| Objective1.To learn the prevalence of H3N2 subtype influenza virus and genome-wide variation at Guangdong province and provide reference for the monitoring,prevention and control of influenza virus,which can provide theoretical basis for the vaccine development of H3N2.2.To analyze the spatial and temporal distribution of different types of avian influenza virus in live poultry retail market and live poultry wholesale market.3.To investigate the seroepidemiology of avian influenza virus in libe poultry market as well as the infection of different types of avian influenza virus in occupationak exposed population.Methods1.The genetic sequence of H3N2 subtype influenza virus was collected by nasopharyngeal swabs of influenza patients in Guangdong flu surveillance output hospital from Jan 2015 to Dec 2017.The influenza virus was separated and cultured by using chicken embryo virus separation method and MDCK cell separation method.The virus nucleic acid was firstly extracted then performed H3 subtype specific detection after positive specimens were detected.The virus was retro-transcribed and amplified by PCR.While the amplification bands were detected by capillary electrophoresis.The amplification products of the target bands were sequenced,20 strains were randomly selected for sequence splicing and analysis each year.2.Avian influenza surveillance and serological study on avian influenza occupational exposure in Guangdong province was conducted by collection the data of environmental samples,live poultry and pharyngeal swabs from six live poultry markets from Feb 2017 to Feb 2019.The influenza virus was separated and cultured by using chicken embryo virus separation method.The virus nucleic acid was firstly extracted and detected the influenza positive samples,and then the subtypes of H5,H7 and H9 were specifically detected.The epidemiological data and nucleic acid test results was collated and analyzed.The serum samples of occupational exposure population in 6 cities of Guangdong province were collected and investigated by questionnaire.HA test and HI test were conducted on serum samples using H5,H7 and H9 subtypes of avian influenza virus of harycoviridae as antigens and positive serum.Results1.Among the H3N2 subtype influenza virus gene sequences in Guangdong province,the number of isolates from Dec 2015 to Dec 2017 was 17,which accounting as the highest proportion,June and July were in the second place.Among the cities,Qingyuan,Heyuan,Zhongshan and Shenzhen account at a higher proportion.The account of children(0-6 years old)and teenagers(7-17 years old)was much higher than that of other age groups.The number of isolates among men were slightly higher than women.The amino acid mutation sites were increased when compared the HA and NA genes of 60 isolates samples with influenza vaccine stains(from 2016 to 2018 and 2015 to 2016).The NA amino acid mutation site that would reduce the sensitivity of the virus to neuraminidase inhibitors did not change,suggesting the isolates in Guangdong province from 2015 to 2017 did not produce drug-resistant strains and were sensitive to neuraminidase inhibitors,such as osektamivir.The dites directly acting on the substrate and catalyzing the enzyme did not undergo the site mutation suggesting that the enzyme actibity center of NA protein remained highly conserved.2.From Feb 2017 to Feb 2019,14958 specimens of serum avian influenza surveillance and occupational exposure population of Guangdong province were collected from 17 live poultry retail market and live poultry trade wholesale market(located at Yangjiang,Guangzhou,Maoming,Meizhou,Chaozhou and Foshan).The specimens include cloacal swabs,pharyngeal swabs and various environmental swabs.The positive rate of type A avian influenza virus and H9 subtype avian influenza virus is higher than other subtypes and the trend is almost consistent.The positive rate of avian influenza virus was different and Foshan has the highest positive rate of avian influenza virus among all types.The positive rate of type A,H5,H7 subtype and unclassified avian influenza virus in the live poultry wholesale market was slightly higher than that in the live poultry retail market.The positive rate of type A avian influenza virus was the highest in Luohu market of Foshan.Among the live poultry wholesale markets,the positive rate of type A H5 subtype,H9 subtype and unclassified avian influenza virus was the highest.The positive rate of H7 subtype avian influenza virus was highest at Yangjing Sanniao wholesale market.The Maoming Maonan district market has the lowest positive rate of each type avian influenza virus.The serum samples for serological investigation were were collected in selected live poultry markets from April and Oct between 2017 and 2018 from occupational exposed people,respectively.All 547 serum samples were negative.ConclusionsThe H3N2 subtype influenza virus gene sequence study in Guangdong Province showed that the H3N2 subtype influenza was characterized by bimodal epidemics and was susceptible to minors.Guangdong Province's 2015 dominant strain is 3c2.a,and the dominant strains in 2016 and 2017 are A2 and 3c2.a,which is slightly different from the overall advantage evolution branch of China.This situation may be due to the different evolutionary choices of viral antigens,which led to the diversification of the H3N2 subtype influenza virus branch in Guangdong Province.This study found that the homology between the isolates of A(H3N2)influenza virus isolates and vaccine strains was generally higher in China from 2015 to 2017,but there were some differences and the branches were not clear enough.The 60 isolates from 5 epitope regions of A-E in this study were compared with the 2015/2016 and 2016-2018 vaccine strains,and the number of amino acid loci in the HA1 region was 38 and 40,respectively,and the mutation rates 11.55%and 12.15%,respectively.In particular,N121K and R142G/K mutations in multiple isolates belonged to the regions key amino acid sites,indicating that the H3N2 subtype influenza virus has a tendency to persist mutations,consistent with earlier reports.No variation in the T128A locus of the HA gene associated with high mortality in children was found in this study.Therefore,we should pay close attention to the variation of key amino acid sites in H3N2 subtype influenza.The H3N2 subtype influenza virus has a lower pathogenicity in recent years than other subtypes of influenza,but it still has the potential to cause a highly pathogenic mass epidemic.We should strengthen and improve the influenza prediction system,timely compare and analyze viral gene sequences,and master the antigenicity and genetic trait variation of A(H3N2)influenza virus in Guangdong Province.Consequently,it provides an important basis for the development of vaccines,and timely prevention and control of the spread and spread of influenza viruses.The monitoring and analusis of avian influenza in Guangdong province showed that the contamination of avian influenza virus in the environment outside the live poultry market was not optimistic and the pollution situation mainly occurs in circulation.The distribution of avian influenza virus of dififerent types was seasonal differnet.Furthermore,the positive rate of detection of A,H5,H7,H9 and other subtypes except H5/H7/H9subtypes is higher in spring and winter.The positive rate of avian influenza virus shows differences among 6 cities and Foshan had the highest positive rate.With the implementation of immunization of H7N9 subtype avian influenza virus since the autumn of 2017,it may be the most impotant reason for the extremely low positive detection rate of H7 subtype.The majority type specimens of external environment and poultry anal swabs were checked out of the bird:flu virus.A type of bird flu virve positive detection rate is higher at specimens of pigeon and quail house wiped sample,surface of scale,transport vehicle,floor wiped sample,duck anal swabs,etc.The results suggest that the contamination was easily caused by weighing and transportation of poultry.The main reason might be related to the different severity of contamination of different types of environmental samples by poultry inspection.Moreover,the HI test results for H5,H7 and H9 subtypes were all negative in this serological investigation,suggesting no recessive infection in the investigation. |
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