| The clade 2.3.4.4 genotype is a new branch of H5 highly pathogenic avian influenza virus(HPAIV)that appeared in 2008,while this branch contains various subtypes such as H5N2,H5N3,H5N5,H5N6 and H5N8.The recent epidemiological surveillance data show that clade 2.3.4.4 has become the dominant epidemic branch in southern and eastern China,while the H5N6 isolates have replaced the H5N1 strains as the dominant epidemic subtype.The host range of H5N6 virus is wide.In addition to avian species including poultry,waterfowl and wild birds,some mammals,such as pigs and cats can also be infected.Furthermore,H5N6 subtype HPAIV is currently the only H5 subtype virus that can infect humans except H5N1,As of March 2020,a total of 24 cases of human infection have been reported,including 8 deaths.One of the key 'factors involved in the ability of avian influenza virus(AIV)to cross the interspecies barrier depends on the receptor binding property,which is mainly determined by the viral hemagglutinin(HA)protein.Most clade 2.3.4.4 viruses bear a T160A mutation in HA protein,resulting in the deletion of the glycosylation site 158,as compared with clade 2.3.4 H5N1 virus.Therefore,exploring whether this mutation is a key factor leading to the interspecies transmission of H5N6 AIV and how it affects the biological characteristics of the virus will help elucidate the pathogenesis mechanism of influenza virus-host interactions.In this study,receptor binding analysis was carried out on H5NX wild strains differing in glycosylation site 158,and recombinant viruses with 160A/T were constructed to identify their receptor binding characteristics and biological characteristics.Further evaluation of the proliferative capacity of both wild and mutant viruses in different cells and the pathogenicity in mice was performed,and the distribution and replication of these viruses in the respiratory tract of mice was also analyzed.Finally,from the host level,high-throughput sequencing technology was employed to analyze the differential gene expressions in A549 induced by infection of the model viruses with different glycosylation site 158.1.Glycosylation site 158 mutation is the key determinant for receptor binding properties of clade 2.3.4.4 H5NX virusesWe analyzed the receptor binding characteristics of the clade 2.3.4.4 H5N2(QD7),H5N5(031),H5N6(HX,Y6)and H5N8(WF1)viruses isolated from the live poultry market and found that the receptor binding properties of the 5 strains were different.Analysis of key amino acids in hemagglutin(HA)proteins found that the receptor binding properties of 031,Y6 and WF1 strains were consistent with those of most human-derived H5N6 viruses,that the HA gene with a T160A mutation deleted the glycosylation site 158.These three strains showed ?-2,3 and ?-2,6 dual receptors binding activity,while HX and QD7 did not change glycosylation site 158 and exhibited only ?-2,3 avian receptor affinity.However,the biological effect of the glycosylation site 158 mutation in the HA gene of clade 2.3.4.4 H5NX virus remains unclear.Therefore,we constructed four recombinant viruses,namely Y6-P-160A,RY6-P-160T,HX-P-160T and RHX-P-160A,with their external genes HA and NA from the wild type H5N6 strains Y6 and HX respectively,and their internal gene cassette from PR8(H1N1).After analyzing the receptor binding characteristics of the four recombinant virases,we found that Y6-P-160A and RHX-P-160A exhibited dual receptor binding,i.e.,both ?-2,3 and ?-2,6 receptor recognition and binding,while RY6-P-160T and HX-P-160T experienced only ?-2,3 avian receptor recognition and binding property.In addition,in the chick embryo fibroblast(CEF)cells,RHX-P-160A displayed the least replication ability,and RY6-P-160T showed the highest replication ability among the four recombinant viruses,while the difference of the highest titers between 4 viruses was not significant.In mammalian cells.Y6-P-160A and RY6-P-160T showed better replication ability than that of recombinant viruses originated from wild H5N6 HX strain,while there is little difference in replication ability between the parental Y6 and HX viruses.Evaluation of the pathogenicity in mice revealed that the both parental Y6 and HX strains had low pathogenicity to mice,while all of the four recombinant viruses showed moderate pathogenicity in mice.However,recombinant viruses Y6-P-160A and RHX-P-160A tended to cause mice death in a shorter period post-challenge and also at a less dose when compared with RY6-P-160T and HX-P-160T strains,in line with the data obtained in receptor binding property analysis.We further constructed recombinant viruses of H5N2,H5N5 and H5N8 harboring the T160A or A160T mutations in HA,and confirmed that the deletion of the glycosylation site 158 is a key molecular marker for recognition and binding of ?-2,6 human receptors.Therefore,we conclude that the deletion of glycosylation at position 158 of HA protein caused by the T160A mutation in the HA protein is a key determinant of the dual receptor binding property of clade 2.3.4.4 H5NX subtype viruses.This molecular marker may help understand the mechanism that HPAIV transmitted cross the interspecies barrier,and to monitor and control their pandemic risk in human population.2.Contribution of glycosylation site 158 modification in H5N6 virus HA protein to the viral biological characteristicsTo explore the effect of glycosylation site 158 in HA protein from H5N6 viruses on viral virulence and heat tolerance,we used two H5N6 viruses Y6 and HX to construct 4 recombinant viruses,respectively named RY6 and RY6-160T,RHX and RHX-160A,with the mutation at residue site 160 in HA protein.In mice challenge model,the pathogenicity of recombinant viruses RY6-160T and RHX with glycosylation site 158 was higher than that of RY6 and RHX-160A strains without glycosylation site 158 respectively.This result is different from that of above mentioned recombinant viruses in the section of receptor binding property study,where the entire internal gene cassette is originated from the PR8 H1N1 virus.In that occasion,the virulence of Y6-P-160A and RHX-P-160A without glycosylation site 158 was higher than that of RY6-P-160T and HX-P-160T with glycosylation site 158 in mice.Further,we found that RY6-160T was more widely distributed in vivo in mouse model than RY6,and the virus could be detected in a