Construction and characterization of a gE/gI/TK-gene-deleted recombinant pseudorabies virus variant expressing the GP5 of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and NADC30-like PRRSV

Porcine reproductive and respiratory syndrome (PRRS) and pseudorabies (PR) are still the major problems of the worldwide pork industry. Previous studies showed that PR virus (PRV) and PRRS virus (PRRSV) commercial vaccines available in China could not provide complete protection against PRV variants and currently prevalent PRRSV strains. In the present study, a recombinant pseudorabies virus rPRV-GP5/HP-GP5/NA expressing the GP5 of the highly pathogenic PRRSV (HP-PRRSV) and NADC30-like PRRSV was constructed by transfecting the transfer plasmid pG-GP5/HP-GP5/NA-EGFP into ST cells inoculated with gE/gI/TK-gene-deleted rPRV NY-gE^-/gI^-/TK^- using homologous recombination and CRISPR/Cas9 gene editing technique. The recombinant virus rPRV-GP5/HP was also constructed. The expression of the GP5 protein was confirmed by Western blot and indirect immunofluorescence assay. These two viruses were similar to the parental virus rPRV-gE^-/gI^-/TK^- in terms of growth curve, morphogenesis and virus plaque sizes, and proliferated in different cell types. The animal test results showed that ELISA antibodies against PRRSV could be detected in piglets immunized with these two recombinant viruses, and the antibody levels were slightly lower than those of commercial vaccines, but these two recombinant viruses elicited high levels of PRV ELISA antibody and neutralizing antibody, as is the case with commercial vaccine. These two recombinant viruses could provide some protection against virulent PRRSV and PRV, and could effectively inhibit virus proliferation in tissues. These findings provide insights that these two viruses need to be optimally engineered as promising bivalent vaccine candidates against PRV and PRRSV for the control and eradication of the variant PRV and currently prevalent PRRSV. IMPORTANT: Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine pseudorabies virus (PRV) can infect pigs of all ages with high mortality. Due to the appearance of the PRRSV variant (HP-PRRSV and NADC30-Like-PRRSV) and the outbreak of PRV variant in China, the current commercial vaccines available cannot provide complete protection against PRV variants and prevalent PRRSV strains, which causes a major economic loss in pig industry worldwide. Therefore, safe and effective new vaccines are urgently developed to simultaneously control and even eradicate the two viruses. This study intends to use the modified attenuated PRV strain as the carrier and the main antigen gene ORF5 of PRRSV as the exogenous gene to construct the recombinant virus strain, and further validate the protective effect of recombinant strains in vitro and in vivo against the challenge of PRRSV and PRV, which is expected to become a candidate vaccine strain.