贵州侗族、苗族和汉族人群乙型肝炎病毒基因型分布

目的　了解贵州侗、苗、汉族HBV感染者的基因型。方法　比较 12 7株各基因型HBV全序列S基因核苷酸序列 ,用DNA分析软件筛选出 3个限制性内切酶。设计 3条HBVS区引物并进行聚合酶链反应扩增 ,产物经MboⅠ、BstNⅠ或BsmAⅠ酶切 ,分析酶切产物电泳图谱 ,建立区分HBV(A～F)基因型的方法。对贵州 16 6份侗、苗、汉族HBV感染者血清进行基因分型 ,5份PCR产物直接测序验证分型方法的准确、可靠。结果　S基因PCR RFLP分型结果准确 ,5份酶切鉴定结果经测序证实。 16 6份标本中 ,B基因型 138份 (83 13% ) ,C型 2 8份 (16 87% ) ,未发现B、C以外的其他基因型。侗族的 4 8例中 ,4 7例为B型 (97 92 % ) ,1例C型 (2 0 8% ) ,苗族的 5 2例中 ,4 9例为B型(94 2 3% ) ,3例C型 (5 77% ) ;6 6例汉族也以B基因为主 (6 3 6 4 % ,4 2 6 6 ) ,但C型有 2 4例 (36 36 % ) ,与侗、苗族HBV感染者相比 ,差异有显著意义 (χ2 =35 0 5 88,P <0 0 0 5 )。结论　贵州地区HBV基因型由B、C 2型构成 ,且以B基因型为主。汉族患者C型较多 ,而侗、苗族患者中B型是占绝对优势的基因型。     

不同基因型戊型肝炎病毒重组抗原p166用于抗体检测的价值

目的探讨不同基因型和亚型戊型肝炎病毒（HEV）ORF2重组蛋白p166用于抗体检测的价值,为研发准确可靠的戊型肝炎诊断试剂提供新的途径.方法用等浓度的不同基因型和亚型HEV p166作为包被抗原,对血清标本进行酶联免疫吸附试验测定.用HEV多基因型通用性引物逆转录套式PCR（RT-nPCR）扩增标本中HEV RNA,并测序、分型.结果8种不同p166抗原对30份健康献血者血清无抗原性,对182份来自世界不同国家和地区的已知HEV抗体阳性血清和7份HEV实验感染动物血清标本均呈阳性反应,但所得血清抗体滴度的高低与所用抗原的基因型有明显关系.RT-nPCR检测的50份中国血清标本中,19份阳性,基因分型均为Ⅳ型,与Ⅳ型中国株p166抗原反应最好.而以同属于第Ⅲ基因型的猪HEV新西兰株和人HEV美国株重组p166检测血清标本,两者结果差异无统计学意义.以多基因型p166混合抗原建立的ELISA抗体检测法与两种市售试剂盒比较,前者敏感性高,特异性好.结论不同基因型和亚型的HEV重组蛋白p166对不同血清标本HEV抗体检测的敏感性高低不同,因此多基因型和亚型p166的混合抗原是HEV抗体检测的最佳抗原.     

