Whole-genome characterization and antigenic analysis of two distinct duck-origin H6 Subtype avian influenza viruses in China.

Avian influenza viruses (AIVs) of the H6 subtype demonstrate broad host tropism and represent a persistent concern for both poultry and public health due to their documented potential for cross-species transmission. This study aimed to isolate and characterize two novel H6 subtype AIVs from duck farms in China, focusing on their genetic evolution, molecular characterization, and antigenic properties. Virus isolation was performed from AIV-positive cloacal swabs. Whole genomes were sequenced and phylogenetically analyzed. Bioinformatic tools were employed to identify critical amino acid motifs, glycosylation patterns, and reassortment events. Antigenic cross-reactivity was evaluated through hemagglutination inhibition (HI) assays. Two strains, designated as H6N6 (SHT) and H6N2 (GXG), were successfully isolated. Phylogenetic analysis classified their hemagglutinin (HA) genes into the ST/2853-like and ST/339-like lineages, respectively. The GXG strain was identified as a triple-reassortant virus, with its matrix (M) gene derived from an H9N2 AIV, and contained a unique arginine insertion at residue 169 of the HA. In contrast, the SHT strain possessed a deletion in the neuraminidase (NA) stalk region (residues 58-68). Asymmetric antigenic cross-reactivity was observed: antiserum against GXG partially inhibited SHT, while SHT antiserum showed no inhibition of GXG. Epidemiological data confirmed the dominance of the ST/2853-like lineage in China, with N6 as the predominant NA subtype from 2015 to 2022. In conclusion, the two H6 AIV isolates exhibit distinct genetic and antigenic characteristics, carry mammalian adaptation markers, and highlight a potential cross-species transmission risk. These findings underscore the necessity for continued surveillance and the development of appropriately matched vaccine candidates.