摘要目的 分析上海市闵行和嘉定区污水处理厂中人类肠道病毒(HEV)的时间分布特点及其基因分型.方法 于2013—2014年,选取闵行和嘉定区两所污水处理厂入水口作为采样点,每月24—28日采集污水标本1份,每份1 L,共48份.经阴离子膜吸附的方法浓缩,再用牛肉汤洗脱,取上清液并接种RD、HEp-2和L20B细胞进行病毒分离,共分离HEV 249株,其中非脊髓灰质炎病毒(NPEV)185株,脊髓灰质炎病毒(PV)64株(均为疫苗相关株).通过RT-PCR鉴定基因型,采用Bioedit 7.0.0对VP1核苷酸序列进行序列同源性分析,使用MEGA 5.0软件,通过邻位法构建系统发生树,分析优势株及病毒传播链.结果 185株NPEV中178株鉴定出基因型,包括:柯萨奇B组病毒(CVB)2、3、5基因型,埃可病毒(ECHO)1、3、6、7、11、13、19、20、24、25、30基因型,柯萨奇A组病毒(CVA)4基因型.CVB5和ECHO6是在NPEV分离株中比例较高的两个基因型,分别占33.5%(56株)和24.9%(43株).闵行区污水水样中HEV分离株主要集中在4—9月份,其中优势株ECHO6主要分布在2013下半年和2014上半年采集水样中,优势株CVB5主要在2013—2014年散在分布.嘉定的ECHO6和CVB5两个优势株的流行趋势与闵行区的基本一致.2013—2014年CVB5形成C6和C8基因亚型2个传播链,而与国外毒株有较大的差异;ECHO6形成C3、C6和D9基因亚型3个传播链,而D9基因亚型在上海属于优势基因亚型,在国内外有一定地域性的传播.结论 2013—2014年上海市污水中的分离到的PV均为疫苗株.HEV优势株为CVB5和ECHO6,其中ECHO6在2013下半年和2014上半年较为流行,CVB5在2014年下半年开始流行.CVB5和ECHO6存在多条传播链的共同流行现象.

abstractsObjective To explore the time and genotype distribution of human enterovirus (HEV) isolated from sewage in Shanghai in 2013-2014. Methods One sewage sample each was collected from two local sewage plants located in Minhang District and Jiading District on the same day at the day 24-28 of every month from 2013 to 2014. Each sample weighed 1 L. The specimens were concentrated by anionic membrane absorption, eluted with beef extract solution, and then used to inoculate RD, HEp-2, and L20B cell lines. A total of 249 enterovirus strains were isolated from sewage samples during the study period, including 185 non-polio enterovirus (NPEV) and 64 poliovirus (PV) strains, which were identified as vaccine strains. RT-PCR and Sanger sequencing were performed to identify HEV genotypes. Homologous analysis of VP1 sequences was conducted using BioEdit (version 7.0.0). Phylogenetic analysis was performed using the neighbor-joining method based on the alignment of VP1 gene sequences using MEGA (version 4.0.2). Results Among 185 NPEV strains, 178 strains were successfully sequenced and classified into 15 genotypes, including coxsackievirus group B (CVB) 2, 3, and 5; enteric cytopathic human orphan (ECHO) virus 1, 3, 6, 7, 11, 13, 19, 20, 24, 25, and 30;and coxsackievirus group A 4. CVB5 and ECHO6 genotypes accounted for 33.5% (56 strains) and 24.9% (43 strains) of NPEV isolates, respectively. During the study period, HEV isolates were mainly isolated in summer and autumn in Minhang District. ECHO6 strains were frequently isolated from June 2013 to July 2014. Thereafter, the number of ECHO6 strains gradually reduced in the second half of 2014. CVB5 strains demonstrated scattered distribution from 2013 to the first half of 2014 and gradually increased in the second half of 2014. The distribution of ECHO6 and CVB5 strains in Jiading District was similar to that in Minhang District. In 2013-2014, CVB5 strains comprised C6 and C8 subgenotypes, which belong to two transmission chains and show large differences compared with foreign strains isolated during the same period. ECHO6 strains comprised C6, C8, and D9 subgenotypes, which belong to three transmission chains. Moreover, ECHO6 subgenotype D9 was a dominant subgenotype in Shanghai, with broad geographical distribution both at home and abroad. Conclusion Poliovirus was identified as a vaccine strain in environmental surveillance from June 2013 to April 2014 in Shanghai. Several transmission strains of ECHO6 and CVB5 were identified, which were the dominant serotypes.