从长春分离的一株急性散发性戊型肝炎病毒部分核苷酸序列分析

目的分析长春一株急性散发性戊型肝炎病毒（ＨＥＶ）的部分核苷酸序列。方法应用逆转录套式聚合酶链反应法检测ＨＥＶＲＮＡ；用荧光法（ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ）直接测序。结果被检的１４例急性散发性戊型肝炎病人中，５例ＨＥＶＲＮＡ阳性。对其中一例病人的ＨＢＶｃＤＮＡ开放读框２区进行核苷酸序列分析并与墨西哥株（Ｍ）和缅甸株（Ｂ）比较，发现长春散发性ＨＥＶｃ－１８７株与ＨＥＶ（Ｍ）核苷酸和氨基酸序列的同源性分别为７９．９％和９３．１％；与ＨＥＶ（Ｂ）散发株和流行株的同源性分别为９２．３％和９６．２％及９３．９％和９７．７％；与新疆株（ＣＨ１．１）同源性分别为９６．５％和９５．４％。结论长春散发性ＨＥＶｃ－１８７株与新疆株（ＣＨ１．１）一样，可能与ＨＥＶ（Ｂ）为同一亚型。     

柯萨奇病毒B组3型中国分离株VP1基因真核表达系统的克隆

目的探讨构建柯萨奇病毒Ｂ组３型（ＣＶＢ３）基因疫苗的可行性。方法应用逆转录ＰＣＲ技术扩增ＣＶＢ３中国分离株ＶＰ１基因,通过ＴＡ克隆法构建ｐＣＲ２．１－ＣＶＢ３ＶＰ１,将ＣＶＢ３ＶＰ１亚克隆至真核表达载体ｐＣＥＰ４,构建真核表达系统ｐＣＥＰ４－ＣＶＢ３ＶＰ１,并进行酶切和测序鉴定。结果发现ＣＶＢ３的中国分离株与Ｎａｎｃｙ株的ＶＰ１基因基本一致,均编码２９３个氨基酸,其中有５９处核苷酸存在变异,但仅改变了１０处氨基酸编码,证实克隆的片段为ＣＶＢ３ＶＰ１基因,表达载体为ｐＣＥＰ４。结论实验成功地构建了ＣＶＢ３中国分离株的真核表达系统ｐＣＥＰ４－ＣＶＢ３ＶＰ１,为ＣＶＢ３基因疫苗的研究奠定了基础     

粪便中戊型肝炎病毒散发株的核酸序列分析

目的了解粪便中戊型肝炎病毒（ＨＥＶ）散发株的核酸序列变异情况。方法用逆转录－套式－聚合酶链反应（ＲＴ－Ｎｅｓｔｅｄ－ＰＣＲ）检测了８例广州地区散发性戊型肝炎患者粪便中的ＨＥＶＲＮＡ，３例阳性。对阳性ＰＣＲ产物的２３８个碱基进行了基因克隆和核酸序列分析，并与已报道的ＨＥＶ序列进行比较。结果本地区３株ＨＥＶ与缅甸株（Ｂ）、巴基斯坦株（Ｐ）、墨西哥株（Ｍ）、中国新疆株（Ｃｈ１．１）和广州血清株（Ｇ－９）的核苷酸和氨基酸的同源性均值分别为８０．６７％和８８．６０（Ｂ）、８１．２５％和８９．２０％（Ｐ）、７７．４５％和８４．８１％（Ｍ）、８１．２５％和８９．２０％（Ｃ）、９７．８５％和９６．２０％（Ｇ）。结论本组３株ＨＥＶ有一定程度的变异。     

我国南方地区急性散发型戊型肝炎病毒基因分析

目的进一步证实我国南方地区存在戊型肝炎病毒（ＨＥＶ）不同的基因序列。方法用逆转录套式聚合酶链反应（ＲＴ－ｎｅｓｔｅｄＰＣＲ）检测８例厦门地区急性散发性成型肝炎患者血清中的ＨＥＶＲＮＡ,其中２例阳性;测定了广州分离株Ｇ９３—２株病毒第５代培养液的ＨＥＶＲＮＡ。对阳性ＰＣＲ产物进行克隆、核苷酸序列分析。结果厦门株Ｘ－Ｓ１与广州Ｇ９３－２株病毒核苷酸和氨基酸序列的同源性为９９２％和９７５％;厦门株（Ｘ－Ｓ１）和广州株（Ｇ９３－２）与缅甸株（Ｂｕｒ—１２１）和我国新疆株（８７Ａ）的核苷酸和氨基酸序列的同源性为７９．９％和８６．３％,与墨西哥株的同源性为７７４％和％．３％。结论经序列分析结果进一步证实我国南方地区确实存在一种不同的ＨＥＶ基因型。     

