HA Genetic And Antigenic Variation Of Influenza A Viruses In Shanghai

Influenza is a highly contagious acute respiratory disease which was caused by influenza virus.Because of high genetic variation of the virus,it infected the population again and again with high incidence,and the older and people with immunodeficiency would die for complications.About 20%of children and 5%of adults develop symptomatic influenza A or B and 250000 to 500000 people were killed worldwide each year.We surveyed the prevalence of influenza in Shanghai from 2004 to 2009,and analyzed the HA genetic and antigenic variation of influenza A viruses.The details as follow:1.By the surveillance of 2004 to 2009,we conclude as follows.Except for the summer of 2004 and winter and spring of 2007,influenza A/H1N1,A/H3N2 and B circulate in all influenza season together,but the dominated type or subtype varied. Influenza B virus was more popular in 2004 to 2005(proportion of positive specimen were 42.8%(24/56) and 56.2%(9/16)),was not isolated in 2006 to 2007,more active in the winter and summer of 2008(proportion of positive specimen were 34.5% (48/139)),disappeared in the summer and autumn of 2008,and only got two in the spring of 2009(proportion of positive specimen were 2.3%(2/88)).A/H 1N1 was more activity in 2005-2006(proportion of positive specimen were 21.4%(12/56),43.7% (7/16),76.4%(26/34)),and less activity in the winter and spring of 2007 and 2008(proportion of positive specimen were 0%(0/44) and 5%(7/139)),but start to act from the July of 2008,proportion of positive specimen in the summer and autumn of 2008 and the spring of 2009 were 87.4%(152/174) and 60.2%(53/88).A/H3N2 was the dominate subtype of influenza(proportion of positive specimen were 46.1% (257/557)).It was more active in the summer of 2004 and spring of 2005 influenza seasons,(proportion of positive specimen were 100.0%(6/6) and 35.7%(20/56), through was less popular in the spring and summer of 2006(proportion of positive specimen were 0%(0/16) and 24%(8/34));started to active again in spring of 2007 and 2008(proportion of positive specimen were 100.0%(44/44) and 61.2%(85/139)); then appeased in the summer of 2008(proportion of positive specimen were 12.6% (22/174);lately resumed active in the spring of 2009(proportion of positive specimen were 37.5%(33/88)).The influenza fastigium of the spring in 2008 was between March and April, which delayed for a month compared to years before.There was another influenza pinnacle in the July and August of 2008,and the peak vaule was higher than the former,which was exceptional to usual.A/H3N2 was the dominated subtype of the winter and spring influenza season(proportion of positive specimen was 61.2%),and A/H1N1 was isolated in the second half now and then(proportion of positive specimen was 5.0%).But A/H1NI domidated in the summer and autumn influenza season(proportion of positive specimen was 87.4%),and A/H3N2 was subordinate (proportion of positive specimen was 12.6%).The transformation of dominate subtype of influenza A might not only correlate to the population immune barrier,but also the boost up of virulence of the virus.We found one patient was infected by both A/H1N1 and A/H3N2,and the ability of replicition of A/H1N1 was stronger than A/H3N2.The influenza fastigium of the winter and spring in 2009 was in the sixth week, which advanced for a month compared with 2008 and similar to usual years.The composing of influenza subtype of the spring 2009 was similar to the summer of 2008. The dominated subtype was still A/H1N1(proportion of positive specimen was 60.2%),but the proportion of A/H3N2 increased(proportion of positive specimen was 37.5%).2.We sequenced the HA gene of influenza A virus and compared with the WHO reference vaccine strains subsequently,to understand the variation and speculate the evolution trend of influenza in Shanghai.In 2005-2009,HA gene of A/H1N1 influenza virus rooted in one trunk,and the sequences of the same year clustered which displayed they were similar to each other,but the branches between years were longer which revealed notable variation.Compared to A/NewCaledonia/20/1999 (H1N1)(recommend as vaccine from 2000 to 2007) and A/Solomon slands/3/2006 (H1N1)(recommend as vaccine from 2007 to 2008),A/Brisbane/59/2007(H1N1) (recommend as vaccine from 2008 to 2010) had shorter branch which