Identification of the Suitable Insertion Site for Expression of Spike Gene of A Porcine Epidemic Diarrhea Virus Variant in A Pseudorabies Virus Vector

Background: The emergence of variant porcine epidemic diarrhea virus (PEDV) strain and pseudorabies virus (PRV) in China in recent years has decreased the effectiveness of CV777 and Bartha K61 vaccines, causing significant loss to the swine industry. Previously, we generated a TK&gE-deleted PRV (PRV TK&gE-AH02 ) based on a virulent PRV AH02LA strain. It was shown to be safe for 1-day-old piglets with maternal PRV antibodies and 4～5 week-old PRV antibody negative piglets and provide rapid and complete protection in weaned pigs against lethal challenge with the PRV variant strain. PRV TK&gE-AH02 may be a promising live vaccine vector for construction of recombinant vaccine in pigs. PEDV spike (S) protein is mainly used in the development of PEDV vaccines. Therefore, the gene-deleted PRV (from PRV variants) vectored vaccine expressing variant PEDV S gene may be viable PEDV and PRV vaccine candidates. However, insertion site is an important factor affecting foreign gene expression and vaccine efficacy. Results: : In this study, we constructed four recombinant PRV-S bacterial artificial chromosomes (BACs) carrying the same S expression cassette 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, RT-PCR and indirect immunofluorescence. We observed higher S gene mRNA expression level in Swine testicular cells infected with PRV-S(UL11-10)ΔTK/gE and PRV-S (UL35-36)ΔTK/gE compared to that of PRV-S(UL46-27)ΔTK/gE at 6 hour post infection ( P < 0.05). Moreover, at 12 hour post infection, cells infected with PRV-S (UL11-10)ΔTK/gE exhibited higher S gene mRNA expression than those infected with PRV-S (UL35-36)ΔTK/gE ( P = 0.097) and PRV-S (UL46-27)ΔTK/gE ( P < 0.05). Recovered vectored mutant PRV-S (UL11-10, UL35-36 and UL46-27) exhibited similar growth kinetics to the parental virus (PRV TK&gE-AH02 ). Conclusions: : The identification and comparison of the insertion sites in PRV genome laids a foundation for future development of recombinant PRV vaccines.