| Background and Objectives:Avian influenza virus is a zoonotic virus that can cause acute respiratory infectious diseases.Since the first human case of the H7N9 strain emerged in 2013,it has caused huge economic losses.Although there have been some reports on the epidemiology of avian influenza in China and antibodies against H7N9 avian influenza at present,the investigation of avian influenza in Jiangsu province in recent years is relatively scarce and reports on the novel antibodies against H7N9 avian influenza are insufficient.In this context,a cross-sectional investigation was conducted to analysis the distribution and trend of avian influenza in poultry and the related external environment in Jiangsu province from 2017 to2019,as well as the epidemiological characteristics and risk factors of the infection of H5N6,H7N9 and H9N2 avian influenza among occupational exposure population.The functions of the whole human H7N9 avian influenza monoclonal neutralizing antibody screened from the whole blood of convalescent patients of natural infection with H7N9 avian influenza virus were verified.Besides,the crystal structure of the complex of the neutralizing antibody and the hemoagglutinin(HA)of H7N9 avian influenza was preliminarily explored and clarified the key binding epitope of the antibody.Therefore,the epidemiological investigation of avian influenza and the study of H7N9 avian influenza antibody have a wide application prospect to achieve the role of emergency prevention or definitive therapy.Methods:Between 2017 and 2019,this study selected live poultry markets,large-scale farms,slaughter and processing house,wild bird habitats,and scattered households to collect the specimens from poultry and related external environments through cluster random sampling in 13 cities in Jiangsu province.After the samples were typed by nucleic acid testing,statistical analysis was performed to explore the infection of avian influenza among avian and the distribution in the relevant environment.In addition,epidemiological survey of the infection among occupationally exposed population were carried out in farms and live poultry markets in Suzhou,Yangzhou,and Huaian.The serum samples and demographic information from those who met the inclusion criteria and filled out questionnaires were obtained and collected.Hemagglutination inhibition(HI)was performed to determine the infection and the data were analyzed to evaluate the epidemiological distribution of different subtypes and the relevant risk factors of occupational exposed populations.This project adopted the flow cytometry to screen specific memory single B cells from the whole blood of convalescent patients.Then we extracted nucleic acid and used the specific light and heavy chain antibody primers to perform RT-PCR amplification and construct antibody sequence.The antibody plasmids were expressed and extracted in large quantities by transforming TOP10 competent cells and kits.Antibodies and antigen proteins were expressed and purified by using 293F cells and insect baculovirus systems,respectively.The neutralization activities were verified through functional experiments such as enzyme linked immunosorbent assay(ELISA).In addition,cryo-electron microscopy was used to analyze the three-dimensional structure of the complex of H7N9 virus HA protein and neutralizing antibody to determine the key amino acid epitope.Results:(1)A total of 14,479 poultry and the external environment samples were collected in this study.1998 were positive for avian influenza A by PCR,and the overall positive rate was13.8%.The positive rates for H9,H7,and H5 avian influenza were 8.8%,3.9%,1.1%,respectively.There was a statistical difference for the positive rates of H9,H7,H5 avian influenza between three years(P<0.001).Besides,the positive rates in winter and spring were generally higher than those in summer and autumn.The positive rate of avian influenza A in southern Jiangsu was higher than those in central Jiangsu and northern Jiangsu cities(x~2=437.959,P<0.001).The positive rates for avian influenza A virus from different surveillance sites were statistically different(x~2=699.173,P<0.001).Among them,live poultry markets in urban or rural and the slaughterhouses with poultry processing were the high risk areas.In addition,the surface wipe specimens from slaughtered or cutting board,the sewage after poultry cleaning and the wipe specimens from the surface of cages were the samples with high risk of infection of avian influenza.(2)Serological monitoring of occupational exposure population collected a total of 1,253serum samples from study subjects.We screened 8 positive samples of H5N6 avian influenza,with an overall positive rate of 0.6%.The serum antibody positive rates for H7N9 and H9N2avian influenza were 2.2%and 0.7%,respectively.Demographic information showed that there was no significant difference between different ages or gender in this study.Huaian was an area with a high risk of H7N9 avian influenza infection among occupationally exposed people,with a seropositivity rate of 5.4%(19/351).There were statistical differences between the positive detection rates in different cities(x~2=22.611,P<0.001).In addition,the H7N9avian influenza infections in different sampling years also had statistical differences(x~2=17.803,P<0.001).The analysis of related risk factors showed that people in live poultry markets are more likely to get infected than other places,and live poultry traders in markets were more susceptible to get infected than those exposed to other situations.In addition,the exposed persons who were exposed to the three types of poultry were at higher risk of H7N9 infection than those exposed to a single specie or two species of poultry(x~2=9.666,P=0.020).(3)In this study,mononuclear cells from the peripheral blood of convalescent patients with H7N9 avian influenza were isolated,and the flow cytometry was used for cell sorting to obtain the single B cells with specificity HA of H7N9 virus.Both ELISA and Western Blot(WB)experiments showed that the fully human monoclonal neutralizing antibodies m Ab910and m Ab6137 bind to the HA of H7N9 avian influenza specifically.The results of HI,microneutralization(MN)and mice challenging experiments showed that two new types of neutralizing antibodies have great neutralizing activity,which could prevent H7N9 infection.After the antigen-antibody incubation and forming the antigen-antibody complex,high performance liquid chromatography(High Performance Liquid Chromatography,HPLC)was used to detect the curve of the complex which appeared earlier and was significantly higher than the peak of the antigen.This largely predicted the formation of the antigen-antibody complex.Conclusions:Avian influenza virus was widespread in 13 cities from Jiangsu province,especially among the live poultry markets and slaughtering factories where were the most severely polluted areas.The infections of H5N6,H7N9 and H9N2 avian influenza virus were common among people with occupational exposure.The risk of infection in Huai’an was high and the H7N9 infection rate has increased.Surveillance sites,specimen types,exposure methods,occupational types,and types of exposure to poultry might be the risk factors related to avian influenza infection.This study screened two novel fully human monoclonal antibodies against H7N9 avian influenza,which had neutralizing activity and could bind to the H7N9 avian influenza HA protein.To further explore the antigen-binding key epitopes of neutralizing antibody,this study will adopt cryo-electron microscopy to analyze the antigen-antibody complex or culture the protein crystals for analysis by X-ray diffraction.This will provide a scientific basis for the design and develop specific targeted immune agents. |
