Identification of four insertion sites for foreign genes in a pseudorabies virus vector.

Abstract

BACKGROUND: Pseudorabies virus (PRV) is a preferred vector for recombinant vaccine construction. Previously, we generated a TK&gE-deleted PRV (PRV) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4&#x2009;~&#x2009;5 week-old PRV antibody negative piglets and provide rapid and 100&#x2009;% protection in weaned pigs against lethal challenge with the PRV variant strain. It suggests that PRVmay be a promising live vaccine vector for construction of recombinant vaccine in pigs. However, insertion site, as a main factor, may affect foreign gene expression. RESULTS: In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same spike (S) expression cassette of a variant porcine epidemic diarrhea virus strain in different noncoding regions (UL11-10, UL35-36, UL46-27 or US2-1) from AH02LA BAC with TK, gE and gI deletion. The successful expression of S gene (UL11-10, UL35-36 and UL46-27) in recombinant viruses was confirmed by virus rescue, PCR, real-time PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in swine testicular cells infected with PRV-S(UL11-10)&#x394;TK/gE and PRV-S(UL35-36)&#x394;TK/gE compared to that of PRV-S(UL46-27)&#x394;TK/gE at 6&#xa0;h post infection (P&#x2009;<&#x2009;0.05). Moreover, at 12&#xa0;h post infection, cells infected with PRV-S(UL11-10)&#x394;TK/gE exhibited higher S gene mRNA expression than those infected with PRV-S(UL35-36)&#x394;TK/gE (P&#x2009;=&#x2009;0.097) and PRV-S(UL46-27)&#x394;TK/gE (P&#x2009;<&#x2009;0.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRV). CONCLUSIONS: This study focuses on identification of suitable sites for insertion of foreign genes in PRV genome, which laids a foundation for future development of recombinant PRV vaccines.