Genetic and biological characterization of a reassortant H3N2 swine influenza virus isolated in China with internal genes from the 2009 pandemic H1N1

Swine influenza virus (SIV) not only causes significant losses to the pig industry but also poses a potential threat to human health due to its ability for cross-species transmission and zoonotic characteristics. In this study, 600 nasal swab samples were collected from pigs in Shandong Province and tested for SIV using RT-qPCR. One sample tested positive, and the virus was successfully isolated in 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs. Subtype-specific RT-PCR and sequencing identified the isolate as H3N2, designated A/swine/Shandong/116/2022 (H3N2). Whole-genome sequencing and similarity analysis showed that PB2, PB1, PA, NP, and M genes were most similar to H1N1 viruses (97.71-99.67%), while HA, NA, and NS genes were closest to H3N2 viruses (96.06-97.85%), suggesting this isolate is a reassortant between H1N1 and H3N2 viruses. Phylogenetic analysis indicated that PB2, PB1, PA, NP, and M genes belong to the 2009 pandemic H1N1 (pdm/09 H1N1) lineage, HA and NA genes belong to the human-like H3N2 (HL H3N2) lineage, and the NS gene belongs to the triple-reassortant (TR) H1N2 lineage. Key amino acid analysis showed a monobasic HA cleavage site (PEKQTR/G), consistent with low pathogenicity, and residues 190V, 226I, and 228S, which may affect receptor binding. PB2 residues 271A, 590S, and 591R may influence viral replication and host adaptation. Compared with the human influenza vaccine strain A/Darwin/9/2021 (H3N2), several amino acid changes were found in HA antigenic sites A, B, C, and E, suggesting possible antigenic drift. In addition, clear differences were found in N-linked glycosylation sites between the isolate and vaccine strain, including loss of several glycosylation sites and the appearance of a new site at position 499, which may change virus antigenicity and immune recognition. Functional studies demonstrated that the isolate efficiently infected MDCK cells and replicated in the respiratory tissues of BALB/c mice, causing mild to moderate lung lesions without mortality or significant weight loss. In summary, the isolated is a multi-source reassortant virus with low pathogenicity, providing valuable insights into the genetic characteristics and epidemiology of H3N2 SIV circulating in pigs in China.