心肌炎和克山病患者分离的肠道病毒核酸序列分析

Sequence analysis of enteroviruses isolated from patients with myocardial diseases

目的 分析自心肌炎和克山病患者分离的肠道病毒的核酸序列 ,探讨病毒病因的分子基础。方法 使用肠道病毒特异引物对从心肌炎和克山病患者分离的肠道病毒RNA进行RT PCR扩增 ,PCR产物经纯化后分别经不同的引物正反向直接核酸循环测序 ,并应用SeqEd和DNA软件及AssemblyLIGN软件对测序结果进行分析。结果 确定 7株病毒 5′端从核苷酸位点 40到 75 0之间710bp基因序列 ;序列分析结果发现 ,尽管为同血清型病毒 ,但其 5′端非编码区基因变异率仍在 15 %左右 ,而血清型为柯萨奇B5病毒则高达 34 0 8% ,与肠道病毒各血清型之间 5′端非编码区基因变异率无明显差异 ;对血清型为CVB3的两分离株病毒 5′端基因特殊位点分析 ,发现与CVB3标准株 (Nancy株 )比较 2 34位由T→C、6 90位由C→A。结论 确定了所分离到的一些肠道病毒 5′端非编码区基因序列 ;肠道病毒 5′端非编码区基因与血清型关系不大 ;CVB3分离株

Objective To characterize the gene sequences of six enteroviral isolates from patients with myocarditis and Keshan disease in selenium deficiency area of Yunnan province and one isolate from patients with myocarditis in Shanghai. Methods The 5′ nontranslated regional sequences and partial VP4 sequences of isolates (serotype: coxsackievirus A9, B2, B3, B5, B6, B6 and one unidentified) were amplified by reverse transcription polymerase chain reaction with enterovirus group specific primers. The nucleotide sequences of individual PCR products were determined by cycle sequencing and compared with known enteroviral sequences (GenBank/EMBOL) using the AssemblyLIGN software package. Results The 710bp fragment sequences from nucleotide position 40 to 750 of 5′ terminal sequences of the isolate (coxsackievirus A9, B2, B3, B3, B5, B6 and B6) were determined and sequencing of the PCR product from isolate with serotype unknown revealed the presence of multiple sequence bands, indicating multiple enteroviruses. Compute comparison showed that coxsackievirus isolate A9, B2, B3 and B6 had 14.51% 16.48% sequence variations compared with published coxsackievirus A9, B2, B3 and B6, respectively, suggesting that intratypic genetic divergence of 5′ nontranslated region among the coxsackieviruses are not difference from intertypic genetic divergence among group B coxsackieviruses, while B5 showed up to 34.08% variations compared with published coxsackievirus B5. Variations at 234(C T) and 690(A C) have been found from 2 isolates of B3 from Selenium deficiency area of Yunnan province in comparison with the prototype coxsackie B3 strain Nancy. The variation at 234 (C T) needs further study. Conclusions The study demonstrates that it is difficult to determine the genetic serotyping of the viruses by sequencing of 5′ nontranslated region. The described method can be applied to the epidemiologic investigation of enteroviral infections.