手足口病和腹泻病患儿肠道病毒71型的基因型检测和分析

Genetype Detection on Enterovirus 71 from Children with Hand-foot-and-mouth Disease and Diarrhea

目的检测和分析手足口病和腹泻病患儿肠道EV71和基因分型.方法收集2007和2008年10～12月30名腹泻患儿和2009年4～6月40名手足口病患儿的粪便标本,同时收集手足口病患儿的临床资料.用试剂盒提取粪便标本病毒RNA,用PCR荧光探针法对上述标本进行EV71的检测;对EV71阳性的标本进行VP1基因节段的RT-PCR,选EV71感染重症和轻症各3份的阳性标本扩增产物纯化后进行核苷酸序列测定和分析,结果与GenBank中的EV71参考毒株进行比较,并依据VP1片段构建种系发生树;应用SPSS13.0软件进行对比统计分析.结果 (1)EV71的感染:40例手足口病患儿的粪便标本,27例EV71阳性,阳性率为67.5%.30例腹泻患儿的粪便标本中,1例EV71阳性,阳性率为3.3%.40例手足口病的患儿中:轻症23例,EV71阳性率52.17%(12/23);重症17例,EV71阳性率88.24%(15/17),两者的差异具有统计学意义(P<0.05).(2)EV71的基因分型:本次EV71均为C基因型,与中国大陆流行的基因型基本一致.与A、B基因型参考毒株的核苷酸同源性分别为39.7%～41.3%和83.3%～84.9%;而与C基因型中的C4亚株代表株的核苷酸同源性高达90.5%～96.4%;在种系进化树上证实本次分离到的EV71毒株属于C4亚型.(3)EV71核苷酸同源性的区域性:本次分析的6毒株其核苷酸同源性达92.6%～100%,与2008年昆明的3株参考株比较,核苷酸同源性为92.6%～99.2%.与2008年安徽、深圳、山东、武汉和北京等省份的分离毒株比较,核苷酸同源性为91.8%～99.2%;与匈牙利、澳大利亚、日本、马来西亚、新加坡等地流行时的毒株比较,核苷酸同源性为80%～86.5%;与2008年广州、浙江和2009年上海的分离毒株比较,核苷酸同源性为45.1%～45.9%.结论 EV71是昆明2009年手足口病的主要病原之一.秋冬季腹泻患儿存在EV71的感染,但感染率低.本次的EV71感染均为C基因型,属C4亚型,其核苷酸同源性与国内外流行时的毒株各有不同.

Objective To detecte and analyze the genetype of enterovirus 71（EV71）from children with diarrhea and hand-foot-and-mouth disease（HFMD）.Methods 70 stool specimens and clinical data were collected from 30 hospitalized children with diarrhea from October to December in 2007 and 2008 as well as 40children with HFMD from April to June in 2009 in Kunming.RNA of stool specimens was extracted by kit,and the genetype of enterovirus 71（EV71）of all specimens were detected by real time RT-PCR.The partial VP1 gene of 6 positive samples of the light and severe HFMD with EV71 were amplified and sequenced,and then compared with references strains from GenBank.A phylogenetic tree was constructed by using the neighbor-joining method of the MEGA program.Moreover epidemiological data were analyzed by using software SPSS13.0.Results（1）EV71 infection：Of the 40 stool samples with HFMD,27 were identified as positive for EV71,the positive rate was 67.5%.Of the 30 stool samples with diarrhea,1 was identified as positive for EV71,the positive rate was 3.3%.Of the 40 patients with HFMD,23 cases were mild and the positive rate of EV71 was 52.17%,17 cases were severe and the positive rate of EV71 was 88.24%.（2）Genogroup on EV71：In the study,genogroup C of EV71 were identified in children with diarrhea and HFMD.Phylogenetic tree suggested that the isolated viruses were similar to the EV71 sequences from China's Mainland and belonged to subgenogroup C4.（3）Nucleotide homologies and fields on EV71：In the study,six isolated virus were similar,the nucleotide homologies were 98.4%～100%.Compared to the three isolated viruses from Kunming City in 2008,the nucleotide homologies were 92.6%～99.2%.Compared to the isolated viruses from Anhui,Shenzhen,Shandong,Wuhan and Beijing in 2008,the nucleotide homologies were 91.8%～99.2%;Compared to the isolated viruses from Hungary,Australia,Japan,Malaysia and Singapore,etc,the nucleotide homologies were 80%～86.5%;Compared to the isolated viruses from Guangzhou and Zhejiang in 2008 and Shanghai in 2009,the nucleotide homologies were 45.1%～45.9%.Conclusions EV71 is one of the major pathogen of HFMD in Kunming in 2009.The infection of EV71 occurres in autumn and winter in Kunming,but infectious rate is very low.In this study,genogroup C of EV71 is identified in children with diarrhea and HFMD and is classified as subgenogroup C4.The nucleotide homologies compared to other isolated viruses at home and abroad,are different.