Characterization of the amantadine-resistant H5N1 highly pathogenic avian influenza variants isolated from quails in Southern China

Highly pathogenic H5N1 avian influenza viruses have spread in poultry and wild birds in Asia, Europe, and Africa since 2003. To evaluate the role of quails in the evolution of influenza A virus, we characterized three H5N1 viruses isolated from quails (QA viruses) in southern China. Phylogenetic analysis indicated that three QA viruses derived from the A/goose/Guangdong/1/96-like lineage and most closely related to HA clade 4 A/chicken/Hong Kong/31.4/02-like viruses. Molecular analysis suggested that QA viruses and clade 4 H5N1 viruses carried consistent residue signatures, such as the characteristic M2 Ser31Asn amantadine-resistance mutation, implying a common origin of these viruses. As revealed by viral pathogenicity tests, these QA viruses could replicate in intranasally infected mice, but were not lethal to them, showing low pathogenicity in mammals. However, they killed all intravenously inoculated chickens, showing high pathogenicity in poultry. Results from amantadine sensitivity tests of wild-type QA viruses and their reverse genetic viruses demonstrated that all QA viruses were resistant to amantadine, and the M2 Ser31Asn mutation was determined as the most likely cause of the increased amantadine-resistance of H5N1 QA viruses. Our study confirmed experimentally that the amino acid at residue 31 in the M2 protein plays a major role in determining the amantadine-resistance phenotype of H5N1 influenza viruses. Our findings provide further evidence that quails may play important roles in the evolution of influenza A viruses, which raises concerns over possible transmissions of H5N1 viruses among poultry, wild birds, and humans.