烟台沿海地区人源和猪源戊型肝炎病毒基因型别的研究

目的 调查烟台市沿海地区人源与猪源戊型肝炎病毒(HEV)基因型别的相关性.方法 应用逆转录一巢式聚合酶联反应( RT-nPCR)方法对当地急性散发戊型肝炎患者、正常人群中抗HEV-IgM阳性者和当地养猪场的猪进行HEV RNA检测,并对HEV RNA阳性标本进行克隆测序和序列分析.结果 16例急性散发戊型肝炎患者中有7例粪便标本HEV RNA阳性;51份lgM阳性正常人群血清标本中有1份HEV RNA阳性;34份猪胆汁标本中有1份HEV RNA阳性.序列分析发现该地区HEV人株与猪株在ORF2部分区域的核苷酸序列同源性为87％～ 98.1％.7株患者的戊肝病毒基因型和1株猪的戊肝病毒基因型均为Ⅳ型,基因序列同源性在87％ ～ 98.1％之间;其中有6例患者和猪的基因序列同源性在93.9％ ～98.1％之间,为Ⅳ型a亚型;1例患者和猪的基因序列同源性为87％,为Ⅳ型d亚型.正常人群的1例戊肝病毒基因型为Ⅰ型d亚型.该地区人与猪HEV的ORF2的部分基因片段与HEV Ⅰ～Ⅳ型的代表株进行比较,核苷酸序列同源性分别是82.5％～100％,81.7％ ～92.9％,81.4％ ～93.9％,84.9％ ～ 100％.结论 该地区人群中流行的HEV存在2个基因型3个亚型,主要以基因Ⅳa型为主,与猪群中流行的HEV基因Ⅳa型同源性较高;HEV Ⅰ型在人群中散在存在.     

烟台沿海地区人源和猪源戊型肝炎病毒基因型别的研究

目的调查烟台市沿海地区人源与猪源戊型肝炎病毒（HEV）基因型别的相关性。方法应用逆转录一巢式聚合酶联反应（RT—nPcR）方法对当地急性散发戊型肝炎患者、正常人群中抗HEV-IgM阳性者和当地养猪场的猪进行HEVRNA检测,并对HEVRNA阳性标本进行克隆测序和序列分析。结果16例急性散发戊型肝炎患者中有7例粪便标本HEVRNA阳性;51份IgM阳性正常人群血清标本中有1份HEVRNA阳性;34份猪胆汁标本中有1份HEVRNA阳性。序列分析发现该地区HEV人株与猪株在ORF2部分区域的核苷酸序列同源性为87％～98．1％。7株患者的戊肝病毒基因型和1株猪的戊肝病毒基因型均为Ⅳ型,基因序列同源性在87％～98．1％之间;其中有6例患者和猪的基因序列同源性在93．9％～98．1％之间,为Ⅳ型a亚型;1例患者和猪的基因序列同源性为87％,为Ⅳ型d亚型。正常人群的1例戊肝病毒基因型为Ⅰ型d亚型。该地区人与猪HEV的ORF2的部分基因片段与HEVⅠ～Ⅳ型的代表株进行比较,核苷酸序列同源性分别是82．5％～100％,81．7％～92．9％,81．4％～93．9％,84．9％～100％。结论该地区人群中流行的HEV存在2个基因型3个亚型,主要以基因Ⅳa型为主,与猪群中流行的HEV基因Ⅳa型同源性较高;HEVI型在人群中散在存在。     

猪戊肝病毒的克隆和部分序列分析

目的：调查戊型肝炎病毒（HEV）对猪的感染情况。方法：用HEV抗体试剂盒检测猪血清中的抗体；用逆转录聚合酶链方法（RT－PCR）检测猪血清中的HEV RNA，对PCR阳性产物进行克隆测序，并对序列进行分析。结果：10份猪血清中有1份为HEV抗体阳性，2份为HEV RNA阳性，其中1份为抗体和RNA均阳性。序列分析显示，从猪中克隆的2株序列（G6和G8）之间在ORF1（102－387bp）和ORF2（6007－6354bp)区域的核苷酸序列的同源性分别为94％和93％，该2株序列在ORF1区在HEVⅠ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ、Ⅶ和Ⅷ型的同源性分别为76％－79％、80％、79％－80％、88％－90％、75％－76％、79％、78％－79％和75％－78％；在ORF2区与Ⅰ、Ⅱ、Ⅲ和Ⅳ型的同源性为75％－77％、74％－77％、77％－78％和84％－99％，与ⅣA亚型的同源性为93％－97％。结论：猪感染的HEV的基因序列与人群中散发性戊型肝炎病毒的ⅣA亚型同源性最高。     

