3.1 Molecular feature and phylogenetic analysis of the virus
Sequencing results showed that the HA cleavage site sequence of the three H6N6 strains was PQIETR/GL, with only a single basic amino acid belonging to the molecular characteristics of low-pathogenic AIV. No mutation occurred at 224, 226, 228, 137, 138, and 190 sites (H3 numbering) of the main receptor binding sites. P186T, H156R, S263G mutation, and amino acid deletion at 158 of HA were found in the ZZ346 virus; these sites were not mutated in the other two viruses (Table 1 ). Whether mutations at these sites of HA would switch receptor specificity remains to be further studied.
The amino acid sequences of NA, PB2, and PB1-F2 of the three viruses were analyzed. The 11aa deletion in the stalk region of NA located at positions 59-69 was found in the ZZ346 virus strain; this deletion was not found in the other two viruses, JX20490 and ZZ1923. In this study, three strains of H6N6 viruses, including 627E, 271T, 701D of PB2-23 (Bussey, Bousse, Desmet, Kim, & Takimoto, 2010; Steel, Lowen, Mubareka, & Palese, 2009), 66N of PB1-F2 (Conenello, Zamarin, Perrone, Tumpey, & Palese, 2007), 38I of PA (Tan et al., 2014) did not mutate but still showed the traits of replication and prevalence in poultry animals (Table 1 ).
The results of gene evolution analysis showed that three H6N6 subtypes of AIV were reassortment virus, and their gene segments were derived from group-Ⅱ (ST2853-like) of Eurasian lineages (Figure1, 2 and Table2 ). For ZZ346 virus, the HA gene came from duck-originated H6N6 subtype in Fujian province in 2007, NA gene from H6N6 swine influenza virus in Guangdong province (A/Swine/Guangdong/K6/2010), while the other internal genes from clade of A/Duck/China/FJ2253/2014 (H6N6) in Fujian province. The HA gene of ZZ1923 came from clade of H6N2 subtype AIV in Sichuan province in 2014, the NA gene from the clade of duck-originated A/Duck/Guizhou/013/2014 (H6N6) in Guizhou province, and the PB2 gene from the A/Duck/Yamagata/061004/2014(H6N6) of Yamagata in Japan, and other internal genes from avian-originated (chicken, duck) and environment-originated H6N1, H6N6 subtype in Guangdong, Fujian, Guizhou, Guangxi, Hunan provinces in China. The HA gene of JX20490 came from clade of duck-originated A/Duck/Jiangxi/10304/2013 (H6N6) in Jiangxi province, the internal gene from clade of A/duck/Jiangxi/10304/2013(H6N6), except PB2 gene from clade of A/Chicken/China/GZ1063/2014 (H6N6).
3.2Receptor-binding specificity of H6N6 viruses
Recently, a screening assay for receptor switching of avian influenza viruses was developed (Suptawiwat et al., 2008). The SAα-2,6 or α-2,3-binding preference could be distinguished by the change of HA titer in reaction with TRBCs and SAα-2,3Gal-specific sialidase-treated TRBCs. Our results showed that H9N2 avian influenza virus GX767 and H3N2 human virus ST602 were preferentially bound to SAα-2,3Gal and SAα-2,6Gal receptors, respectively. Compared with HA titer of untreated TRBC of 1:128, HA titer of ZZ346 strain was 1:8 in SAα-2,3Gal-specific sialidase-treated TRBCs, which only had α-2,6Gal receptors. Nevertheless, ZZ1923 and JX20490 strainses could not agglutinate SAα-2,3Gal-specific sialidase-treated TRBCs (Figure 3 ). Our results indicated ZZ346 strain of the H6N6 virus could bind avian-like SAα-2,3Gal and human-like SAα-2,6Gal receptors.We further confirmed H6N6 viruses preferentially bound to SAα-2,3Gal or SAα-2,6Gal receptors by using a solid-phase binding assay. ZZ346 strain bound to both SAα-2,3Gal or SAα-2,6Gal receptors, although their affinity for the SAα-2,3Gal receptors was higher than that for the SAα-2,6Gal receptors. But ZZ1923 and JX20490 strainses only bound to SAα-2,3Gal receptors (Figure 4 ). The results of the two assays were in agreement, confirming that ZZ346 strain of the H6N6 virus has acquired the ability to recognize the human-like receptors.