Stable non-synonymous substitutions on NS gene (NS1 and NS2 proteins) of Qinghai Lake H5N1 influenza virus (Clade 2.2) after successive passages in Muscovy ducks.

Although worldwide concern has been raised since the large-scale outbreak of highly pathogenic avian influenza in wild birds at Qinghai Lake, China in 2005, the factors responsible for the ability to kill waterfowl remain unclear. The why and how questions of the H5N1 virus species-jump into its reservoir host need to be answered. In this report we test the pathogenicity and adaptation of Qinghai Lake (Clade 2.2) isolate to Muscovy ducks for further understanding of this virus. The isolate was highly pathogenic in ducks and retained its high pathogenicity even after 20 generations of passage in ducks. During the process of serial passages, only the NS gene developed non-synonymous substitutions, which caused two mutations in NS1 protein (Val23Ala and Leu207Pro) and one in NS2 (Phe55Leu). These mutations persisted immutably through all subsequent passages and the pathogenicity remained high, implying that highly pathogenic H5N1 virus remains stable in aquatic birds through oral transmission. Although the exact functions of these mutations are not known, our results provide an important foundation for further understanding the characteristics of the Qinghai Lake isolates.