Effect Of Amino Acid Variation In NA Protein Of H7N9 Subtype Avian Influenza Virus On Neuraminidase Function And Viral Pathogenicity

Since the emergence of the novel H7N9 subtype influenza virus in China in 2013,it has caused great harm to poultry production and public health.Particularly,variants carrying polybasic amino acids at the HA cleavage site has emerged during the fifth wave of H7N9 epidemic period which has made H7N9 evolved from low pathogenic(LP)to high pathogenic(HP)to poultry.In addition,a complex situation once appeared that LP and HP H7N9 coexisted.Notebaly,the isolation rate of H7N9 in both poultry and human decreased significantly with the widely use of H5/H7 bivalent vaccine in poultry,and the further dissemination was effectively limited.At present,LP H7N9 has rarely been isolated but HP H7N9 is still sporadic.Therefore,continuous monitoring of H7N9 virus and the exploration of the variation patten are still important.NA,a complex multifunctional protein,which is second only to HA in abundance of expression on the IAV envelope plays an important role in helping the viruses escape from the mucin-related bait receptors and promoting the release of the viruses from the infected cells.The interaction with host cell surface receptors which NA performs the function of neuraminidase is considered to be a key factor in determining the infectivity and transmissibility of IAV.Besides,NA also carries the Second receptor binding sites(SRBS),which enhance the activity of the nearby enzyme catalytic sites by keeping the substrate in close contact with the site.By this means,SRBS influence the HA-NA balance of viruses and also play an important role in viral replication and pathogenicity.Therefore,it is necessary to study the adaptive amino acid site variation of NA gene of H7N9 subtype influenza virus and its effect on viral neuraminidase function,receptor binding characteristics and pathogenicity,so as to explore the evolutionary mode of H7N9 and master its epidemic trend.1.Analysis of adaptive amino acid variation in NA gene of H7N9 subtype avian influenza virusThe NA genes of H7N9 subtype influenza virus since 2013,which include 1467 sequences of human and 1039 sequences of avian,had been retrieved from the database of Global Initiative on Sharing All Influenza Data(GISAID).Bioinformatics method was used to screening amino acid sites with adaptive variation by compare the sequence of human and avian sources in each year vertically,and the sequence of human and avian sources in the same year horizontally.Particular emphasis was put on the adaptive mutation near the functional domain including neuraminidase catalytic amino acids and the SRBS.The results showed that a total of 11 amino acid sites in the NA gene including A21T(N9 numbering),M72I/T,E73G/K,D121N,R126K,Y166H,T178A,V209Y/I,S242P,D354N and D431G had displayed obviously regular variation in both avian and human H7N9 viruses as year went by.Among them,D121N,R126K and S242P were located near the enzyme catalytic site.Y166H mutated synchronously and stably in both avian and human sequences,and the degree increased gradually.T178A and V209Y/I were located near the framework residues.D354N and D431G were located near SRBS.Besides,D354N might influence the condition of N-glycosylation and D431G was adjacent to the K432 site of SRBS.2.Rescue of H7N9 reassortants with NA point mutation and the determination of viral neuraminidase functionIn order to clarify the influence of the variation of NA adaptive amino acid site that had been screened out in Chapter 1 on the function of neuraminidase of H7N9 viruses,4 amino acid sites including Y166H,S242P,D354N and D431G were selected.A series of point mutated H7N9 reassortants were rescued by modifying the NA gene via site-directed mutagenesis at the HP virus backbone of A/chicken/Guangdong/GD15/2016(rGD15)and its LP virus backbone ?rGD15 lacking the "KRTA" motif in HA gene artificially.Furthermore,the growth curves and neuraminidase function of the 8 recombinant viruses with their parental viruses were measured on different cells.As compared with the wildtype rGD15,the replication level of rGD 15-D431G in MDCK cell was poorer,while that of rGD 15-P242S was stronger.The completely same trend of the effect of D431G and P242S was occurred in the LP backbone.While in human A549 cells,the viral replication level evidently diverged at 72 hours,the growth of rGD15-P242S was significantly better than wildtype rGD15,whereas rGD15-H166Y,rGD15-D354N and rGD15-D431G were weaker than rGD15.The growth of both HP and LP H7N9 strains carring D431G mutation was weaker than the wildtype rGD15 in poultry CEF cells.The result of NA enzymatic activity demonstrated that the enzymatic activities of rGD15-D431G was lower,while that of rGD15-P242S was higher in either HP or LP backbones.Red blood cell(RBC)release assay also showed that the D431G mutation had resulted in the relatively slowest RBC release rate in both HP and LP virus backbones,which was consistent with the results of enzyme activity.3.Receptor binding property and pathogenicity in mice of H7N9 reassortants with NA point mutationFirstly,the receptor binding property of the constructed NA mutants of H7N9 virus above were determined by a solid phase direct binding ELISA assay.The results showed that all of these viruses were consistent with their parental viruses.They retained the binding property to both avian-like ?2,3-SA and human-like ?2,6-SA receptors.Subsequently,the effect of the variation of the adaptive amino acid sites of the NA protein on the pathogenicity of mice was determined.The results showed that rGD15-P242S caused more obvious weight loss in mice,and its ability to replicate in the lung was stronger.The ability of other viruses to cause weight loss was similar or weaker than that of the parental rGD15.The replication titers of rGD15-D431G in lung were significantly lower than that of rGD15 on 3 and 5 days post infection(dpi),the same trend was also observed in spleen on 5dpi.The replication titer of each virus was generally similar in the kidney.The detection of cytokines after challenge revealed that rGD15-P242S had stimulated the production of inflammatory factors TNF-?,IL-6,IL-1?significantly higher than rGD15,while rGD15-D431G had induced the production of TNF-?,IL-1?,INF-? significantly lower than the parental rGD15.In summary,the effects of adaptive amino acid site variation of NA gene on neuraminidase function,receptor binding characteristics and pathogenicity in mice were determined and analyzed in this study using the genetic backbones of HP H7N9 avian influenza virus and the LP mutant.This study could promote our understanding of the evolutionary rules of H7N9 virus to a certain extent.