Genetic Characterization and Pathogenicity of a Triple-Reassortant H1N1 Swine Influenza Virus Isolated in Guangdong

An H1N1 subtype swine influenza virus (SIV) was isolated from nasal swabs collected from pigs with suspected swine influenza (SI) on a farm in Guangdong, China. The virus, designated A/Swine/Guangdong/JJK/2025(H1N1) (abbreviated as JJK), underwent whole-genome sequencing and phylogenetic analysis, which confirmed that it is a triple-reassortant strain. The hemagglutination (HA) and neuraminidase (NA) genes originated from the Eurasian avian-like (EA) H1N1 lineage, the internal genes (PB2, PB1, PA, NP, and M) originated from the 2009 pandemic H1N1 lineage, and the NS gene originated from the triple-reassortant H1N2 lineage. Each of the eight gene segments showed the highest homology to a different reference virus. Analysis of key amino acid substitutions identified multiple mutations in proteins, such as HA, PB2, and PB1, that are associated with enhanced mammalian adaptation, increased virulence, and modulation of host immune responses. Animal pathogenicity testing demonstrated that JJK was significantly pathogenic in female BALB/c mice, with an MLD₅₀ of 10^4.67 EID₅₀/50 μL, and caused severe lung lesions with high viral loads. However, in piglets, infection elicited substantial increases in serum inflammatory cytokines (TNF-α, IFN-γ, IL-6, and IL-10) but produced only mild clinical signs and limited pulmonary inflammation. To address the need for continuous surveillance of these evolving lineages, this study characterizes the genetic features and differential pathogenicity of a reassortant H1N1 SIV in distinct animal models, providing essential evidence to support epidemiological surveillance, risk assessment, and integrated control strategies for SI in China.