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.