Background: Swine influenza viruses (SIVs) is a highly infectious viral disease in pig population. This disease is caused by the influenza A virus. According to the World Organization for Animal Health (WOAH), three subtypes of SIVs have been officially reported, including H1N1, H1N2, and H3N2. However, information about this pathogen circulated in Vietnam is limited, especially during the last 5 years.

Aim: This research aimed to examine the molecular characteristics of SIVs circulating in Vietnam. A total of 213 swab samples from 18 slaughterhouses in Thanh Hoa province, North Central Vietnam, were collected between 2024 and early 2025, and SIVs were detected by realtime RT-PCR. The characteristics of these viruses were further determined based on the phylogenetic analysis of the Hemagglutination (HA) and Neuraminidase (NA) genes.

Methods: SIV subtypes were determined by realtime RT-PCR. The full-length HA and NA genes were amplified by PCR. Phylogenetic and characterization analysis of HA and NA nucleotides and proteins were performed using bioinformatics. Virus isolation and titration were performed in the Madin-Darby canine kidney (MDCK) cell line.

Results: Of 213 samples, 24 were positive for SIVs, representing an 11.27% prevalence at the slaughterhouses (SIVs were confirmed in 5 out of 18 slaughterhouses). These samples included 17 H1N1, 1 H1N2, and 6 H3N2 subtype samples. Phylogenetic analysis of the HA genes showed that four H1N1 subtypes belonged to the H1N1pdm09 lineage, three to the Eurasian "avian-like" (EA) H1N1 lineage, and one to the TR-H1N2 lineage. Additionally, four H3N2 subtypes were classified into clade IV of the recent human-line (RH) H1N1. Based on the NA gene sequences, seven viruses belonged to the EA H1N1 lineage, three to the RH-H3N2 lineage, and one to the TR-H1N2 lineage. Receptor-binding and antigenic site analysis revealed that all isolates could bind to both human- and avian-like receptors. Furthermore, in vitro analysis of the growth kinetics of SIVs showed that 12 SIV isolates could efficiently replicate in MDCK cell lines.

Conclusion: Our study demonstrated that all three WOAH subtypes have been circulating in Vietnam during 2024 and early 2025. H1N1 and H3N2 subtypes are predominant, suggesting a potential threat to human health. This highlights the importance of active surveillance as a critical step for swine influenza prevention and control in Vietnam.