北京地区46例Ⅱ/1b型HCV高变区1的序列变异研究

目的研究北京地区Ⅱ／１ｂ型丙型肝炎病毒（ＨＣＶ）包膜蛋白Ｅ２／ＮＳ１高变区１（ＨＶＲ１）序列变异规律及意义。方法应用逆转录巢式ＰＣＲ技术从４６例北京地区Ⅱ／１ｂ型ＨＣＶ感染病人血清中扩增了ＨＣＶ部分包膜区基因片断（ｎｔ１１１９～１２５８）,纯化后直接采用双脱氧链末端终止法进行序列分析。结果北京地区Ⅱ型ＨＣＶＨＶＲ１位于氨基酸（ａａ）３８４－４０８位,与有关文献报道（３８３－４１０或４１４）略有差异。ＨＶＲ１序列与ＨＣＶ－Ｊ、台湾株、河北株、ＨＢ－１１相应序列比较,核苷酸同源性依次为４４０％～６６．７％（平均５７．７％）,４８．０％～７２．０％（６０．０％）,６０．０％～８５．３％（６９．８％）和５６．０％～８１．３％（６８．２％）,氨基酸同源性依次为２０．０％－５６．０％（３８．２％）,３２．０％～６４．０％（４５．７％）,３６．０％～７６．０％（４９．８％）和４０．０％～７６．０％（５５．６％）。本组ＨＶＲＩ内发现６个较保守的氨基酸位点：３８５位Ｔｈｒ,３８９,３９０,４０６位Ｇｌｙ,４０１位Ｓｅｒ,４０３位Ｐｈｅ。结论对ＨＶＲ１序列变异规律及生物学意义的进一步研究有助于ＨＣＶ疫苗的发展。     

上海地区Ⅱ、Ⅲ型丙型肝炎病毒包膜区cDNA序列分析

目的 分析上海地区丙型肝炎病毒（ｈｅｐａｔｉｔｉｓ Ｃ ｖｉｒｕｗ,ＨＣＶ）Ⅱ、Ⅲ型包膜区ｃＤＮＡ序列变异。方法 采用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）扩增ＨＣＶ包膜区Ｅ１、Ｅ２／ＮＳ１片断（１ ００５ｂｐ）,在３７３ＤＮＡ全自动测序仪上进行了序列分析。结果 ＨＣＶ－Ｅ１、Ｅ２／ＮＳ１区核苷酸和氨基酸同源性比较显示：上海株Ⅱ型、Ⅲ型间同源性分别为６２．４８％和５９．１０％;上海株与加内外同型株间     

丙型肝炎病毒多肽C1,C2,N1·N2合成及其应用

根据丙型肝炎病毒（ＨＣＶ）核苷酸和氨基酸序列合成了ＨＣＶ结构区核心部位Ｃ１、Ｃ２和非结构区Ｎ１·Ｎ２三个多肽。建立了一个敏感和特异的酶联免疫吸附试验（ＥＬＩＳＡ）检测抗－ＨＣＶ，与美国抗－ＨＣＶ第２代试剂盒比较，符合率为９７．７％。检测临床标本１０２５份，抗－ＨＣＶ阳性率在慢性非甲非乙型肝炎（ＮＡＮＢＨ）４３．９％，血透患者３４．４％，肝硬化２５．０％，急性ＮＡＮＢＨ１７．４％，肝癌１６．７％，助     

乙型肝炎病毒的父儿传播与肝外定位

目的研究乙型肝炎病毒（ＨＢＶ）父儿传播与肝外ＨＢＶＤＮＡ的检出。方法以８名ＨＢｓＡｇ阳性而其配偶无任何ＨＢＶ标志的男性携带者与其子宫内受感染的８例胎儿为对象,以巢式ＰＣＲ检测ＨＢＶＤＮＡ,以ＰＣＲ产物直接测序的方法测定父亲血清、精子与胎儿血清ＨＢＶＤＮＡ。６对父儿测定Ｓ区ｎｔ４５１～６６０段,２对测定Ｃ区ｎｔ２０２２－２３２１段序列。结果父几间核苷酸同源性９８％～１００％。Ｓ区测定结果表明６对父儿均感染ａｄｗ亚型病毒。２对父儿此段序列核苷酸有差异。父亲与原型株一致而胎儿在４９１,４９４,５４６,５８１位核苦酸变异致使１１３,１１４,１３１,１４３位氨基酸替代。另外４对父儿此段序列完全一致,其中１对父儿在１２６位均发生氨基酸替代。２对父儿Ｃ区ｎｔ２０２２～２３２１位核苷酸完全一致,父儿共同变异的１１个位点均为无表型变异。在胎儿血清、白细胞、肝脏、心脏、脾脏、肾脏、肺、脐带、肌肉、皮肤、胸腺、睾丸等组织脏器检出ＨＢＶＤＮＡ。结论出生前期存在ＨＢＶ父儿传播,大部分父亲将携带的优势毒株,小部分将少数株传给子代。在胎儿多个脏器与组织检出ＨＢＶＤＮＡ。     

