Molecular Epidemiological Study Of A(H3N2) Influenza Virus In Shandong Province

BackgroundInfluenza is categorized into A,B,C and D.Among them,the A(H3N2)influenza pandemic has been widely popular in the population since 1968.HA and NA proteins are the main H3N2 flu virus surface antigen and could mutate continuously.Especially,the genetic evolution of HA protein is the most active and closely associated with the occurrence of flu and epidemic.The glycosylation sites and receptor binding sites of HA protein may lead to the pathogenicity of strains and change of host.Because NA protein is an important target of drug treatment,the center of the enzyme activity key locus mutation would cause drug resistance.So the structure and function of HA and NA genes in the H3N2 influenza virus and molecular evolution characteristics in the epidemic process as well as patterns of genetic variation is of great significance.ObjectivesTo study situation of etiology surveillance of influenza virus in Shandong province from April,2014 to March,2015,to analyze the characteristics and patterns of genetic variation of HA and NA genes in H3N2 influenza virus in the process of molecular evolution,so as to provide scientific basis for predicting trends of flu and prevention of severe cases.MethodsWe first described the situation of etiology surveillance using database for influenza surveillance in Shandong province.Then 102 H3N2 influenza strains were selected from the laboratory of Shandong Provincial Center for Disease Control and Prevention from April,2014 to March 2015 to be recovered.Among them,95 strains were eventually detected by RT-PCR amplification and sequencing.Homology analysis was conducted by DNASTAR software package(EditSeq program and MegAlign program).ClustalW program of MEGA 5.0(Molecular Evolutionary Genetics Analysis,5.0)software was used to analyze multiple sequence alignment and the Neighbor-Joining method(NJ)was used to construct phylogenetic tree.Results1.There were 21276 samples of Influenza-like Illness collected in Shandong Province from April 2014 to March 3015,which showed that this period was pandemic period and 0-15 year-old persons were the mainly infected population(especially 0-5).A total of 2371 strains of influenza virus were isolated,including 1665 stains influenza A(H3N2)virus and epidemic peak was November 2014.December 2014 and January 2015.2.The homology of HA gene sequences and NA gene sequence of Shandong province were 98.0-100%and 97.7-100%,respectively.HA gene phylogenetic tree analysis showed that most gathered into a gene cluster,with A/Texas/50/2012(H3N2)the distant relatives and with A/Switzerland/9715293/2013(H3N2)the closest relatives.NA gene phylogenetic tree analysis suggested that most aggregated in a large branch,with the genetic distance of A/Switzerland/9715293/2013(H3N2)the shortest and A/Texas/50/2012(H3N2)the longest.Obvious geographical and time characteristics were not observed.3.The number of HA protein amino acid changed was a total of 52,and 17 of them were located in the antigenic determinant and cracking site sequence did not change.Loss of glycosylation sites was NES134-136 and NSS156-158,and NYT170-172 was the new glycosylation site.The number amino acid changed for NA protein was 51.Enzyme active center sites variation included 118(N-D)and 371(V-I)and catalytic sites variations were 156(V-I)and 156(N-T).Lost glycosylation sites included NWS77-79 and NDS320-322 and at the same time new glycosylation sites were NIT84-86 and NAS236-238.Conclusion:1.H3N2 influenza virus was the dominant strain of influenza virus from April 2014 to March 2015 in Shandong province and its epidemic peak time was the spring and winter(November,December and January).2.The homology and phylogenetic tree analysis of HA and NA genes suggested that those genes was close and the heredity distance to the vaccine strains was farther.Amino acid substitutions were observed on a part of the antigen sites in HA and NA protein.The common antigenic drift in HA gene might have happened.Vaccine strains of H3N2 influenza virus from 2014 to 2015 recommended by WHO might protect poorly and the 2015-2016 vaccine strains might have certain protective effect on it.3.Mutations also occurred on glycosylation sites of HA and NA genes.The influence of glycosylation sites change on strain gene characteristics might not be the same among different genes and different areas of the same gene.4.No mutation was observed on inhibitor sites of enzyme activity centers of NA and NA proteins,thus the viruses were might be sensitive to oseltamivir.