Molecular Characterization
All of the HA genes contained 1742 nucleotides and most of the H9N2
strains possessed a conserved amino acid sequence motif ofPSRSSR↓GLF at the cleavage site, except for EnAH 04713/18
(PSKSSR↓GLF motif), which was a typical feature of LPAIVs. We
also examined the amino acid substitutions at the receptor-binding site
(RBS) of the HA protein, especially 183N and 190T. Among the 33 strains
in this study, 29 isolates possessed an A190T mutation, whereas all
isolates contained the H183N mutation. The amino acids at positions
226–228 were “LMG” in all strains. The potential N-linked
glycosylation sites (PNLGSs) in the HA proteins of the 33 isolates were
predicted by the NetNGlyc 1.0 web server. The results showed that
positions 29, 141, 298, 305, 313, 492 and 551 were conserved.
Non-conserved PNLGSs at positions 145 and 218 were located in the HA
protein globular domain. Only EnAH09183/14 virus possessed a PNLGS at
position 145, whereas the EnAH 08826/13, EnAH 08829/13, EnAH 09189/14
and EnAH 09180/14 viruses possessed a PNLGS at position 218. The seven
PNLGSs in the H9N2 viruses in this study appeared to be relatively
stable between 2015 and 2018
(Table
1).
Table 1. Molecular characteristics of the HA amino acid
sequences in H9N2 AIVs in this study Dashes (-) indicate the absence of
a potential glycosylation site
The NA gene from EnAH08829/13 virus did not cluster into one branch with
other viruses in this study. The hemadsorbing sites (HBSs) of
EnAH08829/13 virus contained “IRNGSRSG” and “DSENW” motifs at
positions 366–373 and 399–403, whereas “PQE” was observed in
position 431–433 in all of the strains. With the exception of
EnAH08829/13, the other viruses in this study possessed the “IKNGSRSG”
motif at position 366–373. Regarding the active center, the 33 strains
possessed six different patterns, with mutations mainly evident at
positions 141, 143, 149 and 155. All of the isolates in this study
possessed an amino acid deletion in the stalk region, unlike viruses
belonging to the G9-like lineage (Table 2).
Table 2. Molecular characteristics of the NA amino acid
sequences in the H9N2 AIVs analyzed in this study
Some critical residues in the internal genes were associated with
pathogenicity and infectivity, for example 627E and 701D in PB2 were
characteristic of LPAIVs and were observed in 27 of the 33 strains in
this study. Residues 100A, 356R and 409N in PA were human
adaptation-related markers that were observed in 3 of the 33 strains.
All of the strains possessed the I368V mutation in PB1 protein, but the
H99Y substitution was not detected in any of the strains. The 588I and
591K mutations in PB2 were also not detected in these strains. Residues
95K, 224N and 242N were found in the M1 protein of 31 strains, and 37A
was observed in this protein in all isolates. Residue 21G was detected
in the M2 protein of 26 strains, all of which showed increased
infectivity. The NS1-42S mutation was observed in all of the strains
analyzed in this study, but 92E was only found in EnAH 19204/15 virus.
The S31N mutation responsible for amantadine resistance was found in the
M2 protein of all H9N2 isolates, but substitutions in the NA protein
related to antiviral resistance to neuraminidase inhibitors were not
detected (Table 3).
Table 3. Molecular characteristics of the internal amino acid
sequences
in the H9N2 AIVs analyzed in this study