摘要
目的利用课题组前期分离获得的EV71临床分离毒株,对其全基因组序列测定及分析,建立EV71感染的动物模型,分析其基因序列与致病性的关系。方法利用RT-PCR和cDNA末端快速扩增技术(RACE)分段扩增EV71全长基因,对各扩增片段进行克隆和测序;用软件MEGA 5、RNAstructure 5.3进行基因组结构及特点分析;腹腔注射毒株建立EV71感染的ICR乳鼠模型。结果得到3个部分相互重叠并涵盖全基因组的重组质粒,测序后拼接成全基因序列,命名为EV71 BC08株存入GenBank(JQ514785)。序列分析表明BC08株为EV71C4a基因亚型,其5′非编码区(5′UTR)A627点突变可能影响病毒5′UTR二级结构。BC08株能感染乳鼠并导致其出现神经系统症状,病理学研究表明受染乳鼠肌肉和大脑组织有病毒VP1蛋白表达。结论 BC08毒株基因组全长7 406bp,为C4a亚型,与近年我国EV71流行株有很高的同源性(>95%),但A627点突变可能影响病毒5′UTR二级结构及功能。BC08毒株可感染乳鼠并致病,这对该临床分离株的致病能力提供了直接证据,也为建立EV71感染的乳鼠模型奠定基础。
Objective To sequence the whole genome of a clinically isolated enterovirus 71（EV71） strain BC08,investigate its pathogenicity and develop an EV71-infection mouse model.Methods Full length of viral RNA was amplified from our previously isolated EV71 strain BC08 by RT-PCR and 5′/3′ RACE-PCR.Three overlapped amplified fragments covering the full genome were cloned into pGEM-Teasy vector for sequencing and data were analyzed by software MEGA5 and RNAstructure 5.3.At the same time,imprinting control region（ICR） mice were intraperitoneally challenged with BC08 strain and its pathogenicity was evaluated by anatomopathology and the mortality rate.Results The full-length of EV71 BC08 genome is 7406 nucleotides（GenBank accession：JQ514785）.BC08 isolate is classified into sub-genotype C4a of EV71 by phylogenetic analysis.Sequence analysis showed that the A627 in 5′ UTR may lead its secondary structure to form a short stem and loop structure in the viral internal ribosome site（IRES）.The infected ICR mice demonstrated neurological complications and high morbidity rate.Immunohistochemistry detected positive VP1 capsid protein expression in both brain and muscle tissue.Conclusions Full genome sequencing analysis identified the BC08 isolate as the EV71 C4a sub-genotype and highly homology in nucleotide sequence with other recently prevalent EV71 strains in mainland of China.The A627 in the 5′ UTR of this isolate might be important for the high pathogenicity in both infected infants and IRC mice.
出处
《中国病毒病杂志》
CAS
2012年第6期419-425,共7页
Chinese Journal of Viral Diseases
基金
国家高技术研究发展计划(863计划)项目(2007AA02Z317)
国家自然科学基金(21172012/b020601)
