摘要
目的了解2007年5—6月北京市儿童中流行的手足口病的病原。方法于2007年5、6月间采集来首都儿科研究所附属儿童医院门诊就诊的手足口病患儿咽拭子标本6份和1份病情危重的手足口病并发神经系统症状而急诊收住院患儿(编号F4243,年龄9岁8个月,女性)的经气管插管呼吸道抽吸液、脑脊液及血清标本。将咽拭子和气管插管抽吸液标本接种Hep-2、MDCK和Vero细胞进行病毒分离。同时设计位于肠道病毒5’非编码区(UTR)的通用引物2对(巢式PCR)、肠道病毒71型(EV71)VP1基因不同位置的特异性引物2对半(PCR和半巢式PCR)和柯萨奇病毒A组16型(CA16)的特异性引物1对检测标本中的病毒核酸。首先用随机引物将标本中的RNA逆转录成cDNA,然后分别用上述引物进行PCR扩增肠道病毒5’UTR和EV71/CA16的VP1基因片段;对EV71阳性的标本还扩增了VP1全基因,扩增产物直接进行序列测定和分析。结果6例门诊就诊的普通手足口病患儿和F4243的呼吸道标本中均扩增到肠道病毒5’UTR基因片段,有2例门诊患儿和F4243的标本中还扩增到EV71的VP1基因片段,提示该组手足口病患儿均为肠道病毒感染,其中3例为EV71感染。序列测定和分析表明有1例普通手足口病患儿(F4211)和F4243标本确定为EV71。所有标本接种Hep-2和MDCK细胞均未出现病变,说明常见的呼吸道病毒为阴性。除1份咽拭子标本(F4283)外,其余6份接种Vero细胞的标本均出现细胞病变。用肠道病毒通用引物和EV71特异性引物对分离到的病毒株核酸的扩增结果显示这6株病毒均为肠道病毒,其中F4211和F4243为EV71,与直接从呼吸道标本中扩增的结果相符;其余4株为CA16。结论北京市儿童中流行的手足口病与肠道病毒EV71和CA16感染有关;EV71可以在5岁以上儿童中引起严重的神经系统并发症,应引起注意。
Objective To reveal the etiological agent of hand, toot and mouth disease in children in Beijing. Methods Throat swabs were collected from 6 infants and young children with hand, foot and mouth disease who visited the affiliated Children＇s Hospital from May to June 2007. Aspirated fluid from tracheal intubatton, serum and cerebral spinal fluid （CSF） were collected from a 9 years old girl （No. 4243） having central neural system complication of severe hand, foot and mouth disease and admitted to the hospital from the Emergency Department. Throat swab and aspirated fluid were inoculated into the cell lines Hep-2, MDCK and Vero for virus isolation. RNAs were extracted by Trizol from 6 throat swab specimens and aspirated fluid, serum while CSF was from that severe case （No. 4243）. The gene fragment from 5＇ UTR of enterovirus was amplified from throat swabs and aspirated fluid by reverse transcription-polymerase chain reaction （RT-PCR） with the primer pairs located at the untranslated region of all enterovirus. EV71 was identified by RT-PCR with the 2 and half primer pairs located at different parts of VP1 gene of EV71. The PCR products for VP1 encoding gene of EV71 from the specimens were sequenced and sequence analysis was performed by comparing those published VP1 genes of EV71. EV71 and CA16 specific primers were used to identify the isolates by RT-PCR and the sequences were directly determined from PCR products. Results Gene fragments with expected molecular weight were amplified from all 6 throat swabs and the aspirate by the primer pairs universal for the 5＇ UTR of entemvirus, suggesting that these patients with hand, foot and mouth disease were infected by entorovirus. Out of these 7 specimens, 2 throat swabs and the aspirate were also showing positive for the VP1 of the EV71 by different primer sets. Sequence analysis revealed that the sequences for the amp｜icons from 1 throat swab （No. F42ll ） and the aspirate shared highest homology with those published EV71, indicating that these specimens were truly positive for EVT1. The sequences amplified from these specimens shared 100% and 98.9% homology to each other and were closer to the sequences of EV71 identified from Zhejiang province than those from Taiwan and strain BrCr. Gene fragments for 5＇ UTR of enterovirus were obtained by RT-PCR after CPE appeared in 6 out of 7 inoculations including that aspirate fluid in Vero cell, indicating that enteroviruses were isolated from these specimens. Virus isolates from one throat swab （No. F4211 ） and the aspirate （No. 4243） were positive by RT-PCR with the primer pairs for EV71, which was consistent with RT-PCR amplification directly from specimens. Virus isolates from other 4 specimens were CA16 by RT-PCR and sequence analysis. Conclusion These data suggested that hand, foot and mouth disease recently appeared in children in Beijing was related with EV71 and CA16. EV71 could cause severe clinical manifestations with central nerve system complications even in the child older than 5 years.
出处
《中华流行病学杂志》
CAS
CSCD
北大核心
2007年第10期1004-1008,共5页
Chinese Journal of Epidemiology
