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