Development of molecular diagnostic assays for equine respiratory viruses and analysis of the role of equine arteritis virus envelope proteins in the early events of virus entry.

摘要

There is an urgent need for detection of viral respiratory pathogens to identify the causal agent(s) involved and to prevent the spread of related diseases. The first part of this dissertation focuses on development, optimization and validation of Real-time reverse transcription polymerase chain reaction (rRT-PCR) assays for the detection of several common equine viral pathogens: equine arteritis virus (EAV), equine influenza virus and equine rhinitis viruses A and B. Emphasis of the second part of this dissertation is on studying the role of EAV envelope proteins in virus attachment and entry. Using an infectious cDNA clone of EAV and reverse genetics, a panel of chimeric viruses was generated by swapping the N-terminal ectodomains and full-lengths of the two major envelope proteins (GP5 and M) from porcine reproductive and respiratory syndrome virus (PRRSV). The recombinant viruses expressing the N-terminal ectodomain of PRRSV GP5 or M or both (GP5ecto, Mecto, and GP5&Mecto, respectively) in an EAV backbone were viable and genetically stable. Compared to the parental virus, these three chimeric viruses produced lower titers and smaller plaque sizes indicating that they have a crippled phenotype. Interestingly, the three chimeric viruses could only infect EAV susceptible cell lines but not the PRRSV susceptible cell line. Therefore, the exchange of GP5 and/or M protein N-terminal ectodomains from PRRSV did not alter the cellular tropism of the chimeric viruses. We also investigated the role of one of the minor envelope proteins (E) of EAV in virus attachment and entry. The results showed that EAV infection of equine endothelial cells is heparin-dependent and the C-terminus of the E protein contains a putative heparin-binding domain. We generated a panel of arginine to glycine mutations in the conserved region of both the full-length EAV infectious cDNA clone and individual E protein expression vectors. The triple mutation R52,60,65G construct grew significantly slower and produced much smaller plaques. The double mutant R52,60G completely blocked the interaction between E protein and heparin. Taken together, these data indicated that E protein interacts with heparin to facilitate virus attachment and plays a major role in EAV infection.;KEYWORDS: Molecular Diagnostics, Real-time RT-PCR, Equine Viral Respiratory Disease, Equine Arteritis Virus, Viral Envelope Protein.