Molecular Epidemiology Of Human Infection With H7N9 Avian Influenza Virus In Liaoning Province

Objective: We comprehensively explore the molecular epidemiological characteristics of human infection with H7N9 avian influenza virus in Liaoning Province,and analyze the whole gene sequence characteristics of H7N9 avian influenza virus to provide a reference for the future source of genes for human infection with H7N9 avian influenza in Liaoning Province.Meanwhile,which contribute to provides molecular epidemiological data for prevention and control H7N9 avian influenza virus in Liaoning province.Meantime the study will help enrich the diversification of the prevention and control methods of human infection with H7N9 avian influenza in Liaoning Province in the future,in order to provide a scientific basis for the prevention and control of human infection with H7N9 avian influenza in Liaoning Province in the future.Methods: Firstly,we extract the viral nucleic acid from the sample according to the standard operating procedures,and use the q Real-time PCR to detect the extracted viral nucleic acid.the sample is positive if CT ≤ 37.Virus isolation and sequencing were assisted and completed by the Influenza Laboratory of the Chinese Center for Disease Control and Prevention.Virus isolation and whole genome sequencing were assisted and completed by the influenza laboratory of the Chinese Center for Disease Control and Prevention.The DISTANCE in MEGA-X was used to calculate the genetic distance between sequences.the reference sequence of A(H7N9)virus was downloaded from the Global Initiative on Sharing Avian Influenza Data(GISAID),The ALIGNMENT in MEGA-X compares the reference sequence and the sequence of the virus in this study,then uses the NeighborJoining(NJ)method to construct a phylogenetic tree,and uses the N-glycosylation prediction software Net NGlyc1.0 Server to analysis HA and NA amino acid sequence glycosylation site on online.Results: The nucleotide homology of the two surface genes of human infected with H7N9 avian influenza virus in Liaoning Province is 97.9%-100.0%,the amino acid homology is98.7%-100.0%;the nucleotide homology of the internal gene is 93.9%-100.0%,the amino acid homology is 95.7%～100.0%.All genes encoded by the virus have the highest homology with the vaccine strain A/Hunan/02650/2016 recommended by World Health Organization(WHO).The phylogenetic tree of the surface gene and internal gene of H7N9 avian influenza virus shows different characteristics.The phylogenetic tree of HA and NA segments shows that all the strains of Liaoning Province were clustered together with the Yangtze River Delta region(Jiangsu,Zhejiang)in the same period,belonging to the Yangtze River Delta lineage.But the phylogenetic tree of internal genes shows that the strains in Liaoning Province are not clearly geographically divided,and they are scattered between the Yangtze River Delta lineage and the Pearl River Delta lineage strains;and it can be found that all the virus strains in Liaoning Province are differentiated on the evolutionary tree Out of sub-branches,and farther from the strains in the early stage of the epidemic.There is no continuous multiple amino acid insertion(KRTA)at the cleavage site 338-339 on the HA protein of all strains in Liaoning Province isolated,which is a molecular feature of low pathogenic avian influenza virus.We find that HA of all strains from Liaoning Province included S138 A,G186V,Q226 L,and T221 P mutations on receptor binding sites,which may enhance the human receptor binding affinity of the virus.All strains have a deletion of "QISNT" amino acids at positions 69-73 in the stem region of the NA protein,and there is no resistance site mutation on the NA protein,but the S31 N mutation appears on the M2 protein.The virus is resistant to amantadine drugs.The study indicates that all strains in Liaoning Province had mutations of E627 K on the PB2 protein,which lead to enhance the adaptability of mammals.Some strains also have mutations related to enhanced virulence in other gene-encoded proteins,which enhances the virulence and replication power of the H7N9 avian influenza virus.Through the analysis of HA and NA N-glycosylation sites,it is found that the number and position of Nglycosylation sites in the HA protein have not changed compared with previous H7N9 avian influenza viruses;but the amino acid sequence NCSH→NCTH mutation of Nglycosylation sites in the NA protein was observed at position 42 in some strains,compared with previous strains.Conclusion: The human infected H7N9 avian influenza strains have high homology and close relations in Liaoning Province from 2016 to 2017;All strains have the highest homology with the vaccine strain A/Hunan/02650/2016 recommended by WHO of the year.The human infection H7N9 avian influenza strains belong to the Yangtze River Delta lineage in Liaoning Province from 2016 to 2017.The study finds that the H7N9 avian influenza virus is constantly evolving as the epidemic cycle goes on.The human infection H7N9 avian influenza strains in Liaoning Province from 2016 to 2017 do not have the molecular characteristics of the highly pathogenic H7N9 avian influenza virus,and belongs to the low pathogenic avian influenza virus(LPAI).Some key amino acid variations were observed in the HA protein receptor binding sites,The virus has dual receptor binding characteristics of avian and human.The virus has strong replication,virulence and host adaptability;the H7N9 avian influenza strain in Liaoning Province is sensitive to neuraminidase inhibitors,but has shown to be resistant to adamantane drugs.