Rescue and characterization of a recombinant HY12 bovine enterovirus carrying a foreign HA epitope in the 3A nonstructural protein

Abstract

Full-length infectious cDNA clones for recombinant HY12 bovine enteroviruses designated as rHY12-3A-2-HA, rHY12-3A-3-HA, and rHY12-3A-9-HA were constructed by the insertion of an epitope from influenza virus hemagglutinin (HA) at the N-terminus of the HY12-encoded 3A protein at amino acid positions 2, 3, and 9. The recombinant HY12 viruses expressing the HA epitope were rescued and characterized using immunoperoxidase monolayer assay, western blotting, and electron microscopy. The three rescued recombinant marker viruses showed similar characteristics, such as TCID 50 titer, plaque size, and growth properties, to those of parental rHY12 virus. Comparative analysis of the nucleotide sequences demonstrated the three recombinant marker viruses remained stable for 15 passages with no genetic changes. The recombinant viruses remained viable in various permissive cell lines, including BHK-21, Vero, and PK15 cells, suggesting that the insertion of the HA epitope tag had no effect on virus infectivity. Mice infected with the recombinant marker viruses and the parental virus produced anti-HY12-virus antibodies, while the recombinant marker viruses also produced anti-HA-epitope-tag antibodies. Taken together, these results demonstrate that HY12 viruses containing genetic markers may be useful tools for future investigations of the mechanisms of viral pathogenesis and virus replication, as well as for vaccine development.

Experimental immunization of mice with a recombinant bovine enterovirus vaccine expressing BVDV E0 protein elicits a long-lasting serologic response

“…The BEV genome contains a large open reading frame encoding four structural proteins and seven nonstructural proteins. Previous studies explored various potential insertion sites for exogenous genes in BEV vectors, including the junction of the VP1 and 2A genes within the 2A, 2C, 3A, and VP1 genes, and in the VP1 B-C or D-E loop [15][16][17]. Early studies showed no adverse effects on viral biology or replication after insertion of exogenous epitopes at these sites.…”

“…Chu et al [16] inserted the main antigen neutralization epitope (residues 141-160) of the FMDV (vaccine strain O1/Manisa/Turkey/69) VP1 gene into the junction of VP1/ 2A of BEV (LC-R4 strain). Liu et al [17] constructed a recombinant infectious BEV clone by insertion of the epitope of influenza virus hemagglutinin (HA) into the 3A or VP1 gene of BEV (HY12 strain), respectively. These studies indicated that the biological characteristics of recombinant BEV are similar to those of the parental virus, and experimental infection animal models could produce immune responses to the exogenous genes.…”