Biological Effects Of Hemagglutinin Glycosylation Patterns Of G57 Genotype Of H9N2 Subtype Avian Influenza Virus

Since its first isolation from turkeys in North America in 1966,the H9N2 subtype of avian influenza has not only caused huge economic losses to the poultry industry,but also poses a serious threat to public safety as it can be transmitted across hosts to infect mammals and humans.The hemagglutinin(HA)protein,one of the major surface capsid proteins of influenza viruses,not only plays a key role in the binding of the virus to the sialic acid(SA)receptor of the host cell,but is also the main antigenic protein of influenza viruses,stimulating the production of neutralizing antibodies in the body.In this paper,we analyzed the sequences of H9N2 subtypes of avian influenza viruses in the NCBI and GISAID databases and found that the prevalent H9N2 avian influenza viruses in China are mainly of the G57 genotype.We analysed the glycosylation pattern of the full-length sequence(n=5,271)of the HA gene of the G57 genotype virus and found that the glycosylation pattern of its HA gene sequence showed polymorphism: the first strain isolated in 2007 had seven N-linked glycosylation sequences(NGS),while after 2010 there was a succession of viruses containing six to three N-linked glycosylation sequences.The first strain isolated in2007 had seven N-linked glycosylation sequons(NGS),and since 2010,glycosylation patterns containing six to three NGS have emerged.It is interesting to note that the prevalence of viruses with different HA glycosylation patterns varies significantly.Among these five glycosylation patterns,the prevalence of viruses containing 6 NGS has been maintained to date,and is the glycosylation phenotype with absolute prevalence among the G57 genotypes.The study of the amino acid site variation accumulated in the HA gene of H9N2 subtype avian influenza viruses over evolution and its effects on viral proliferation ability,receptor affinity and antigenicity are important for further revealing the adaptive evolution of viruses and vaccine design.In order to investigate the biological effects of the hemagglutinin glycosylation pattern of H9N2 subtype avian influenza viruses,five strains of viruses with different HA glycosylation patterns,named re HA3,re HA4,re HA5,re HA6 and re HA7,were rescued in this study using gene targeted mutagenesis and reverse genetic manipulation.growth kinetic curves of the five strains on CEF cells showed that,after infection The growth kinetic curves of the five viruses on CEF cells showed that the titres of re HA6 were significantly higher than those of the other four viruses from 2 to10 h post-infection(p<0.01);the titres of re HA7 were significantly higher than those of re HA6 at 48 h post-infection(p<0.05),while there was no significant difference in the titres of the viruses at 72 h post-infection(p>0.05).p H stability test results showed that re HA5,re HA6 and re HA7 were more stable under different The results of the p H stability test showed that re HA5,re HA6 and re HA7 were more stable under different p H conditions,while re HA3 and re HA4 were less acid-resistant.The results of the thermal stability test showed that re HA6 was able to maintain its hemagglutinating activity for a longer period of time at 56°C and 50°C,while the remaining four viruses maintained their hemagglutinating activity for a shorter period of time.The overall evaluation showed that re HA6 had the best thermal and acid stability,re HA7 and re HA5 were the next best,while re HA4 and re HA3 were the weakest.The results of the erythrocyte elution test showed that re HA7 was the slowest to dissociate from the erythrocyte surface,re HA6 and re HA5 were the next best,while re HA4 and re HA3 were the fastest.The results of the agglutination assay with erythrocytes treated with different concentrations of RDE showed that re HA7 maintained full hemagglutinating activity at the highest concentration of RDE used,with re HA6 being the second and re HA3 the lowest.The solid binding assay showed that all five strains could bind to both ?2,3SA and ?2,6SA,indicating a dual receptor binding property.In addition,re HA7 had the strongest affinity for both receptors,followed by re HA6 and re HA5,while re HA3 was the weakest.The results of the cross-hemagglutination inhibition test showed that the antigenic difference between re HA3 and re HA4 and re HA7,re HA5 and re HA6 gradually increased with increasing glycosylation,and re HA5 gradually increased with re HA6 and re HA7.The above results indicated that the glycosylation pattern of hemagglutinin affected the stability,proliferation ability,receptor affinity and antigenicity of H9N2 subtype avian influenza virus,and the most significant effects were found at the glycosylation sites of 210 and 305.The results of this study provide new insights into the adaptive evolution of H9N2 subtype avian influenza viruses.