深圳地区HIV-1流行毒株的env基因序列测定和亚型分析

目的了解深圳地区ＨＩＶ－１各亚型毒株的流行情况和传播方式。方法用套式－聚合酶链反应（ｎｅｓｔｅｄ－ＰＣＲ）对５份在深圳市发现的ＨＩＶ－１感染者外周血单个核细胞（ＰＢＭＣ）中的ＨＩＶ－１膜蛋白基因进行扩增，并对Ｃ２－Ｖ３及其邻区３５０－４５０个核苷酸序列进行测定和分析。结果５份样品中存在Ｂ和Ｅ两种亚型的ＨＩＶ－１毒株序列，其中Ｂ亚型２份，彼此间的基因离散率为１０．２％；Ｅ亚型３份，彼此间的基因离散率为２．７％。Ｂ亚型株与欧美Ｂ亚型毒株序列十分接近，Ｅ亚型株与泰国Ｅ亚型共享序列非常相似。结论深圳地区ＨＩＶ－１Ｂ亚型为经性途径由境外传入，ＨＩＶ－１Ｅ亚型经血途径在静脉吸毒者及部分献血员中传播     

日本血吸虫副肌球蛋白全基因克隆、测序及体内表达

目的： 将日本血吸虫大陆株副肌球蛋白（ Ｓｊｃ９７） 编码区全基因克隆及测序, 并研究 Ｓｊｃ９７ 全基因核酸疫苗在小鼠体内的表达。方法： 抽提日本血吸虫成虫总 Ｒ Ｎ Ａ, 逆转录 聚合酶链反应 （ Ｒ Ｔ Ｐ Ｃ Ｒ） 扩增编码 Ｓｊｃ９７的全基因ｃ Ｄ Ｎ Ａ, 双脱氧链末端终止法测 Ｄ Ｎ Ａ 序列。构建含 Ｓｊｃ９７ 全基因的质粒表达载体 （ｐ Ｃ Ｍ Ｖ Ｓｊｃ９７） , 并用免疫荧光染色法研究ｐ Ｃ Ｍ Ｖ Ｓｊｃ９７ 在小鼠体内的表达特性。结果与结论： 用 Ｒ Ｔ Ｐ Ｃ Ｒ 扩增获得了 Ｓｊｃ９７ 的编码区全基因ｃ Ｄ Ｎ Ａ, 并测定了该基因编码区的全序列。与日本血吸虫菲律宾株、日本株及曼氏血吸虫副肌球蛋白编码区全基因比较, 核苷酸序列同源性分别为： ９９４ ％ 、９９２ ％ 和９１０ ％ ; 推导的氨基酸序列同源性分别为：９９７ ％ 、９９８ ％ 和９６０ ％ 。ｐ Ｃ Ｍ Ｖ Ｓｊｃ９７ 核酸疫苗能在注射的小鼠局部肌肉组织表达 Ｓｊｃ９７ 。     

Complete nucleotide sequence of an attenuated hepatitis A virus: comparison with wild-type virus.

The complete nucleotide sequence of an attenuated hepatitis A virus, HAV HM-175/7 MK-5, was determined from cloned cDNA. This virus was derived from wild-type HAV HM-175 after 32 passages in African green monkey kidney cells. The resultant cell culture-adapted virus is attenuated for chimpanzees. This virus was passaged an additional three times in monkey kidney cells to obtain sufficient virus for molecular cloning and was designated HM-175/7 MK-5. Three overlapping cDNA clones were obtained that together spanned the entire genome. Comparison of the nucleotide sequence of cDNA from wild-type virus (propagated in marmoset liver in vivo) with attenuated virus (grown in cell culture) showed 24 nucleotide changes distributed throughout the genome. Five base deletions occurred in the 5' noncoding region, and 12 of the 16 base substitutions in the coding region resulted in amino acid changes. Amino acid changes occurred in viral capsid proteins VP1 and VP2 and several of the nonstructural proteins. Thus, a small number of nucleotide changes are responsible for adaptation to cell culture and attenuation of HAV strain HM-175.     

