DATA AVAILABILITY
Genome sequences generated in this study are publicly available in the
GenBank database under the accession numbers: MW494902-MW494909;
MW494931-MW494938; MW495024-MW495031.
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Table 1. Important amino acid sequences in HA, NA, PB2, PB1-F2 and PA of
viruses
Table 2. Phylogenetic lineage of interal genes of ZZ346, ZZ1923 and
JX20490
Table 3. Viral protein distribution in the respiratory tract of BALB/c
mice inoculated with H6N6 influenza A viruses
The symbols -, +, ++, and +++ indicate that the numbers of cells with
viral NP positive signal were 0, 1 to 20, 20 to 100, and >100,
respectively, in each section.
Figure 1. The phylogenetic tree based on the HA gene of H6 influenza
viruses. The viruses obtained in this study were highlighted in blue
while the representative strains were highlighted inred. Each group was
labeled to the right of the phylogeny. The length of scale bar
represents the nucleotide substitutions per site. The values for the
nodes are the consensus percentage support of SH-aLRT, aBayes, and
UFBoot, respectively.
Figure 2. The predicted receptor-binding site (RBS) structure of H6N6
ZZ346 virus. The 130-loop, 190-helix and 220-loop were highlighted in
red. The important amino acids in the RBS pocket were labeled
individually.
Figure 3. Characterization of the
receptor-binding properties of influenza viruses was determinated by
Hemagglutination test with 0.6% TRBCs treated and untreated with
SAα-2,3Gal-specific sialidase. The dashed line indicates the detection
limit.
Figure 4. Receptor-binding specificity of H6N6 viruses using a
solid-phase direct-binding assay. A: The human influenza A virus
A/ST/602/05(H3N2) and avian influenza virus DK/GX/767/2010 (H9N2) were
used as controls. B Receptor-binding properties of the representative
AIV strains to human-like SAα2-6 and avian-like SAα2-3 receptors were
tested using the solid-phase direct binding assay with trisaccharide
receptors. Red and green lines represent human-like and avian-like
receptors, respectively. Two replications presented similar results and
the mean values were shown.
Figure 5. Pathological changes and virus replication in the trachea and
lungs of mice inoculated with H6N6 viruses. Hematoxylin and eosin
staining of the trachea (A1 to A3) and lungs (C1 to C3),
immunohistochemical staining of the trachea (B1 to B3) and lungs (D1 to
D3). The trachea and lungs of mices were inoculated with ZZ346 strain
(A1 to D1), ZZ1923 strain (A2 to D2) and JX20490 strain (A3 and D3). The
influenza NP antigen staining appeared as brown color (B1, B3 and D1).
Scale bars, 50μm.
Figure 6. H6N6 viruses replication in the lungs of human inoculated with
H6N6 viruses in vitro . The immunohistochemical method was used to
detect virus NP protein in lungs inoculated with ZZ346 strain (A),
ZZ1923 strain (B2) JX20490 strain (C) and ST602 strain (D). The
influenza NP antigen staining appeared as brown color (A and D). Scale
bars, 50μm.