| Since the emergence of the reassortant H7N9 influenza virus in 2013,it has gradually evolved from low pathogenicity(LP)to highly pathogenicity(HP)in poultry,and has caused at least five epidemic waves in human population in China.At present,LP H7N9 avian influenza virus(AIV)is rarely isolated,while HP H7N9 AIV is continuous in circulation and variation in some areas of the country.HA is the most abundant glycoprotein and antigenic component expressed on the surface of influenza virus envelope,and plays an important role in the viral life cycle.With continuous evolution and variation of the viruses,the substitution of key amino acid sites of the HA protein could also alter the N-linked glycosylation(NLG)state.The addition or deletion of the NLG site and its nearby amino acid variation may also have a combined effect with the sugar chain,thereby affecting the function of the HA protein and the biological properties of the virus such as antigenicity and pathogenicity,and may enable the viruses to acquire the ability to spread efficiently,with the potential to cause a pandemic.Therefore,it is imperative to closely monitor the adaptive amino acid variation and the resulted NLG status in HA protein of H7N9 subtype AIV.1.Analysis of the amino acid variation in HA protein of H7N9 influenza virus from 2016 to 2021 and the construction of HA mutated reassortantsFirstly,the statistical analysis of the amino acid site variation of the HA gene of HP H7N9 influenza virus from 2016 to 2021 was carried out by searching the GISAID database and combining the data from the laboratory,while the NLG site variation was analyzed by the online tool of DTU Health Tech.We revealed that 4 amino acid sites in the receptor binding pocket region of HA protein head showed regular variation:T126K,V135T,S145P,A160T(H3 numbering),among which V135T and A160T also led to the addition of potential NLG sites at 133 and 158.Then,HP H7N9 AIV(GX110)without glycosylation at HA positions 133 and 158 was selected as the parental virus,and 4 point-mutated viruses with different NLG patterns were constructed by site-directed mutagenesis and reverse genetics technology,respectively named rGX110,rGX110133N,rGX110-158N and rGX110-133N?158N.Western-blot results showed that the molecular weights of the HA0 and HA1 proteins of the rGX110-133N,rGX110-158N and rGX110133N?158N viruses were increased,as compared with the rGX110 virus,which indicated these two sites indeed glycosylated.The EID50 and TCID50 results showed that the three HA mutants each had a higher replication ability in chicken embryos and cells than rGX110 virus.Next,we further selected rGX110 and rGX110-133N?158N as backbone viruses,introduced T126K and S145P single-point and double-point mutations into their HA genes,and six HA point-mutated viruses were successfully rescued,respectively named rGX110-126K,rGX110-145P,rGX110126K?145P,rGX 110-13 3N?15 8N-126K,rGX110-133N?158N-145P and rGX110133N?158N-126K?145P.We found that S145P mutation can significantly enhance the replication ability of the viruses in cells and chicken embryos.And it was worth noting that the value of hemagglutination titers,EID50 and TCID50 of rGX110-133N?158N and its HA mutants were generally higher than those of rGX110 backbone viruses,which again showed that the H7N9 virus with double NLG modification at positions 133 and 158 in the HA protein may have certain competitive advantages to promote its prevalence.2.Effects of glycosylation at positions 133 and 158 in HA protein on the biological properties of HP H7N9 avian influenza virusesTo determine whether the NLG modification at positions 133 and 158 in HA protein head of HP H7N9 virus enhances the competitive advantage of the viruses,we compared the biological properties of four HA point-mutated viruses:rGX110,rGX110-133N,rGX110-158N,and rGX110-133N?158N.By treating the viruses in buffers with different pH values for certain time or at 56? for different time,we revealed that glycosylation at positions 133 and 158 could enhance the acid and thermal stability of the viruses.Western blot results showed that rGX110133N?158N virus had significantly earlier HAO cleavage and higher level of HA1 protein expression when compared to the other three HA mutants.The growth curve of the viruses on cells in different species showed that glycosylation at positions 133 and 158 in HA protein could improve the replication ability of the viruses on CEF,MDCK and A549 cells,among them rGX110-133N?158N virus had the strongest increase.Receptor-binding assay results showed that the four viruses still retained dual receptor properties of ?2,3-SA receptor??2,6-SA receptor,but the affinity of rGX110-133N?158N virus for ?2,6-SA receptor was significantly reduced.Further determination of virus adsorption ability showed that the addition of glycosylation at site 158 inhibited viral attachment to the surface of CEF and A549 cells.The results of guinea pig erythrocyte coagulation test revealed that that with the same NA enzyme activity,the HA-NA functional balance of rGX110-133N,rGX110-158N,and rGX110-133N?158N,containing glycosylation at site 133 and 158,were stable,but that they could bind to guinea pig red blood cells weaker.In the SPF chicken model,the rGX110-133N and rGX110-133N?158N viruses showed increased replication and shedding levels in vivo,while rGX110-158N virus resulted a decreased effect.In the BALB/c mouse model,the replication of the three point-mutant viruses with single or double NLG modification in HA protein was enhanced,while the pathogenicity was weakened to varying degrees.Among them,rGX110-133N?158N caused the most evident decrease in virulence,which might be somewhat correlated with the reduced binding ability to?2,6-SA receptor.3.Effects of T126K,S145P and the combination with 133?158 glycosylation in HA protein on the biological properties of HP H7N9 avian influenza virusesIn order to clarify the effect of 133N?158N glycosylation and the combination with T126K and S145P on the properties of HP H7N9 viruses,the single-point or double-point mutant on the backbone of rGX110 and rGX110-133N?158N were further analyzed in this study.The results of the cross-hemagglutination inhibition test showed that a single point mutation of S145P in HA protein can alter antigenicity of HP H7N9 virus,while T126K mutation needs to be combined with 133?158 glycosylation to have the effect.The results of goose hemagglutination assay and receptor-binding assay showed that T126K and S145P mutation were critical to the enhanced binding to ?2,6-SA receptors of rGX110-133N?158N.The results of the hemagglutination assay with red Wood cells of guinea pigs showed that the rGX 110-13 3N?15 8N backbone viruses had a high HA-NA functional match,while the rGX110-126K and rGX110-133N?158N-126K viruses,with T126K in HA protein,had a low HA-NA functional match,and which shows that they have stronger receptor-binding ability,which is consistent with the results of receptor-binding assay.The growth curve of the viruses on mammalian cells showed that the position 145 is the key site of the replication ability of the rGX110,rGX110-133N?158N viruses,while the position 126 needs to be combined with 133?158 glycosylation to improve the virus replication ability on mammalian cells.In the BALB/c mouse model,the results showed that no matter in the rGX110 or rGX110-133N?158N viruses,the T126K mutation in the HA protein can lead to a significant decrease in the body weight or even death of the mice;the mouse organ virus titer results showed that T126K,S145P mutation can promote the replication level of rGX110,rGX110-133N?158N viruses in the lungs,and acquire the ability to replicate in the brains. |
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