| In the spring of 2013,a novel H7N9 subtype influenza virus broke out among chickens and people in eastern China for the first time,posing not only a heavy blow to the poultry industry,but also a serious threat to human health.Currently,the virus has continuously triggered at least five epidemic waves in the population in our country.Comparing with any of the previous four,the fifth epidemic wave during 2016-2017 especially possessed a much earlier starting time,a sharply increased number of infected people,and a much wider infecting area.In addition,highly pathogenic(HP)variants with the insertion of m?Ltiple basic amino acids at the cleavage site of the HA gene also emerged in that period.As one of the major envelope proteins on the surface of influenza virus,the HA gene coded hemagglutinin not only plays an important role in the process of virus binding to sialic acid(SA)receptors on the host cell and mediating the fusion of virus envelope with cellular membrane,but also enables the stimulation of neutralizing antibodies as a main antigenic protein of influenza virus.Moreover,hemagglutinin is yet critical for virus pathogenicity.Therefore,study the amino acid site variation in HA gene of H7N9 subtype avian influenza virus during viral evolution since 2013 and the corresponding effect on viral receptor binding specificity,antigenicity and pathogenicity,is of great significance to further reveal the viral evolutionary dynamics and guide the vaccine design for more effective prevention and control of H7N9 outbreaks.1.Analysis of adaptive amino acid variation in HA gene of H7N9 subtype avian influenza virusThe HA genes of H7N9 subtype avian influenza virus since 2013 had been retrieved from the database of Global Initiative on Sharing Avian Influenza Data(GISAID)for multiple sequence alignment,with a particular emphasis on the adaptive amino acid variation near the receptor binding domain(RBD).Additionally,variated amino acid sites were also matched with the human H7N9 isolates for comparative analysis.The results showed that a total of 20 amino acid sites in the HA gene displaying obviously regular variation were screened out from both avian and human H7N9 viruses as year went,including the 6 site mutations in the RBD region of A121T/P,S127N,A134V,R139K,L177I and M236I(H3 numbering).Besides,the vast majority H7N9 strains either of avian or human origin bear 138C,186V,193K,221P,226L and 228G among the key amino acids that had been recognized as closely related to viral receptor binding property.However,226Q possessed an evidently raised proportion in both avian and human H7N9 viruses in 2017.2.Rescue of highly and low pathogenic H7N9 subtype avian influenza viruses and construction of HA mutated reassortantsTo study the effect of the above screened adaptive amino acid variation and the addition or elimination of glycosylation near site 158 of HA gene on the biological characteristics of H7N9 virus,the eight plasmid systems of a low pathogenic(LP)strain TM24 and a HP strain GD15 were constructed based on reverse genetics,respectively.Subsequently,we successf?Lly rescued the two parental viruses rTM24(157-&160-)and rGD15(157-&160+),and a series of HA mutants were also constructed through modification of the HA gene via site-directed mutagenesis.The EIDso and TCID50 of each rescued H7N9 reassortants were determined.The results showed that neither the single-site mutations of S127N,R139K,L177I and M236I nor the combined four-site mutations of NRII and NKII introduced on the HA backbone of LP rTM24(157-&160-)had obviously changed the replication ability of those HA mutants,except for rTM-NRII with decreased propagation on chicken embryos and cells when compared with the parental virus.As for the reassortants of modified HA glycosylation sites,the proliferation of rTM-1(157-&160+)was weakened on chicken embryos while both the rTM-2(157+&160-)and rTM-3(157+&160+)got enhanced in vitro.Alternatively,on the backbone of HP rGD15(157-&160+),HA mutants of rGD-1(157-&160-)and rGD-2(157+&160+)both got inferior reproductive performance in vitro while just rGD-3(157+&160-)acquired higher titers on chicken embryos than the parental virus3.Receptor binding property,antigenicity and mice pathogenicity of HA mutated H7N9 reassortantsFirstly,the receptor binding property of the above constructed HA mutants of H7N9 virus were determined by a solid phase direct binding ELISA assay.The results showed that when LP rTM24(157-&160-)was used as the HA backbone,the 9 reassortant viruses either with mutations in RBD region or addition of glycosylation sites rarely changed the receptor characteristics in contrast to their parental virus.They retained the binding property to both avian-like ?2,3-SA and human-like a2,6-SA receptors.However,based on the HA backbone of HP rGD15(157-&160+),all the 3 mutated reassortants still coincided with the dual receptor binding specificity of the parental virus but exhibiting an improved affinity to a2,6-SA receptors.Subsequently,antiserum of each HA mutated H7N9 reassortants were prepared to analyze the antigenicity by cross hemagglutinin inhibition(HI)assay.The res?Lts showed that antiserum against rGD-1(157-&160-),rTM-1(157-&160+)and rTM-S127N manifested good cross-reactivity with both LP and HP viruses,with the HI titers of 10 ± 0.8 log2,9.2 ± 0.8 log2 and 8.9 ± 0.7 log2,respectively.However,antiserum of rTM,NKII,rTM-L1771 and rTM-NRII displayed relatively poor reactivity,just with HI titers of 6.1±1.3 log2,6.3 ± 1.4 log2 and 6.4±0.9 log2,respectively.In addition,the analysis of the calculated antigenic R value showed that both the HA mutants of rTM-1(157-&160+)and rTM-NRII bearing minor antigenic difference against their parental strain with the HA backbone of LP H7N9(R=0.5).In contrast,among the HA mutants with the HA backbone of HP H7N9,both rGD-1(157-&160-)and rGD-3(157+&160-)yet possessed antigenic divergence from the parental strain rGD15(157-&160+),especially that significant antigenic variation was detected between GD-3(157+&160-)and rGD15(157-&160+)(R=0.35).Finally,we determined the effect of the increase or decrease of glycosylation sites near amino acid 158 of HA gene on the pathogenicity of H7N9 reassortants in mice.The results showed that among the LP H7N9 reassortants,rTM-1(157-&160+)induced not only a more evident declinein mice body weight but also strong viral replication and severe invasionin mice lung.As for the HA mutants of HP H7N9 viruses,rGD-1(157-&160-)couLd cause severe pneumonia in infected mice,with significantly higher virus titers in mice lung on day 3 post challenge when compared with the parental rGD15(157-&160+)and the mutated rGD-3(157+&160-).In summary,based on the backbones of LP and HP H7N9 subtype avian influenza viruses,we determined and analyzed the effect of the key amino acid variations in the RBD region and the increase or decrease of the glycosylation near site 158 in HA gene on the receptor binding property,antigenicity and mice pathogenicity.Our results may provide theoretical references for further exploration of the evolutionary rules,pathogenic mechanisms and vaccine design of H7N9 virus. |
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