云南大理猪戊型肝炎病毒分子流行病学调查

目的：了解云南大理农村的猪群中戊型肝炎病毒（ hepatitis E virus, HEV）感染情况,为HEV的防控提供理论依据。方法利用逆转录巢式聚合酶链反应（ RT?nested PCR）技术,对所采集大理41份猪粪便样品进行HEVORF2基因部分片段扩增,经琼脂糖凝胶电泳后对目的条带进行回收纯化及克隆测序,序列利用DNAStar和MEGA4．0软件进行同源性比较和进化树构建。结果 RT?nested PCR方法扩增出348 bp目的基因序列,系统进化树显示该病毒属于基因4型h亚型HEV。此次实验共检测41份猪粪便样品,其中23份为阳性,阳性率为56．10％。结论大理猪群存在较高的HEV感染率,存在HEV跨种间感染人的风险,应该加强防控以免HEV爆发流行。     

急性肝炎恢复期血清检测抗戊型肝炎病毒抗体及RNA的临床诊断意义

目的 在急性非甲-戊型肝炎患者中进一步追踪检测不同时期血清抗戊型肝炎病毒(HEV)IgG，以明确临床诊断。方法 用美国Genelabs公司和北京万泰公司抗HEV诊断试剂检测抗HEV，用PCR方法检测HEVRNA，并进行基因序列分析。结果 95例入院首次血清学诊断为急性非甲-戊型肝炎患者中，在住院后11～25d、25～35d分别测定血清抗HEV,16例血清抗HEV阳性(万泰公司)，急性期血清HEVRNA检测10例HEVRNA阳性，经核苷酸序列分析证明，其中4例为Ⅰ型HEV感染，6例为Ⅳ型HEV感染。GenekLbs诊断试剂检测12例抗HEV阳性，7例HEVRNA阳性，其中4例是HEVⅠ型病毒感染，3例是HEV Ⅳ病毒感染。结论 对非甲-戊型肝炎患者进行急性期HEV RNA检测和恢复期抗HEV检测可以进一步明确病原学诊断，在这部分患者中存在HEV不同基因型感染。可能是HEV感染漏诊的原因之一。     

广州地区小儿无症状乙肝病毒携带者的基因型研究

目的：扩增HBV DNA S基因，建立HBV DNA的分型方法，研究广州地区小儿无症状乙肝病毒携带者（AsC）的基因型。方法：利用PCR－RFLP基因分型方法，扩增HBV DNA S基因，以限制性内切酶AvaⅡ、MboI酶切分型。结果：60份AsC血清样本，B型37份（62．7％），C型20份（33．3％），B、C混合型2份（3．3％），未分型1份（1．7％），经序列分析证明为C型，C型共35％。结论：PCR－RELP分型方法经济实用，分型率达98．3％，广州地区小儿乙肝病毒的基因型以B型和C型为主，其中携带者中B型占优势。     

中国北方汉族人细胞色素P4501 A1基因MspⅠ多态性的研究

目的　探讨中国北方汉族人细胞色素P(cytochromeP ,CYP) 4 5 0 1A1基因MspⅠ多态性。方法　用聚合酶链反应 限制性片段长度多态性 (PCR RFLP)技术 ,分析了 172名北方汉族正常健康成人CYP1A1基因 3′端限制性内切酶MspⅠ位点的3种基因型 (A、B、C)的分布频率。结果　MspⅠ等位基因m1、m2分别占 6 0 8%、39 2 %。MspⅠ基因型A占 34 9% ,基因型B占 5 1 7% ,基因型C占 13 4 %。结论　本研究结果提示中国北方汉族人解毒酶CYP1A1基因存在MspⅠ多态性。     

载脂蛋白E基因的PCR快速分型法的建立

目的建立载脂蛋白E基因的PCR快速分型法.方法采用以PCR扩增为核心的载脂蛋白E(apoE)基因型分析方法,扩增其中含有112位和158位两个氨基酸位点之间的编码序列,产物为267bp apoE基因经扩增后,扩增产物经HhaI 内切酶消化后,然后进行5%琼脂糖凝胶电泳检测,快速鉴定apoE的基因型.结果本法检测了85名健康人apoE 基因型,求出三个等位基因E2、E3、E4的频率分别为0.0824、0.8412、0.0764.结论该方法简单、准确,适合于一般实验室开展.     