showed it had best protection to the population of Shanghai.The influenza sequences of the summer (July to September) were farther to the influenza sequences of the winter and spring of 2008 and 2009,yet closer and clustered to the influenza sequences of the summer of 2006.Like R145K,R188M,A189T,I417V,S450N,N495S,these amino acid deduced from RNA variation only found in the influenza sequence of summer of 2006 and 2008,and R188M,A189T were in the antigenic site Sb.The phylogenetic tree of the A/H3N2 influenza virus in 2004～2009 was characterized as with one trunk and very short brunches.The trunk of the phylogenetic tree was very long and directed along with the time of isolated influenza, but the short branch hint the alternation of the virus is rapid.Phylogenetic tree of HA showed that A/H1N1 isolates in the same year clustered,and most A/H3N2 isolated were homologous in the same year while some were inserted to the clusters of near years.The influenza viruses in Shanghai of 2004～2009 also tried to evolve in the diverse ways,but conformed to the other countries worldwide and were similar to WHO recommend vaccines in the same period under the population immune stress. The interlaced influenza viruses isolated from different years made the more remarkable successive connection compared to A/H1N1.A/Brisbane/10/2007(H3N2) recommended as vaccine of 2008-2010 had shorter branch which showed whose sequence was similar to most influenza viruses and it had best protection to the population of Shanghai.Some influenza viruses isolated in 2008 were found had more sites variance,through most of which weren't in the antigenic areas and didn't reach the rules of new mutation,whether they would dominate and affect the epidemic need more time to survey.3.HA antibody was the main protection antibody and the variation of HA gene was the fastest of influenza virus genome,so the production,selection and effect of vaccine met a lot of problems.We chose golden hamster as the animal model and select influenza viruses isolated from sentry hospitals and influenza outbreaks of Shanghai area(include Shanghai,Ningbo,Wuxi) in 2005-2008 to get strain specific antiserum and do cross HAI experimentation so that to under the variation of influenza HA antigenic.The HAI value of strain specific antiserum to antigenic in the research were between 64 to 1024 and the similar research in which golden hamster were also chosen as the animal model got the HAl value that was between 16 to 256. The HAl value was between 8 to128 in one study which infected BALBc mice with several kinds of influenza vaccines.So the quality of antiserum in the research was sastisfactory.The analysis of HA antigen of A/H3N2 influenza viruses in 2005～2009 showed the antigen between 2005 and 2007 changed,but the antigen between 2007 and 2008 seemed resemble.The time from A/Sydney/5/1997(H3N2) was isolated to now was such a long period that the antigen evolved a lot and through they belonged to the same subtype,the cross HAI reaction and the cross protection between it with the influenza virus of 2005 to 2008 was feeble.The antigen of A/Fujian/411/2002(H3N2) recommended as vaccine of 2004 to 2005 was similar to the influenza virus of 2005, so the cross protection was subsistent,but it was obvious different from the influenza virus of 2007 to 2008,so the cross protection was faint.The antigen of A/Brisbane/10 /2007(H3N2) recommended as vaccine of 2008 to 2010 was similar to the influenza virus of 2008,so the cross protection was prominent.The analysis of HA antigen of A/H1N1 influenza virus in 2005-2008 showed the antigen between 2005 and 2006 were similar,but the antigen between 2005 and 2006 with 2008 both changed.The antigen of A/Newcaledonia/20/1999(H1N1) recommended as vaccine of 2000 to 2007 was similar to the influenza virus of 2005, so the cross protection was remarkable,but it started to be little different from the influenza virus of 2006,so the cross protection declined,and was almost heterogeneous to the influenza virus of 2008,so the cross protection slender.The antigen of A/Brisbane/59/2007(H1N1)-like recommended as vaccine of 2008 to 2010 was similar to the influenza virus of the winter and spring of 2008,so the cross protection was prominent,but it was little different from the influenza virus of the summer of 2008,and the cross protection reduced.