Molecular characterization of H3N2 and H4N6 subtypes avian influenza viruses isolated from mallards in Poyang Lake, China in 2010

Poyang Lake is the largest inland freshwater lake in China and contains many species of wild birds and waterfowls. We conducted a survey of avian influenza viruses in nine semi-artificial waterfowl farms in Poyang Lake during January to March of 2010. Out of 1036 cloacal swabs collected, three H3N2 and one H4N6 influenza viruses were isolated from healthy mallards. All the isolates were genetically and phylogenetically characterized. The analysis of putative HA cleavage sites showed that all the four isolates possessed the molecular characteristics (QTRGL for H3N2 viruses, PEKASR for H4N6 virus) of lowly pathogenic avian influenza (LPAI) virus. The phylogenetic analysis of the viral genomes showed that all four virus isolates clustered in the Eurasian clade of influenza viruses. The M gene of the viruses possessed the highest homology with highly pathogenic H5N1 influenza viruses. In addition, co-infection of H3N2 and H4N6 in the same farm was observed. And interestingly, we isolated two subtypes viruses (H3N2 and H4N6) and their progeny virus (H3N2) with evidence of genome reassortment from the same farm, in which the PB1 and PB2 gene segments of H4N6 replaced those of the H3N2 strain. The results of animal infection experiments showed that all the four isolated viruses were lowly pathogenic to chickens and not pathogenic to mice, which was consistent with the results of genetic analysis.