Genetic analysis of hepatitis A virus protein 2C in sera from patients with fulminant and self-limited hepatitis A

BACKGROUND/AIMS: To examine whether genetic differences in hepatitis A virus (HAV) are responsible for the range of clinical severities, we analyzed the HAV 2C genome, whose mutations have previously been shown to be important for enhanced replication in cell culture systems and to be related to virulence in simians. METHODOLOGY: Serum samples from 45 Japanese patients with sporadic hepatitis A, comprising 9 patients with fulminant hepatitis (FH), 10 with severe acute hepatitis (AHs), and 26 with self-limited acute hepatitis (AH), were examined for HAV RNA. RESULTS: Compared with the sequence of wild-type HAV strain HM-175, the nucleotide sequences of 2C had homology of 89.0 +/- 0.6% in FH, 88.6 +/- 0.9% in AHs, and 89.0 +/- 1.6% in AH. Differences were not statistically significant among the three groups. Deduced amino acid sequences had homology of 97.6 +/- 0.4% in FH, 96.5 +/- 1.9% in AHs, and 96.8 +/- 1.7% in AH. The difference between FH and AH was statistically significant (p < 0.05), although there were no specific nucleotide or amino acid substitutions. CONCLUSIONS: Fulminant hepatitis patients had fewer amino acid substitutions in 2C, indicating the association between severity of hepatitis A and amino acid variations in 2C of HAV.     

Studies of prototype live hepatitis A virus vaccines in primate models

We have prepared two prototype live hepatitis A virus (HAV) vaccines by serial passage of the HM-175 strain of HAV in African green monkey kidney cells. Passage 21 (P-21) HM-175 virus shows evidence of attenuation for chimpanzees but not for marmosets; passage 32 (P-32) HM-175 virus shows evidence of attenuation for both species. Animals that received P-32 HAV had fewer elevations in levels of liver enzyme activity and evidence of less virus replication in the liver and excretion of virus in stool than did those that received wild-type virus. The P-21 and P-32 viruses were highly immunogenic in both species. The information provided by this study will aid in the development of a live HAV vaccine.     

家养野猪脑心肌炎病毒的分离、鉴定及全基因组序列分析

目的 分离家养野猪脑心肌炎病毒(Encephalomyocarditis virus,EMCV),作全基因组序列测定及与相关物种同源性比较。方法 用改进的“细胞接种与RT-PCR方法相结合”技术,成功分离到国内首株家养野猪EMCV JZ1202株,并对其全基因组序列进行测定和分子特征分析。结果 分离毒的基因组全长为7 735 bp,与国内外不同动物源EMCV参考毒株的核苷酸同源性为81.2%～99.9%,与国内猪源EMCV分离毒的同源性高达99.4%以上。基于EMCV全基因组、ORF和VP1基因序列绘制的系统发育进化树显示,EMCV可分为 G1、G2和G3 3个群,JZ1202株与其他国内参考毒株同属于G1群。结论 结果证实,家养野猪可感染EMCV并引起发病,提醒作野生动物养殖要考虑EMCV的传播;EMCV存在较大的地域差异,在家养野猪和鼠之间可能存在着交叉感染;EMCV在感染家养野猪时可能发生个别氨基酸突变,以适应不同的猪种。     

Primary structure and gene organization of human hepatitis A virus.

The RNA genome of human hepatitis A virus (HAV) was molecularly cloned. Recombinant DNA clones representing the entire HAV RNA were used to determine the primary structure of the viral genome. The length of the viral genome is 7478 nucleotides. An open reading frame starting at nucleotide 734 and terminating at nucleotide 7415 encodes a polyprotein of Mr 251,940. Comparison of the HAV nucleotide sequence with that of other picornaviruses has failed to reveal detectable areas of homology. However, a computer analysis of the putative amino acid sequence of HAV and poliovirus demonstrated the existence of short areas of homology in virion protein 3 (VP3) and throughout the carboxyl-terminal portion of the polyproteins. In addition, extensive protein structural homologies with poliovirus were detected.     

Hepatitis A virus genotype IA-infected patient with marked elevation of aspartate aminotransferase levels

We describe a case of acute liver failure (ALF) without hepatic encephalopathy with marked elevation of aminotransferase due to hepatitis A, according to the revised Japanese criteria of ALF. This liver biopsy of the patient showed compatible to acute viral hepatitis and she immediately recovered without intensive care. She had no comorbid disorders. Of interest, phylogenetic tree analysis using almost complete genomes of hepatitis A virus (HAV) demonstrated that the HAV isolate from her belonged to the HAV subgenotype IA strain and was similar to the HAJFF-Kan12 strain (99% nucleotide identity) or FH1 strain (98% nucleotide identity), which is associated with severe or fulminant hepatitis A. Careful interpretation of the association between HAV genome variations and severity of hepatitis A is needed and the mechanism of the severe hepatitis should be explored.     

