Abstract
Background:
H6N6 subtype avian influenza viruses are widely circulating in waterfowl and exhibit considerable genetic diversity. Their frequent reassortment and potential host adaptation highlight the need for molecular surveillance in waterfowl populations.

Aim:
This study aimed to investigate the genetic characteristics and molecular evolutionary relationships of waterfowl-origin H6N6 subtype avian influenza viruses isolated in Shanwei, Guangdong Province, China.

Methods:
Whole-genome sequencing was performed on one duck-origin and one goose-origin H6N6 subtype avian influenza virus isolate. Sequence homology of the eight gene segments was analyzed using BLAST. Phylogenetic trees were constructed based on the HA and NA gene sequences. In addition, key amino acid residues were analyzed to evaluate molecular features associated with receptor binding, mammalian adaptation, drug susceptibility, and pathogenicity.

Results:
Both isolates were identified as H6N6 subtype avian influenza viruses. BLAST analysis showed that the closest reference strains differed among the gene segments of the two isolates. The HA and NA segments of the duck-origin isolate showed the highest similarity to H6N6 viruses isolated from ducks in Fujian Province, whereas several gene segments of the goose-origin isolate were closely related to H6N6 viruses isolated from geese in Guangxi Province. Both isolates retained 226Q and 228G at the HA receptor-binding sites, 627E and 701D in the PB2 protein, and 31S in the M2 protein. The goose-origin isolate possessed 66S in the PB1-F2 protein, whereas the duck-origin isolate had a deletion at positions 62–73 in the NA protein.

Conclusion:
These findings indicate the circulation of H6N6 subtype avian influenza viruses among waterfowl in Shanwei. Overall, the two isolates retained molecular characteristics typical of avian-origin low-pathogenicity influenza viruses. However, their complex genetic origins and several notable molecular markers highlight the need for continuous surveillance and further biological risk assessment.

Key words: Avian influenza virus; Genetic evolution; H6N6 subtype; Waterfowl; Whole-genome sequencing.