variety of organs from the challenged mice.On the contrary,RY6 could only be detected in the lungs and hearts of the infected mice,coinciding with their pathogenicity evaluated in mice.The growth kinetic curves in different cells indicated that the replication ability of recombinant virus RY6-160T was better than that of RY6 strain.To clarify the effect of glycosylation site modification on antigenicity of the recombinant viruses,the reactivity of the recombinant viruses with positive antiseras was determined,all four viruses exhibited no difference in hemagglutination inhibition(HI)titers against the antisera specific to the vaccine strain Re-8,implying that the antigenicity of the recombinant viruses did not change significantly.Furthermore,the thermal stability of the recombinant viruses was assessed,RY6 and RHX-160A kept infectivity in cells after treating at 560 C for 60 minutes,while RY6-160T and RHX lost their infectivity after the same treatment,suggesting a survival advantage of HPAIV strains without glycosylation site 158 circulated in natural environment.Although both RY6-160T and RHX showed more virulent in mice than RY6 and RHX-160A,RY6 and RHX-160A exhibited stronger binding ability to both guinea pig red blood cells and A549 cells.These results showed that the deletion of the glycosylation site 158 does not significantly alter the viral pathogenicity in mice,suggesting that the virulence enhancement of H5N6 strains with the glycosylation site 158 modification might have to cooperate with other HA protein mutations and/or virus internal genes.However,the deletion of the glycosylation site 158 in HA protein did confer the H5N6 viruses with both the robust heat tolerance and the binding ability to host cell.These advantages make the H5N6 strains with the glycosylation site 158 deletion easily adapt to the natural environment and readily circulate in hosts among clade 2.3.4.4 H5NX isolates.3.Influence of the glycosylation site 158 modification in H5N6 virus HA protein on the host immune responseSince the distribution of influenza virus receptors in the respiratory tract of mammals is different from that of poultry,avian influenza viruses replicate more efficiently in the lower respiratory tract of mammals.In the above study,we demonstrated that the glycosylation site 158 in HA protein of H5N6 viruses directly affects the receptor binding properties of the tested viruses,and the viral load of RY6 in both the turbinate and trachea was significantly higher than that of RY6-160T,indicating that RY6 replicated more effectively in the challenged mice when compared to the RY6-160T strain.The host cytokines stimulated by these two viruses at different time points post-infection were investigated.The inflammatory cytokines,such as HMGB1,IL-10 and TNF a were triggered more quickly,and expressed higher in RY6 strain inoculated mice,than those in RY6-160T strain challenged ones Meanwhile,the expression of these cytokines gradually decreased with the infection time lapse in both the virus inoculated mice.However,the level of cytokines triggered by RY6-160T strain in the late stage of infection was significantly higher than that in RY6 strain challenged mice,leading to the enhancement of pathogenicity in mice.In A549 cells,inoculated with high-doses,RY6 strain replicated to higher titers at 3 hours and 6 hours post-inoculation than the RY6-160T strain did when the challenge dose was high.However,this replication superiority for RY6 strain disappeared in the later stage of infection.The replication superiority of RY6 strain in the early stage accounts for the superior ability of RY6 strain to bind A549 cells when compared to that of RY6-160T strain.On the other hand,the efficiency of RY6-160T strain in both assembling and budding of viral-like particles was significantly higher than that of RY6 strain,and an apoptosis induced by RY6-160T strain was also stronger than that induced by RY6 strain.The results of differential transcriptomics analysis applied to the A549 cells infected with two strains showed that the differential expression genes in A549 cells induced by RY6 strain overwhelmed those by RY6-160T strain,indicating that RY6 triggered a wider range of host innate immune response pathways than RY6-160T strain after infection.Further biological function analysis revealed that RY6 strain infection of A549 cells caused a series of differential expressions of host genes that negatively regulated viral replication,interfering with viral invasion,replication,and transcription.The KEGG pathway analysis also confirmed the observations mentioned here.During RY6 strain infection cycle,NOD-like receptor pathway,p53 signaling pathway,NF?B pathway and other related pathways were triggered and up-regulated simultaneously.In addition,we found that the two host proteins EGFR and MYH9 might contribute to the virus invasion of cell after the virus binding to the sialic acid receptors on the cell surface Therefore,RY6 strain with deletion of glycosylation site 158 triggered the host immune response mechanism to antagonize the viral infection,making its pathogenicity to be milder and favor virus spread.In contrast,RY6-160T strain tended to stimulate the excessive expression of proinflammatory factors in the host,resulting in high pathogenicity in mice,in accordance with the pathogenicity judging by the mouse challenge model.In summary,major conclusions based on this study are as the following:(1)The deletion of glycosylation at position 158 caused by the T160A mutation in the HA protein of HPAIVs is a key determinant of the dual receptor binding property of clade 2.3.4.4 H5NX subtype viruses;(2)The deletion of the glycosylation site 158 does not significantly alter the H5N6 HPAIV viral pathogenicity in mice,however,this modification confer the H5N6 viruses with both the robust heat tolerance and the binding ability to host cell;(3)H5N6 virus with the deletion of glycosylation at position 158 of HA protein trigger the host immune response mechanism to antagonize the viral infection,making its pathogenicity tend to be milder.In contrast,H5N6 viruse with the glycosylation at position 158 of HA protein tends to stimulate the host to express excessive proinflammatory factors,resulting in higher pathogenicity in mice. |
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