长沙市家禽市场环境中H5N1亚型禽流感病毒传播风险研究

Risk related to the transmission of H5N1 subtype avian influenza virus in the environment of poultry markets in Changsha, China

目的对长沙市家禽市场职业暴露人群进行禽流感病毒（AIv）H5N1亚型抗体水平和环境AIV核酸检测，并对环境中AIVH5N1亚型的血凝素（HA）基因进行测序分析。方法抽取长沙市1个区和1个县，各选择2个城区或乡镇家禽市场进行职业暴露人群H5N1抗体和环境AIV核酸检测。利用单放射免疫扩散溶血实验（SRH）对102份家禽市场职业暴露人员血清标本进行H5N1抗体检测，real-time PCR方法检测160份家禽市场环境标本（污水、禽类粪便、羽毛和禽类笼具表面涂抹标本）AIV核酸，对4份污水H5N1亚型AIV核酸阳性标本进行HA基因RT-PCR扩增和TA克隆测序，测序结果利用Lasergene和Mega5．0软件进行氨基酸比对和进化树构建。结果AIVH5N1抗体监测结果显示，家禽市场职业暴露人群血清H5N1抗体阳性率为25．5％（26／102），其中乡镇和城区家禽市场职业暴露人群阳性率分别为50．0％（9／18）和25．4％（17／67），乡镇家禽市场职业暴露人群阳性率高于城区。长沙市家禽市场环境中H5亚型AIV核酸阳性率为3113％（50／160），其中乡镇家禽市场阳性率为37．3％（31／83），高于城区家禽市场24．7％（19／77）；不同标本H5亚型AIV核酸阳性率不同：污水（50．0％，24／48）、羽毛（44．5％，4／9）、禽类粪便（29．8％，14／47）和禽类笼具表面涂抹（14．3％，8／56）；差异均有统计学意义（P〈0．01）。TA克隆测序得到4个AIV H5N1亚型HA基因序列，进化树显示4个AIV H5N1亚型HA基因与中国内地和香港禽来源的AIV分离株为同一分组，属于欧亚分支；4个AIVH5N1亚型HA基因受体结合位点氨基酸序列仍然为禽源（QSG）、HA1和HA2蛋白之间连接肽为多个碱性氨基酸序列（RERRRKK或RERRGKK），与人源AIV H5N1亚型具有相同的受体结合位点和高致病性的分子特征。结论长沙市家禽市场环境中存在较多数量的AIV H5N1亚型，是导致职业暴露人群抗体阳性率达25．5％的原因之一；环境中存在的AIV H5N1亚型HA基因表现出来的高致病性分子特征，增加了家禽市场环境中发生AIV传播的风险。

Objective To investigate the risk of H5N1 subtype avian influenza virus （AIV） transmission in the poultry market environment in Changsha city. H5N1 antibody levels among the groups related occupational exposure and AIV nucleic acid in the environment of poultry markets were detected. The characteristics of hamagglutinin （HA） genes of H5N1 AIV in the environment were analyzed. Methods One district and one county from Changsha city were selected randomly and two poultry markets at inner city or township levels were selected in the same district or county respectively. H5N1 antibody of the occupational exposure groups in the poultry market was tested and AIV nucleic acid in the poultry market environment monitored. One hundred and two blood samples of the occupational exposure groups were tested for H5N1 antibody with single radioimmunoassay diffusion hemolysis （SRH） while 160 environment samples （from sewage, birds stools, feathers and smearing samples of poultry cages） in the poultry market were also detected for AIV nucleic acid with real-time PCR method. Four sewage samples of H5N1 subtype AIV were collected from poultry markets in Changsha, and the HA genes of H5N1 subtype AIV amplified by RT-PCR and then sequenced with TA cloning. Amino acid sequence alignment and phylogenetic tree analysis were conducted by Lasergene and Mega 5.0 software. Results The results through H5N1 antibody monitoring program showed that H5N1 antibody positive rates from workers were 25.5% （26/102）, 50.0% （9/18） and 25.4% （17/67） respectively in the poultry markets of township and inner cities. H5N1 antibody positive rate in the township poultry markets was higher than in the inner cities poultry markets. Results from the surveillance on AIV nucleic acid showed that the overall H5 subtype positive rate in Changsha poultry markets was 31.3% （50/160）, and the positive rate of townships poultry markets was 37.3% （31/83）, which were both higher than those from the inner cities poultry markets （24.7%, 19/77）. H5 subtype AIV positive rate was different in the tested specimens, with ranking of positive rates were sewage （ 50.0%, 24/48）, feathers （44.5%, 4/9）, birds stools （29.8%, 14/ 47） and smearing samples of poultry cages （14.3%, 8/56） , with statistically significant differences （P〈0.01）. Four H5N1 HA genes TA cloning were successfully constructed and identified as Eurasian branch, similar to viruses isolated in China's Mainland and Hong Kong in the same group, according to genetic analysis. Sequence data of the four HA genes showed the same feature of high pathogenicity, compared to the H5N1 AIV from China's Mainland of human origin. The receptor specificities were still with avian influenza origin （QSG）and the connecting peptide between HA1 and HA2 possessing the polybasic motif （RERRRKK or RERRGKK）. Conclusion One of the reasons for H5N1 antibody positive rate of 25.5% among poultry markets workers was that there were large numbers of H5N1 subtype AIV detected in the environment of poultry markets and HA genes of H5N1 subtype AIV in the poultry markets environment carried molecular characteristics of highly pathogenic which could increase the risk for H5N1 subtype AIV transmission in the environment of poultry markets.