Hepatitis E virus genotyping based on full-length genome and partial genomic regions

Some genomic regions for hepatitis E virus (HEV) genotyping have been reported to correlate well with the results from the phylogenetic analyses on the basis of the complete genome. However, few studies have systemically investigated the genomic regions for HEV genotyping using a combined phylogenetic and statistical approach. A consensus region for HEV genotyping has not been determined. In this study the nucleotide identities and genetic distances of 24 partial genomic regions and the complete genome sequences of 37 HEV strains were compared statistically. It was demonstrated with both one-way ANOVA and two-way ANOVA that only one genomic region in RNA-dependent RNA polymerase domain (4254–4560 nt) for which there were no significant differences when compared with the full-length genome (P > 0.05). The same four genotypes were identified by phylogenetic analysis based on this statistically predicted region identified as for the complete genome. RT-PCR amplification of HEV strains from all four genotypes confirmed conservation of the flanking primer sites of this region. Serum samples from 20 patients with a clinical diagnosis of hepatitis E were further analyzed by PCR using the same primers, 13 were positive and could be classified into genotype 4. These data strongly suggested that this newly identified region could be used for future HEV genotyping.     

Geographical distribution of hepatitis E virus genotypes

Hepatitis E virus (HEV) is the major agent of acute hepatitis in developing countries where the infection occurs sporadically or in large waterborne epidemics. HEV, classified in the Caliciviridae, is not culturable. The detection of HEV RNA by RT-PCR in serum and stool samples is reliable during the 7 to 15 days following the onset of the disease. Restriction endonuclease analysis, cloning and sequencing of PCR products allow a phylogenetic analysis of HEV isolates. Although they belong to a single serotype, strains recovered from different geographical regions display a significant genetic heterogeneity. Sequencing data from ORF1 and ORF2 regions has led to the characterization of 3 distinct genotypes: genotype I gathering the Asian and African subgenotypes; genotype II gathering swine and human US strains; genotype III limited to the Mexico prototype. Novel variants are currently described from Africa (Nigeria), China and Europe (Greece and Italy). Each genotype appears to be related to a well defined geographical area. Nevertheless, a genetic variability is observed within endemic regions such as Asia or Africa. Nigerian endemic isolates especially could represent an intermediate stage in the evolutionary process towards genetic diversity. The animal reservoir, proved by the detection of HEV sequences by PCR among pigs in Nepal and in the USA, could help to resolve unanswered questions about the origin of HEV genotypes, their spread and evolution.     

Hepatitis E virus isolated from rabbits is genetically heterogeneous but with very similar antigenicity to human HEV

Rabbit hepatitis E virus (HEV) in China may represent a novel HEV genotype, although no consensus has been reached. It is unclear whether the ORF2 capsid protein containing the immunodominant epitopes from rabbit HEV differs from those of human HEV. In this study, 661 bile samples collected from domestic rabbits in Jiangsu province, eastern China were amplified by RT‐nPCR using a set of HEV universal ORF2 primers. All 42 (6.4%) positive PCR products were sequenced. Phylogenetic analysis using the ORF2 sequences of 557 bp in length showed the Jiangsu isolates were separate from HEV genotypes 1, 2, 3, 4, avian HEV and rat HEV, and clustered together with rabbit HEV sequences. These 42 isolates were divided into five branches including two newly identified in the present study. Comparison with rabbit HEV sequences from China available in GenBank, using a 298 bp ORF2 segment, showed these sequences clustered together into a unique rabbit HEV clade, and were divided into eight sub‐branches with high genetic heterogeneity. In addition, 267 serum samples collected from domestic rabbits, serial serum samples from two rhesus monkeys experimentally infected with HEV genotype 1 or 4, and serial serum samples from two New‐Zealand rabbits infected experimentally with rabbit HEV were tested simultaneously by EIA using recombinant truncated ORF2 capsid proteins derived from rabbit and human HEV. The virtually identical results obtained suggest that rabbit and human HEV ORF2 antigens contain very similar immunodominant epitopes. All these data are helpful to identify the biological characteristics of the newly identified rabbit HEV. J. Med. Virol. 85:627–635, 2013. © 2013 Wiley Periodicals, Inc.     

