Abstract
The wild-type H1N1 and H3N2 swine influenza virus (SIV) strains are unsuitable for vaccine production because of high lethality in chicken embryos and low reproductive titers. This study developed recombinant H1N1-Re1 and H3N2-Re1 strains via HA and NA genes from the wild-type H1N1 SW/GX/755/17 and H3N2 SW/GX/1659/17 strains combined with six internal genes from the H1N1 A/PR/8/34 strain. The recombinant viruses demonstrated typical cytopathic effects in MDCK cells, and the presence of viral particles was confirmed via electron microscopy. Growth curve analysis revealed titers of 10(8.31) and 10(8.17) EID(50) per 100 µL for H1N1-Re1 and H3N2-Re1, respectively, within 72-96 h postinoculation. Virus stocks were used to produce a bivalent inactivated vaccine. After two immunizations, hemagglutination inhibition titers in piglets were significantly greater than those induced by commercial vaccines and were sustained from 5 to 29 weeks postimmunization. Upon challenge with virulent wild-type SIV strains, viral isolation occurred in all pigs in the PBS group (5/5 protection), whereas no virus was detected in the bivalent vaccine group (0/5). In contrast, the commercial vaccine group had a viral isolation rate of 1/5. Pathological examination revealed severe pulmonary lesions in the PBS group, mild changes in the commercial vaccine group (1/5), and normal lung morphology in the bivalent vaccine group. This study demonstrated the successful application of an eight-plasmid reverse genetics system to develop recombinant vaccine strains with enhanced immunogenicity and replication efficiency. The bivalent inactivated vaccine provides prolonged and complete protection against H1N1 and H3N2 SIV strains, offering a robust tool for controlling evolving SIV variants.