中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2001年
1期
21-24
,共4页
彭天庆%杨英珍%牛存龙%张红毅
彭天慶%楊英珍%牛存龍%張紅毅
팽천경%양영진%우존룡%장홍의
心肌炎%克山病%肠道病毒%序列分析
心肌炎%剋山病%腸道病毒%序列分析
심기염%극산병%장도병독%서렬분석
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750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34.08%，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234位由T→C、690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5′端基因特殊位点变异与致病的关系值得进一步探讨。
目的 分析自心肌炎和剋山病患者分離的腸道病毒的覈痠序列，探討病毒病因的分子基礎。方法 使用腸道病毒特異引物對從心肌炎和剋山病患者分離的腸道病毒RNA進行RT-PCR擴增，PCR產物經純化後分彆經不同的引物正反嚮直接覈痠循環測序，併應用SeqEd和DNA軟件及AssemblyLIGN軟件對測序結果進行分析。結果 確定7株病毒5′耑從覈苷痠位點40到750之間710bp基因序列；序列分析結果髮現，儘管為同血清型病毒，但其5′耑非編碼區基因變異率仍在15%左右，而血清型為柯薩奇B5病毒則高達34.08%，與腸道病毒各血清型之間5′耑非編碼區基因變異率無明顯差異；對血清型為CVB3的兩分離株病毒5′耑基因特殊位點分析，髮現與CVB3標準株（Nancy株）比較234位由T→C、690位由C→A。結論 確定瞭所分離到的一些腸道病毒5′耑非編碼區基因序列；腸道病毒5′耑非編碼區基因與血清型關繫不大；CVB3分離株5′耑基因特殊位點變異與緻病的關繫值得進一步探討。
목적 분석자심기염화극산병환자분리적장도병독적핵산서렬，탐토병독병인적분자기출。방법 사용장도병독특이인물대종심기염화극산병환자분리적장도병독RNA진행RT-PCR확증，PCR산물경순화후분별경불동적인물정반향직접핵산순배측서，병응용SeqEd화DNA연건급AssemblyLIGN연건대측서결과진행분석。결과 학정7주병독5′단종핵감산위점40도750지간710bp기인서렬；서렬분석결과발현，진관위동혈청형병독，단기5′단비편마구기인변이솔잉재15%좌우，이혈청형위가살기B5병독칙고체34.08%，여장도병독각혈청형지간5′단비편마구기인변이솔무명현차이；대혈청형위CVB3적량분리주병독5′단기인특수위점분석，발현여CVB3표준주（Nancy주）비교234위유T→C、690위유C→A。결론 학정료소분리도적일사장도병독5′단비편마구기인서렬；장도병독5′단비편마구기인여혈청형관계불대；CVB3분리주5′단기인특수위점변이여치병적관계치득진일보탐토。
Objective To characterize the gene sequences of six enteroviralisolates 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.
