Study On Influenza Epidemiology And Molecular Evolution Of The H₃N₂ Subtype HA Genes In Hubei From 2007?2012

ObjectiveThe influenza virus was one of the main pathogens which causing the acute respiratory infection disease,it was widely transmitted?for example:droplets and aerosols?,rapidly spread,and easily variated.It can not only cause people?pigs? birds?dogs and horses spreading across species,but also lead to viral pneumonia,multiple organ function damage,respiratory failure and even death when the crowds infected severely.According to the sentinel hospital monitoring,we could systematically identify the epidemical features from 2007 to 2012 in Hubei province,meanwhile through the understanding of the HA gene sequencing of seasonal H₃N₂ subtype flu,we could provide theoretical basis for the prevention and control of influenza in Hubei province.Methods1.The descriptive epidemiological analysisCollected the monitoring data of flu virus in Hubei province from 2007 to 2012,then established database by Excel 2013,and drew epidemiological chart,used the SPSS17.0software to process data while the inspection standard was equal to 0.05 or less,then we could come to the conclusion about the age distribution?the seasonal distribution?the trendy intensity and the subtype change of the influenza virus.2.The molecular biology research?1?The antigenicity analysis methodThe virus strains were inoculated in MDCK cells to be revived,then measured the virus suspension with erythrocyte agglutination test and hemagglutination inhibition test to know whether the antigen was changed.?2?Genetic analysisWe used the method of magnetic beads to extract the virus nucleic acid,and the method of RT-PCR to amplify the HA genes,then measured the RT-PCR products by 1%agarose gel electrophoresis,finally the products were sequenced by Huada genomical company of Wuhan branch.We used the DNA Star software to splice the sequences,usedthe Bioedit software to compare the nucleotide and amino acid,used the Net NGlyc 1.0server software to analyze the glycosylation sites,and used the MEGA5.2 software to construct the evolutionary tree,with N-J method.Results?1?The ILI cases monitoring features:The result showed that:the ILI % had obvious summer and winter peaks in the years of 2007 and 2008,and had a single peak in the fall-winter season in 2009,but non epidemic peak in 2010,2011 and 2012.The ILI% in teenagers under the age of 15 years was up to 92%,especially in infants and young children under the age of five,the ratio was 66%,and they were the main infected people.?2?The flu epidemic features:In the year of 2007,the seasonal flu H₃N₂ subtype was the main epidemical flu virus,in the year of 2008,the seasonal flu H1N1 was the main epidemical flu virus,in the year of 2009,the H1N1 virus was the main epidemical flu virus,in the year of 2010,seasonal H₃N₂ was the main epidemical flu virus,in the year of2011,H1N1 virus was the main epidemical flu virus,and in the year of 2012,seasonal H₃N₂ was the main epidemical flu virus.In the years of 2007?2008?2009?2010 and2012,there were two typical peaks-winter and summer peaks,and there was one peak-winter peak in 2011.?3?The H₃N₂ antigenic variation analysis:Choosing A/Hubei/Jingzhou/0779/2012?H₃N₂?as the standard antigen,if the measured strain titer was four times different compared with standard antigen titer,it meant to mutate.The measured strains of nucleotide sequence compared with the same period vaccine standard strains?2007-2012?had higher homology,which were 97% ? 99%.?4?The glycosylation analysis of H₃N₂ virus HA gene:The strains in Qiaokou and Jiangan areas had nine conservative glycosylation sites,the strains in Jingzhou and Dangyang areas also had nine conservative glycosylation sites,but the sites were different from Qiaokou and Jiangan areas.The strains in Yidu area had 12 glycosylation sites,the amounts and sites were all different from the other ares in Hubei province in our study.?5?The mutation analysis of amino acids in H₃N₂ virus HA gene:The number of the mutated amino acids was 50 in HA1 ares,including the sites of A zone?140?142?144?145?,B zone?157?158?159?189?194?198?,C zone?53?278?,D zone?207?and E zone?81?,There were 2 mutations in receptor site,which were 194 and 225,among them,the 194 was in the antigenic determinant area B and 225 in the non-antigen section.Conclusion?1?The ILI % fell after the novel influenza H1N1?2009?epidemic in Hubei province and the popular peak was not obvious and the ILI % should be monitored throughout the year,infants and young children under the age of 5 years were the main monitoring population,they were susceptible of flu,and they should be timely vaccination to enhance the protection.?2?In flu season there were two mainly popular peaks,summer and winter peaks in hubei province from 2007 to 2012,which were in conformity with the traditional popular seasonal flu.The advantage strains was given priority in flu season.A variety of mixed flu virus infected in non-epidemic season.The H₃N₂ subtype influenza was the dominant strain which caused flu epidemic every other year,we should pay more attention to the epidemical trend of it.?3?The antigenic variation happened in the H₃N₂ subtype influenza strains during the six years in Hubei province from 2007?2012.Combined with the result of the evolutionary tree analysis,the strains of nucleotide sequence had high homology with the same time vaccine strains.But in the same year,gene variations existed between different strains.?4?The glycosylation sites of H₃N₂ subtype influenza virus in different regions in Hubei province had very big differences in the amount and site,and the increasing or missing of the glycosylation sites impacted on the variation of influenza.?5?A large amount of mutations in amino acid appeared in the H₃N₂ subtype influenza in Hubei province,and the antigenic determinant mutations were mainly concentrated in A and B areas.In our study,we had a certain knowledge of variation trend of influenza through a combination of epidemiology and molecular biology research in Hubei province from2007?2012,which provided theoretical basis for future prevention and control of hubei province,as well as provided the reference for the other parts of China.