Identification of numerous hepatitis C virus genotypes in Montreal, Canada.

Hepatitis C virus genotypes were determined for 358 viremic individuals in Montreal, Canada, by restriction endonuclease analysis of PCR products and phylogenetic analysis of core gene sequences. Types 1, 2, and 3 occurred in 62.8, 14.2, and 13.7%, respectively; types 4 and 5 were found in 3.9 and 4.5%, respectively; and genotypes 6a and 7c and a novel genotype each occurred in 0.3%. Types 4, 6, and 7 and the novel genotype were mostly from persons who had immigrated to Canada.     

Hepatitis E virus ORF3 antigens derived from genotype 1 and 4 viruses are detected with varying efficiencies by an anti-HEV enzyme immunoassay

The function of the hepatitis E virus (HEV) open reading frame 3 (ORF3) protein product remains unclear but it is able to induce a strong antibody response following HEV infection. Therefore, it has been used in some enzyme immunoassays (EIAs) for detecting anti-HEV antibody. In order to evaluate the difference in antigenicity of HEV ORF3 polypeptides derived from genotypes 1 and 4, two EIAs were developed, based on ORF3 polypeptides from genotypes 1 and 4 HEV. Serial weekly serum samples from two rhesus monkeys vaccinated with ORF3 antigens derived from the genotype 4 ORF3 protein and nine rhesus monkeys experimentally infected with genotypes 1 and 4 HEV were tested for anti-HEV using the assays. HEV ORF3 antigens derived from viruses of genotypes 1 and 4 showed different patterns of reactivity with sera obtained from monkeys immunized with ORF3 antigens or infected experimentally with HEV. The genotype 1 ORF3 polypeptide exhibited stronger reactivity with the sera from monkeys infected with genotype 1 than the genotype 4 ORF3 polypeptide. The genotype 4 ORF3 polypeptide demonstrated stronger reactivity with the sera from monkeys infected with genotype 4 than did the genotype 1 ORF3 polypeptide. The HEV ORF3 polypeptide contains genotype-specific antigens and the antigen-antibody reactions between the same genotypes were stronger than those between different genotypes.     

Genotyping of hepatitis C virus by Taqman real-time PCR.

BACKGROUND: Genotype of hepatitis C virus (HCV) is of major importance for the outcome of treatment. The response rate is considerably lower for genotype 1, the predominant genotype in western countries. OBJECTIVES: To develop and evaluate a new, simple method for genotyping of HCV based on real-time polymerase chain reaction (PCR) and Taqman probes targeting the 5' non-coding region. STUDY DESIGN: The method was compared with Innolipa on 220 serum samples representing genotypes 1-4, and was applied on a further 614 clinical samples. RESULTS: Taqman typing of the 220 samples showed genotype 1 in 69, genotype 2 in 58, genotype 3 in 57 and genotype 4 in 19, while 17 were non-reactive. There was a complete concordance with Innolipa with the exception of seven samples, which were of genotype 1 by Taqman, but genotype 4 by Innolipa. Sequencing of these samples showed a subtype 4 variant which differed at two positions compared with subtypes 4b/c/d, which are targeted by the probe. By adding a modified probe, these genotype 4 variants could also be identified. Out of 614 consecutive clinical samples, 524 could be typed by the Taqman assay; 45.2% were genotype 1, 19.3% genotype 2, 33.8% genotype 3 and 1.7%, genotype 4. CONCLUSION: The method was overall accurate and provides an attractive alternative for genotyping because processing time and costs are significantly reduced. Inclusion of probes targeting genotypes 5 and 6 is required for the method to be useful in areas where these genotypes are present.