Abstract

Porcine rotavirus (PoRV) is a significant pathogen that causes diarrhea in piglets, with the G9, G5, and G4 genotypes being the most prevalent in China. Although vaccination is the most effective strategy to prevent PoRV infections, the currently available G5 genotype-based vaccine offers limited cross-protection against other circulating PoRV genotypes in pig farms. In this study, we developed an entirely plasmid-based reverse genetics (RG) system for the PoRV strain NJ2012 (G9P[7]) and generated two recombinant reporter viruses expressing the fluorescent UnaG and NLuc proteins, respectively. Furthermore, we successfully constructed multivalent recombinant PoRV strains by inserting the VP7 gene of G4 genotype into the gene segment 7 (NSP3) and/or the VP7 gene of G5 genotype into the gene segment 5 (NSP1) within the backbone of rNJ2012-WT strain. These multivalent recombinant viruses efficiently expressed G4 and/or G5 genotype of the VP7 protein in infected cells and elicited robust immune responses in mice. The adult mice immunized with the trivalent recombinant PoRV (rNJ2012-fG5-VP7/haG4-VP7), which simultaneously expressed VP7 proteins from G4, G5, and G9 genotypes, conferred passive protection to suckling mice against infections caused by multiple G genotypes of PoRV. In summary, these findings established a platform for efficient generation of multivalent recombinant PoRV, offering a scalable methodology to facilitate the development of next-generation PoRV vaccines.