| Objectives:To investigate the mutation and migration of H9N2 in recent years,the evolutionary characteristics of global H9N2 genes from 2014 to 2018 were analyzed,which will provide evidence for the control of avian influenza.To explore new methods of treatment and detection,monoclonal antibodies of H9N2 were made and the specificity,cross-activity and neutralizing activity were explored.Methods:1.Genetic evolution:NA sequences of H9N2 from 2014 to 2018 were downloaded from GISAID and NCBI.The evolutionary tree was built in BEAST and the rate of genetic evolution,changes in common ancestors and major popular branches were analyzed.The dynamic migration process of NA was reconstructed with Asymmetric model.Migration Rate,Bayes Factor,Posterior Probability and Markov Jumps Counts in different regions were calculated to show the dynamic migration pathways.The homology,antigenic sites,sialic acid binding sites,glycosylation sites and enzyme active sites were analyzed by MEGA software.The selection pressure and epidemic trends of NA were analyzed with SLAC and FEL models by datamonkey online software.2.Antibody analysis:The BALB/c mice were immunized with purified H9N2 virus.After four immunizations,the mice spleen cells which had higher titer were fused with Sp2/0 myeloma cells.The monoclonal antibodies against HA/NA were screened by ELISA.The cross-activity and specificity of antibodies were tested,and the neutralization activity was preliminary explored in cell experiments.Results:1.A total of 432 sequences of NA were obtained from 2014 to 2018.The rate of evolution was(4.12±0.20)×10-3(95%HPD:3.73×10-3?4.52×10-3)time/site/year,and the time of common ancestor was 1983(95%HPD:1980?1987).The nucleotide homology of NA was 74.68%?99.93%,amino acid homology was 79.56%?99.93%.Among all regions,East Asia had the lowest nucleotide and amino acid homology(nucleotides 77.73%?99.93%,amino acids 79.16%?99.93%),while Southeast Asia had the highest amino acid homology(amino acids 90.48%?99.93%).2.The phylogenetic results showed that the NA sequences of H9N2 avian influenza virus from 2014 to 2018 were divided into North American lineage and Eurasian lineage.Eurasian lineage was futher divided into Korea-like,G1,G9 and Y280branches.In the distribution of countries,sequences separated from China,Japan,Russia and Vietnam were scattered in various branches and have different degree of mutation.3.The results of genetic migration showed that there were two decisive migration routes in the global spread of NA,including China to East Asia and China to Southeast Asia;Secondly,the migration routes from China to South Asia and Southeast Asia to East Asia played an important role.Overall,NA frequently migrated from China to other regions,while East Asian were mainly immigrant.There were few signs that NA sequences migrated from Europe and Americas to other countries.4.The characteristics of genetic molecular showed that 0.46%(2/432)of the NA stems were deleted at 38 and 39 sites.Otherwise,deletion at 63?65 sites accounted for56.25%(243/432).In terms of antigenic sites,153,328,331 and 346 sites were the most active.In comparison,China had the largest antigenic variation in NA sequences,followed by Africa.Among the sialic acid binding sites,367?369?399?401?432?433sites were active.Compared with other countries,China had the most significant variation in sialic acid binding site,followed by East Aisa.Among the enzyme active sites,no mutations in neuraminidase resistance site was found.In terms of glycosylation sites,the site at 264 was completely disappeared and the sites at 44,61,69,365 and402 were significantly mutated.5.The pressure of genetic selection showed that global NA sequences had positive selection sites at 3,8,19,61,77,86,93 and 312 in SLAC model(P<0.1),at 3,8,9 and19 in FEL model(P<0.1).6.In terms of monoclonal antibodies,splenocytes from two mice were fused and a total of 43 96-well plates were plated.Eventually,nine m Abs of NA(1E4-E11,6A9-F10,6C2-C11,6G11-C8,7F5-A7,8C1-D4,9G12-1C4,19C2,21E10)and four m Abs of HA(1B5-D11?7D5-F6?13E10-G8?13H7-G12)were obtained.Among the m Abs,the titer of 7D5-F6 was the highest(1×10-6),while the titer of 1E4-E11 and 8C1-D11was the lowest(1×10-3),others were ranged from 1×10-5to 1×10-4.7.The specific test showed that there were four m Abs of NA(7F5-A7,9G12-1C4,19C2,21E10)which could all bind to H9N2 virus in different years.The cross-reactivity results showed that no m Ab could bind to H1N2,H3N2,H5N8,H6N6 and H7N9.8.Cell neutralization experiments showed that the TCID50of the H9N2 virus was10-5.857.Two m Abs of HA(1B5-D11,13H7-G12)and one m Ab of NA(9G12-1C4)had neutralizing protective activity,the PD50 was 2-8.833,2-8.167 and 2-7.833.Conclusion:1.The evolution of global NA gene accelerated from 2014 to 2018.G1 and Y280were the dominant branches in evolution.Further more,China to East Asia and China to Southeast Asia were two main genetic migration routes.Countries need to pay more attention to international controls on live poultry trade to prevent further spread of H9N2 avian influenza virus.2.The enzyme activity sites of global NA sequences were stable,while the stem had some deletions.The antigenic sites,sialic acid binding sites,enzyme active sites had different degree of mutations.The positive selective pressure sites were constantly emerging.It is suggested that countries need to strengthen the surveillance of relevant mutation sites and update the vaccine strains in time.3.In the preparation of antibodies,mice were immunized with purified live H9N2virus.This method ensured the purity and complete spatial structure of antigen,which was significant for the success of influenza monoclonal antibody.Two HA and one NA monoclonal antibodies in this study had the neutralization activity in cells.In the future,the key antigen regions can be further located and the antibodies can be futher humanized,which is significant to improve the effect of targeting therapy.4.The four NA monoclonal antibodies in this study could bind to H9N2 from different years,but could not bind to other subtypes of influenza viruses.On that basis,those four antibodies could be further used in specific detection kits of N2 in the future,which is a new directon for rapid detection of avian influenza. |
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