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目的了解引起青海省高原地区儿童病毒性脑炎肠道病毒的流行病学特征和病原谱构成。方法选择青海省监测点2010-2015年报告的病毒性脑炎患儿以及临床诊断病毒性脑炎患儿共256例,采集患儿脑脊液标本,提取核酸,采用Real-timePCR方法检测肠道病毒通用型(EV)核酸阳性脑脊液标本均做分型试验,分别检测肠道病毒71型(EV71),肠道病毒柯萨奇16型(CA16)和肠道病毒埃可30型(ECHO30)病毒的核酸。将肠道病毒未分型脑脊液标本接种人横纹肌肉瘤细胞(Rhabdomyosarcoma cell,RD)进行培养。提取核酸,采用半巢式聚合酶链反应(Sn-PCR)对肠道病毒VP1区进行特异性扩增和基因测序,通过BLAST确定肠道病毒类型。结果 256例病毒性脑炎患儿中男169例,女87例,男女比例为1.94∶1;肠道病毒通用核酸阳性22例,检出率8.95%,其中以5-10岁居多(17例),占81.82%。青海省高原地区肠道病毒性脑炎病例主要集中在夏秋季(7-11月),占67.19%。肠道病毒通用阳性的22份标本中ECHO30核酸阳性17份,占77.27%;EV71和CA16核酸阳性各1份。3份未分型标本经VP1区基因序列测定分析,相关序列同源性为97%~99%,分别确定为COXB4、COXB5和ECHO6。结论引起青海省高原地区儿童病毒性脑炎的肠道病毒以ECHO30为主,EV71、CA16、COXB4、COXB5、ECHO6感染病例散发存在。
Objective To understand the epidemiological characteristics and pathogenic spectrum of enterovirus in children with viral encephalitis in Qinghai Plateau. Methods A total of 256 children with viral encephalitis and children with viral encephalitis reported from 2010-2015 in Qinghai were selected. Cerebrospinal fluid (CSF) samples were collected from children and nucleic acids were extracted. Real-time PCR was used to detect enterovirus (EV) nucleic acid positive cerebrospinal fluid samples were typed to test the nucleic acid of enterovirus 71 (EV71), enterovirus Coxsackie 16 (CA16) and enterovirus ECHO30 . Enteroviral undifferentiated cerebrospinal fluid samples were inoculated with human rhabdomyosarcoma cells (RD) for culture. Nucleic acids were extracted and the specific amplification and gene sequencing of VP1 of enterovirus was performed by semi-nested polymerase chain reaction (SN-PCR). The enterovirus type was determined by BLAST. Results Among 256 children with viral encephalitis, 169 were males and 87 were females, the ratio of male to female was 1.94:1. The positive rate of enteric viruses was 22.95%, with a prevalence of 8.95% ), Accounting for 81.82%. The cases of enterovirus encephalitis in Qinghai Plateau are mainly concentrated in summer and autumn (July-November), accounting for 67.19%. Among the 22 specimens positive for enterovirus, 17 were positive for ECHO30, 77.27% for ECHO30, and 1 for EV71 and CA16. Three un-typed specimens were sequenced and analyzed for the sequence of VP1 gene. The sequence homology was 97% -99%, which was identified as COXB4, COXB5 and ECHO6, respectively. Conclusion ECHO30 is the predominant enterovirus in children with viral encephalitis in Qinghai Plateau, and the EV71, CA16, COXB4, COXB5 and ECHO6 infections are existent.