Generation of G9 genotype porcine rotavirus using reverse genetics system and its application for antiviral screen and vaccine development.

Group A rotaviruses (RVs) continue to be one of the most important pathogens causing severe acute gastroenteritis in infants and young animals worldwide. Recently, the prevalence of porcine RV (PoRV) from pig farms has strikingly increased, adversely affecting the swine industry, particularly with the G9 genotype of PoRV VP7 emerging as the predominant genotype spreading in China. Current vaccines against PoRV fail to provide sufficient protective immunity, necessitating urgent development of effective vaccines and antiviral drugs against PoRV. Here, we successfully established and improved the entirely plasmid-based reverse genetics (RG) system to rescue a G9 genotype of recombinant PoRV AHFY2022 strain (G9P[23]). Using the improved RG system, we rescued recombinant AHFY2022 harboring the fluorescent UnaG protein or nano-luciferase (NLuc) reporter within gene segment 7 that encodes non-structural protein 3 (NSP3). Furthermore, we adopted the UnaG reporter virus to screen anti-PoRV drugs and identified two promising antiviral drugs, C8 and C9. Moreover, we generated the recombinant PoRV (rAHFY2022-G5-VP7) containing G5 genotype of VP7 from PoRV-positive samples in the backbone of AHFY2022 strain. The reassortant strain exhibited efficient replication and genetic stability. Mouse models were utilized to evaluate the immune responses elicited by rAHFY2022-G5-VP7 strain in vivo, revealing similar neutralizing antibodies and cellular immune response compared to the parental AHFY2022 strain in mice. Together, this study provides an important tool for screening potential anti-PoRV drugs and developing novel vaccines against prevalent PoRV strains.