Study On The Epidemical And Molecular Characteristics Of Influenza Virus Circulating In Shenzhen In 2007

ObjectiveTo study the epidemiological characteristics of influenza in Shenzhen in 2007 and the relationship between molecular and genetic characteristics of HA1 domain of influenza virus and influenza epidemic so as to provide scientific evidence for making preventive strategies for influenza.Meterials and methodsInfluenza-like illness(ILI),outbreak surveillance data and reference line were analyzed using EXCEL and SAS 8.0 software,which was collected from the influenza surveillance network of Shenzhen.Naso-pharyngeal swabs from cases of ILI were propagated both in the allantoic and amniotic cavity of 9-to 11-day-old embryonated chicken eggs and Madin-Darby canine kidney(MDCK) cells. Hemagglutinin Inhibition(HI) test was used to identify type A(H1N1 and H3N2) virus and B virus.The sera were collected by simple cluster sampling method before and after influenza season in order to survey the antibody level of influenza in Shenzhen population.RNA of all the influenza viruses isolated in 2007 were extracted using High Pure Viral RNA Kit(Roche).The HA1 region was amplified by RT-PCR and subsequently sequenced to analyze the HA1 genetic evolution by DNASTAR, Simmonic and Mega 3.1 software.Phylogenetic analysis was confirmed for the homology of nucleitide comparing with the reference viruses of vaccines recommended by WHO and representative virus confirmed by China CDC so as to gain insight into the relationship between genetic,molecular characteristic of influenza virus and influenza epidemics.Results1.The epidemiological characteristics of influenza in Shenzhen in 2007The percentage of ILI in Shenzhen shows one peak mainly distributed in April to July,which reaches peak at 11.22%exceeded the alert line of 10.86%during the week ending of June 24,2007(week 25).The majority of ILI distributed in people aged below 15 and accouting for 72.97%.The alert line was with good sensitivity and seasonality,being timely in finding unusual value.50 suspected influenza outbreaks were reported in Shenzhen in 2007 and 35 outbreaks were confirmed by PCR.Of which,29(58.0%) outbreaks were caused by influenza A virus,while 6(12.0%) outbreaks were caused by influenza B virus.Most of the outbreaks occurred in primary and middle schools and frequently distributed from March to June, accounting for 92.0%.2831 specimens were tested for influenza virus in Shenzhen in 2007,and 343 were positive.The isolating rate was 12.12%.The proportions of influenza H1N1,H3N2 and B virus were 3.50%,62.68%and 33.82%,respectively. The predominant strain was influenza H3N2 virus from January to June and B(Yamagata) was during July to Devember.The monthly isolating rate shows an increasing trend and reaches the peak in June(28.38%),and a decreasing trend in the latter half year.With the baseline information we found that the predominant strain appeared by turns.The sera from 828 and 872 individuals were collected in Shenzhen in March and September,respcetively.The level of H3N2 antibody was high,and the antibody positve rate were 65.82%and 72.94%in March and September,respectively. While the positive rate of influenza H1N1,B(Victoria),and B(Yamagata) virus in March and September were 46.98%,43%,43.84%,21.90%,41.18%and 21.67%, respectively.The level of GMT of influenza H1N1,B(Victoria) and B(Yamagata) virus were low besides influenza H3N2 virus.2.The genetic variation of HA1 of the influenza virus circulating in Shenzhen in 2007Phylogenetic analysis of influenza H3N2 virus was consistent with the homology of nucleitide comparing with the reference viruses.Strains isolated from January to April clustered with A/JXDH/312/2006(H3N2),which was recommended by China CDC.The viruses isolated from May to December(accounting for 89.30%) show a closer relationship to A/Brisbane/10/2007(H3N2)(vaccine strain for 2008～2009 season) than to A/Wisconsin/67/2005(H3N2)(vaccine strain for 2006-2008 season), which was clustered with viruses of 2005～2006.A/Brisbane/10/2007-like viruses were a antigenic variant which evolved from,but were antigenically distinct from,A/ Wisconsin/67/2005-like viruses.Compared with A/Wisconsin/67/2005(H3N2),all the influenza H3N2 viruses in 2007 had the amino acid substitutions of D122N and I223V.Strains clustered with A/JXDH/312/2006(H3N2) showed the amino acid substitutions of R142G and N144D(located at antigenic site A),while the virus isolated after May had the amino acid substitutions of G50E,K140I(located at antigenic site A).While the amino acid substitutions of D122N,N144D resulted in the deletion of glycosylation site.The sequences at receptor-binding sites were conserved.HA1 phylogenetic analysis of influenza H1N1 virus shows that there were at least three clades circulated in Shenzhen from 2005 to 2007.Viruses isolated from 2006 to 2007 were in the same clade with A/GDLH/219/2006(H1N1).Viruses,during Januray to April of 2005,were clustered with A/New Caledonia/20/1999(H1N1). Viruses,in the latter months of 2005,were clustered with A/Solomon Island/3/2006 (H1N1).Compared with vaccine strain A/New Caledonia/20/1999(H1N1),the virus isolated after May of 2005 occurred T82K,Y94H,R146K,R209K and T267N amino acid substitution,while some virus after May 2006 took place the amino acid substitutions of A190T,H193Y,E195D(located at antigenic site B)and R146K (antigenic site A).The sequences at the receptor-binding sites and glycosylation sites were conserved.Phologenetic trees of the HA1 domain shows that all the influenza B virus falled into two lineages,Yamagata and Vicroria lineage.Influenza B(Victoria) virus isolated form 2006 to 2007 clustered with B/Malaysia/2506/2004(vaccine strain for 2006～2008 season) and B/Shenzhen/155/2005.Strains of B(Yamagata) lineage of 2007 reveal a closer relationship to B/FJXL/54/2006 than to B/Florida/4/2006(vaccine strain for 2008～2009 season).Compared with vaccine strains,influenza B(Victoria) virus had the S134P amino acid substitution.Virus belonging to the B(Yamagata) lineage shows the amino acid substitution of R48K,P108A,L131P,and D197N.The glycosylation site of 197-199 shows high rates of variation.76.66%of the B B(Yamagata) virus shows the amino acid substitution of D197N,and resulted in the deletion of one glycosylation site. ConclutionsThere is one epidemic peak of influenza in Shenzhen,which was mainly distributed in summer.The alert line has good sensitivity and seasonality,which could help to find out the abnormal value in time.The predominant strain circulating in Shenzhen appears by turns.Children are easily infected by influenza virus,so the school surveillance is quite important.The genetic and antigenic characteristics of influenza H3N2 virus isolated in 2007 had changed and resulted in the increasing influenza activity of influenza H3N2 virus.The variation of influenza virus and the level of antibody of people play an important role in the influenza activity.It is necessary to bring the molecular analysis into influenza surveillance and more attention should be paid to the emergence of novel influenza virus.