Recombinant EHV-1 Vector Expressing Immunodominant Hemagglutinin Protein of Equine Influenza Virus H3N8 (Sub-Lineage...

Equine herpesvirus type 1 (EHV-1) and equine influenza virus (EIV) are major respiratory pathogens in horses, causing significant economic losses in domesticated horses. Bacterial Artificial Chromosome (BAC) technology can be used to precisely manipulate the EHV-1 genome for the development of live-attenuated vector vaccines. Earlier, our group developed a live-attenuated EHV-1 vaccine by deleting virulence-associated genes using this technology and the mutant EHV-1 has been exploited for expressing foreign gene in the current study. Specifically, in this study, a mutant EHV-1 virus expressing the hemagglutinin (HA) gene of H3N8 EIV (sub-lineage: Florida clade 2) was generated and characterized in vitro.

The HA gene of EIV (Florida clade 2) was used for antigen gene cloning. The expression cassette for the HA gene was commercially synthesized and inserted into the backbone of EHV1∆IR6 BAC using an En passant mutagenesis strategy. Recombinant clones were selected using antibiotic selection, PCR, and RFLP. Further, the recombinant virus was regenerated in RK-13 cells via transfection and characterized in vitro for plaque size, growth kinetics and immunofluorescence antibody test (IFAT).

PCR and RFLP confirmed the successful insertion of the HA gene into pEHV1∆IR6/gE BAC. The recombinant virus, vEHV1∆IR6/gE-HA(FC2), was successfully rescued in RK13 cells and demonstrated expression of the EIV haemagglutinin proteins by immunofluorescence assay. Although plaque size was reduced in the generated mutant virus in comparison to parental virus, the growth kinetics of the recombinant viruses were comparable to those of vEHV1∆IR6/gE.

These findings demonstrate the successful expression of immunodominant hemagglutinin protein of EIV by recombinant EHV-1 and indicate the potential suitability of EHV-1 BAC as a vector platform for foreign gene expression.