Mutations of 127, 183 and 212 residues on the HA globular head affect the antigenicity, replication and pathogenicity of H9N2 avian influenza virus

Abstract

H9N2 avian influenza virus (AIV), one of the predominant subtypes devastating the poultry industry, has been circulating widely in the poultry population and causing huge economic losses. In this study, two H9N2 viruses with similar genetic backgrounds but different antigenicity were isolated from a poultry farm, namely A/chicken/Jiangsu/75/2018 (JS/75) and A/chicken/Jiangsu/76/2018 (JS/76). Sequence analysis revealed that their surface genes differed in three amino acid residues (127, 183 and 212) on the head of hemagglutinin (HA). To explore the differences between the two viruses in their biological features, six recombinant viruses, including the wild‐type or mutant HA and NA of JS/75 and JS/76 were generated with A/Puerto Rico/8/1934 (PR8) backbone via reverse genetics. The chicken challenge study and HI assay data indicated that r‐76/PR8 showed the most obvious antigen escape due to 127 and 183 amino acid substitutions in HA gene. Further studies verified that the 127N site was glycosylated in JS/76 and its mutants. Receptor‐binding assays showed that all the recombination viruses were prone to bind the human‐like receptors, except for the mutants which glycosylated 127N was deleted. Growth kinetics and mice challenge experiments indicated that 127N‐glycosylated viruses showed less replication in A549 cells and lower pathogenicity in mice compared with wild‐type viruses. Therefore, the glycosylation site and two amino acid alternations in the HA globular head were responsible for the differences in antigenicity and pathogenicity between the two H9N2 isolates. This study is significant in the research of the antigenic variation and vaccine updates for the H9N2 AIV. Also, highlighted the critical functions of glycosylation in the influenza virus on the pathogenicity against mammals.

Hemagglutinin glycosylation pattern-specific effects: implications for the fitness of H9.4.2.5-branched H9N2 avian influenza viruses

“…This means that alterations in the glycosylation pattern of a given strain may lead to antigenic drift, enabling both the parental and mutant strains to escape immunity to one another. Consistent with the findings of Boyoglu-Barnum et al [ 53 ], our study also suggests that glycosylation of the HA stem contributes to viral antigenicity, despite the fact that glycosylation of the HA globular head is the primary driver of IAV antigenic drift [ 48 , 54 ]. It should be emphasized that the degree of HA glycosylation and the type of glycans attached to HA are host – or cell-dependent [ 55 ].…”

Effects of the Glycosylation of the HA Protein of H9N2 Subtype Avian Influenza Virus on the Pathogenicity in Mice and Antigenicity

“…One of the key factors that can evade vaccine immunity is the HA gene [19]. These phenomena are closely related to the glycosylation of the H9N2 AIV HA protein, as the deletion or addition of GMS to the HA protein affects antigenicity, pathogenicity, and even receptor binding [20][21][22]. Collectively, these findings suggest that we must be mindful of the potential interspecies transmission ability of H9N2 AIV and investigate the changes in antigenicity and pathogenicity caused by the glycosylation of the H9N2 AIV HA protein during the evolution of the virus [23,24].…”

“…Glycosylation of the HA protein blocks antibodies from binding, thus protecting against viruses [29]. The GMS on the HA globular head has been revealed to be particularly important for the antigenicity and receptor-binding characteristics of influenza virus [22,30]. GMS differences in H3N2 subtype influenza viruses impact the antibody neutralization response to influenza vaccine strains, decreasing the efficacy of seasonal influenza vaccines [31].…”

“…The introduction of glycosylation at site 158 (H9: position 152) of the H5N1 subtype influenza virus can promote viral production and intensify the host response, thus increasing its pathogenicity in mice [33]. The presence of N127D on the HA of H9N2 AIV suggested that the glycosylation at 127 was eliminated, which decreased the pathogenicity of the virus in mice [22]. The HA glycosylation of H3N2 subtype influenza viruses has been shown to increase annually, and the increase in GMS has resulted in a decrease in its pathogenicity in mice.…”

The molecular determinants of antigenic drift in a novel avian influenza A (H9N2) variant virus

“…These residues identified by mAbs were important for understanding the viral antigenicity; however, many residues are completely conserved among circulating viruses. While one or two amino acid changes can cause sufficient antigenic drift in some situations, simultaneous substitutions linked with unselected mutations at several sites is most generally observed [ 18 – 20 ]. Hence, an improved understanding of the molecular basis of antigenic differences between two related strains could ultimately facilitate the selection of more effective vaccine components to control the circulation of influenza in poultry.…”