Phylogenetic Analyses of the Internal Genes
The other six internal genes (polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), polymerase acidic protein (PA), nucleocapsid protein (NP), matrix protein (MP) and non-structural protein (NS)) shared 94.4%–99.6%, 95%–98.7%, 96.1%–98%, 94%–99%, 96.6%–98.8% and 94.6%–98.9% nucleotide identity, respectively.
The topological structures of the phylogenetic trees of MP and PB2 were highly similar (Figure 2C and 2H). All of the MP and PB2 genes from the strains analyzed in this study belonged to the G1-like lineage, and clustered into two phylogenetic subgroups. The phylogenetic tree revealed that the PA, PB1, NP and NS genes of all of the strains clustered together with the A/chicken/Shanghai/F/98-like (F/98-like) viruses (Figure 2F, 2G, 2D and 2E). Taken together, these findings indicated that the six internal genes of the H9N2 viruses isolated in Anhui province from 2013 to 2016 had undergone dramatic evolution and reassortment.
To understand the relationship between these internal genes and some novel AIV infections in humans in recent years, we added the internal gene sequences of the early H7N9 (LP and HP), H5N6, H10N8 and H7N4 AIVs into the phylogenetic tree. Surprisingly, all of the internal genes of the early H7N9 and H10N8 AIVs, even HPAI H7N9, clustered together in one group with the strains analyzed in this study. Intriguingly, the early H5N6 AIVs from Guangdong, Changsha and Sichuan did not form one branch with these strains, but did cluster into one group with Anhui H5N6 AIVs. A similar phenomenon occurred for H7N4 AIVs, with only their PB2 genes belonging to the G1-like lineage forming a different subgroup.