Simultaneous expression of three G genotypes of VP7 proteins in a recombinant porcine rotavirus confers protective immunity against multiple rotavirus infections.

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.IMPORTANCEPorcine rotavirus (PoRV) is a primary etiological agent of diarrhea in swine, posing significant challenges due to the diversity of circulating genotypes and the limited cross-protection offered by existing PoRV vaccines. To address this, we developed multivalent recombinant vaccine candidates capable of eliciting robust immunity against multiple genotypes of PoRV strains. Using the established entirely plasmid-based reverse genetics (RG) system based on the G9 genotype of PoRV strain NJ2012 (G9P[7]), we further engineered the multivalent recombinant virus simultaneously expressing VP7 proteins from G4, G5, and G9 genotypes. Immunization of adult mice with this trivalent recombinant virus conferred broad-spectrum passive protection to their suckling mice against infections by multiple PoRV genotypes. Our findings established a novel platform for efficiently developing multivalent PoRV vaccines, offering a promising strategy for the prevention and control of PoRV outbreaks.