Multiplex serological profiling reveals diverse avian and mammalian influenza A virus exposure in swine

Animal-origin influenza viruses pose significant pandemic threats, with swine serving as key hosts. Serological surveillance in pigs remains limited in regions with intense human-animal-avian contact. Between March 2020 and July 2022, we collected 4,069 pig serum samples in Cambodia, of which 1,321 (32.5%) were influenza A virus (IAV) seropositive by ELISA. We developed a multiplex microsphere immunoassay comprising a broad panel of hemagglutinin (HA) and neuraminidase (NA) antigens. Seroprevalence was highest for pandemic H1N1/pdm09 (35.3-45.7%) and lower for classical swine H1 (16.4-17.7%) and European avian-like swine H1 (∼15%) lineages. Lineage-specific or shared exposures to multiple lineages were observed, indicating complex infection histories. H3 responses varied by clade, while antibodies to avian H5, H7, and H9 were detected in <8% of IAV-positive sera. These findings underscore the ongoing zoonotic risk posed by diverse IAVs circulating in pigs and highlight the need for integrated serological and genomic surveillance.