(5):1097-1106.
Broaddus, C. C., Balasuriya, U. B. R., White, J., Timoney, P. J., Makloski, C., Torrisi, K., and Holyoak, G. R. 2011. Infection of embryos following insemination of donor mares with equine arteritis virus infective semen. Theriogenology 76:47-60.
Miszczak, F., Shuck, K. M., Lu, Z., Go, Y. Y., Zhang, J., Sells, S., Vabret, A., Pronost, S., Fortier, G., Timoney, P. J., and Balasuriya, U. B. R. 2011. Evaluation of Two Magnetic Bead-Based Viral Nucleic Acid Purification Kits and Three Real-Time RT-PCR Reagent Systems in Two TaqMan Assays for Equine Arteritis Virus Detection. J. Clin. Microbiol. 49(10):3694-3696.
Go, Y. Y., Bailey, E., Cook, D. G., Coleman, S. J., MacLeod, J. N., Chen, K. C., Timoney, P. J. and Balasuriya, U. B. R. 2011. Genome-wide association study among four horse breeds identifies a common haplotype associated with the in vitro CD3+ T cell susceptibility/resistance to equine arteritis virus infection. J Virol. 85(24):13174-84.
Miszczak, F., Legrand, L., Balasuriya, U. B. R., Ferry-Abitbol, B., Zhang, J, Hans, A., Fortier, G., Pronost, S., Vabret, A. 2011. Emergence of novel equine arteritis virus (EAV) variants during persistant infection in the stallion: origin of the 2007 French EAV outbreak was linked to an EAV strain present in the semen of a persistently infected carrier stallion. Virology (Epub ahead of print).