Molecular characterization of hepatitis-A-virus infections,in the context of two outbreaks in southern Thailand

As hepatitis A virus (HAV) is usually transmitted through the faecal-oral route, hepatitis A is a communicable disease. In countries of intermediate to low endemicity, sudden outbreaks of human infection with the virus may occur. Between September 2001 and April 2002, there were two outbreaks of HAV infection in the Ruso and Yeengor districts of Narathiwas province, in southern Thailand. Isolates of HAV were recovered during these outbreaks, from 14 in-patients with acute hepatitis in Ruso (12 positive for anti-HAV IgM and all positive for HAV RNA), 16 children with asymptomatic infection in Yeengor (14 positive for anti-HAV IgM and nine for HAV RNA), and four isolated cases in Bangkok (all positive for anti-HAV IgM). Molecular characterization of the VP1-P2A region of each isolate was followed by phylogenetic analysis. All of the isolates from Narathiwas province were found to be of genotype 1a, to have the same VP1 nucleotide sequence, and to show a high level of sequence homology (>/= 99.5%) with the isolates from Bangkok and with previous Thai isolates. These results should facilitate further research into HAV transmission and genotype identification in community outbreaks.     

Serum neutralizing antibody response to hepatitis A virus

Serum neutralizing antibody to hepatitis A virus (HAV) was measured in experimentally infected primates and naturally infected humans by means of an assay based on the autoradiographic detection of viral replication foci in vitro. Infection of primates with either PA-33 or HM-175 strains of HAV elicited antibody capable of neutralizing either strain. Sequential testing of two monkeys showed that neutralizing antibody correlated closely with antibody detected by immunoassay, developed before liver enzyme elevations, and was associated with a substantial reduction in fecal shedding of viral antigen. In tests performed on human subjects involved in an outbreak of hepatitis A, neutralizing antibody was present three to five days before the onset of symptoms and was found in both 19S and 7S immunoglobulin fractions. Immunity to HAV is probably due primarily to neutralizing antibody, and the ability to quantitate this antibody will be helpful in the evaluation of new HAV vaccines.     

中国大陆地区柯萨奇病毒A组4型VP1区基因特征分析

目的 分析中国大陆地区柯萨奇病毒A组4型(Coxsackievirus A4,CV-A4)流行毒株基因特征和进化规律。方法 利用Mega6.0软件对GenBank核苷酸数据库收录的分离于中国大陆地区CV-A4毒株VP1区基因序列进行分析,构建系统进化树,并计算分离毒株核苷酸和氨基酸同源性。结果 纳入中国大陆地区CV-A4毒株合计116株,中国大陆地区16个省份CV-A4流行毒株潜在的优势基因型为F。与原型株High Point相比,中国大陆地区CV-A4毒株核苷酸和氨基酸同源性分别为79.9%~84.1%和92.5%~98.7%,收录中国大陆地区CV-A4毒株间核苷酸和氨基酸同源性分别为82.6%~100.0%和91.5%~100.0%。结论 针对中国大陆地区CV-A4毒株流行特征和进化规律,采取有效措施防控手足口病。     

北京市西城区手足口病柯萨奇病毒A5型VP4区基因特征分析

目的分析2009年北京市西城区手足口病柯萨奇病毒A5型(CoxA5)的VP4区基因特征,了解其变异情况。方法采集手足口病患儿咽试子,提取病毒RNA,用肠道病毒VP4区分型引物进行半巢式RT-PCR(RT-nPCR),通过测序和GenBank Blast比对确定肠道病毒型别,并对其VP4区进行序列和进化分析。结果共鉴定出5株CoxA5,GenBank登录号为JN981017-JN981021。在VP4区,5株CoxA5与中国株HQ728261核苷酸及氨基酸序列同源性分别为96%～99%和98%～100%。进化分析表明,5株CoxA5与中国株HQ728261遗传距离为0.005～0.045,共同构成一个独立的进化树分支。5株CoxA5在VP4区无氨基酸的缺失和插入,未发现特有的氨基酸置换。结论 2009年北京市西城区手足口病病原体CoxA5VP4区与中国株HQ728261在进化上密切相关,其序列未发生明显变异。