Multiple amino acid substitutions involved in the adaptation of H6N1 avian influenza virus in mice.

H6N1 avian influenza viruses (AIVs) are one of the most abundantly detected avian influenza virus subtype, and a human H6N1 infection case has been reported in 2013. H6N1 AIVs may pose a potential human risk, however, the factors that promote the replication of H6N1 viruses in mammals remain poorly understood. Here, we generated mouse-adapted variants of a H6N1 virus (A/Mallard/SanJiang/275/2007) to identify adaptive changes that confer enhanced virulence to H6N1 viruses in mammals. After eight sequential passages in mice, the mouse lethal doses (MLD50) of the variants were reduced >1000-fold compared to the parental virus. We found that the variants displayed the greatest enhancement of replication in vitro and in vivo, and also were capable of replicating in the brains of infected mice. These observations suggest that enhanced growth characteristics and modified cell tropism may contribute to increased virulence of H6N1 AIVs in mice. Sequencing of the variants revealed amino acid changes in the PB2 (E627K), PA (T97I), and HA (N394T) proteins. Our results suggest that these mutations involved in the enhancement of the ability of H6N1 virus to efficient replicate and cause severe disease in mammals.