肝病患者中庚型肝炎病毒感染的检测

目的探讨临床肝病病人中庚型肝炎病毒（ＧＢＶ－Ｃ／ＨＧＶ）感染情况及临床特点。方法应用庚型肝炎病毒基因组５’ＵＴＲ两对寡核苷酸作为引物，建立逆转录套式聚合酶链反应，检测１６９例不同肝病患者血清标本中ＧＢＶ－Ｃ／ＨＧＶＲＮＡ，并对其中１例ＰＣＲ扩增产物进行克隆及测序。结果１６９例各型肝病病人ＧＢＶ－Ｃ／ＨＧＶＲＮＡ总的检出率为９５％（１６／１６９）。在２９例有手术输血史患者中，３１０％（９／２９）ＧＢＶ－Ｃ／ＨＧＶＲＮＡ呈阳性，明显高于无手术输血史组（５％，Ｐ＜００１）。序列分析显示１株庚肝病毒５’ＵＴＲ部分基因片段与已知庚肝病毒株核苷酸同源性在８９１４％～９８９１％之间。结论ＧＢＶ－Ｃ／ＨＧＶ感染普遍存在于临床肝病患者中，病人感染ＧＢＶ－Ｃ／ＨＧＶ的临床表现未发现有特殊性，ＧＢＶ－Ｃ／ＨＧＶ可能不是非甲～戊型肝炎的主要致病因子。     

中国人丙型肝炎病毒基因组3′端非编码区的研究

目的分析中国丙型肝炎病人ＨＣＶ基因组３′端非编码区（３′ＮＣＲ）,以促进对ＨＣＶ基因组复制机制的研究。方法采用两种方法,从上海地区感染ＨＣＶ的病人血清中,扩增获得ＨＣＶ基因组３′端非编码区：一是用套式ＰＣＲ直接扩增,二是先分别获得ＨＣＶ３′ＮＣＲ的前半部分和后半部分,再将两片段进行融合ＰＣＲ。ＰＣＲ产物进行测序后作同源性分析。在此基础上,建立了针对３′端非编码区的ＲＴＰＣＲ方法,并与基于５′端非编码区的ＲＴＰＣＲ方法检测ＨＣＶＲＮＡ的特异性和灵敏度比较。结果序列分析表明,中国丙型肝炎病人ＨＣＶ基因组３′非编码区由４部分组成：高度变异区、Ｐｏｌｙ（Ｕ）区、Ｐｏｌｙ（Ｕ／Ｃ）区和９８碱基区。同源性分析显示,９８碱基区在不同分离株间高度保守并与国外报道株一致,而Ｐｏｌｙ（ＵＵＣ）区存在较大差异。３′端非编码区和５′非编码区ＲＴＰＣＲ检测血清ＨＣＶＲＮＡ有较高符合率（９５％）。结论ＨＣＶ基因组３′端非编码区的３′末端（９８碱基）,在不同分离株间的高度保守性提示,该区在ＨＣＶ基因复制中起重要作用。基于３′非编码区的ＲＴＰＣＲ方法,将有助于ＨＣＶ感染的诊断。     

丙型肝炎病毒血清学分型与基因分型研究

为了解某农村单采浆供血员人群所感染丙型肝炎病毒（ＨＣＶ）的血清型和基因型构成，并对两种分型方法进行比较，本工作以ＨＣＶＣ区型特异性多肽对抗－ＨＣＶ进行血清学分型，对已确定血清型的血清进行５′非编码区（ＮＣＲ）逆转录套式聚合酶链反应（ＲＴ－ｎＰＣＲ）和限制性片段长度多态性（ＲＦＬＰ）分析以确定基因型，并对６份扩增产物进行序列测定。结果显示１４０份抗－ＨＣＶ阳性血清中，血清Ⅰ型和Ⅱ型分别为４４份（３１．４３％）和１２份（８．５７％），余８４份未能分型。４４株已知血清型的ＨＣＶ中，１ｂ、２ａ和３ｂ等３种基因型分别为３４株（７７．２７％）、９株（２０．４５％）和１株（２．２７％）。两种分型方法一致率为９３．１８％（４１／４４）。对６株ＨＣＶ５′ＮＣＲ的序列分析证实了ＲＦＬＰ分型的正确性。结论认为该人群ＨＣＶ感染以血清Ⅰ型或基因１ｂ型为主；Ｃ区型特异性多肽血清学分型法与ＲＦＬＰ基因分型法符合率高，具有一定应用价值。     

丙型肝炎病毒1b型NS5 A区基因结构变异与α干扰？…

目的 观察丙型肝炎患者丙型肝炎病毒（ＨＣＶ）１ｂ型基因组部分ＮＳ５ Ａ区核苷酸,氨基酸的变异情况并探讨其与α干扰素疗效的相关性。方法 患者干扰素治疗前,中,后留血标本,用聚合酶链反应（ＰＣＲ）扩增ＨＣＶ病毒ＮＳ５ Ａ区部分基因片段并用直接测序法测序。与ＨＣＶ－Ｊ株及ＨＣＶ－河北株（ＨＣＶ－ＨＢ）比较核苷酸及氨基酸序列的同源性,根据α干扰素疗效分析ＨＣＶ１ｂ是否存在干扰素敏感决定区。     

多重引物聚合酶链反应扩增丙型肝炎病毒基因及基 …

利用聚合酶链反应（ＰＣＲ）技术对丙型肝炎病毒（ＨＣＶ）的５’－非编码区（５’－ＮＣＲ）、Ｃ及ＮＳ４基因区的３对引物分别及同时扩增，检测８０例抗－ＨＣＶ阳性患者的血清ＨＣＶ ＲＮＡ，并进行了ＨＣＶ基因分型研究。各不同引物所介导的ＰＣＲ检出ＨＣＶ ＲＮＡ的结果为：５’－ＮＣＲ基因区６０％（４８／８０），Ｃ基因区３７％（３０／８０），ＮＳ４基因区３０％（２４／８０）。以上３对引物同时扩增仅４２％（３４／     

中国人丙型肝炎病毒基因组3‘端非编码区的研究

目的 分析中国丙型肝炎病人ＨＣＶ基因组３‘端非编码区,以促进对ＨＣＶ基因组复制机制的研究。方法 采用两种方法,从上海地区感染ＨＣＶ的病人血清中,扩增获得ＨＣＶ基因组３’端非编码区：一是用套式ＰＣＲ直接的增,二是先分别ＨＣＶ３‘ＮＣＲ的前半部分和后半部分,再将两片段进行融合ＰＣＲ。ＰＣＲ产物进行测序后作同源性分析。     

从基因分型看血液透析及肾移植患者丙型肝炎病毒感染状况

采用国产和美国Ｏｒｔｈｏ公司第２代抗丙型肝炎病毒（ＨＣＶ）试剂对１００例维持性血透及肾移植患者进行血清丙型肝炎病毒抗体（抗－ＨＣＶ）对比检测。阳性标本用聚合酶键反应（ＰＣＲ）法检测ＨＣＶＲＮＡ并采用型特异的ＨＣＶ亚基因探针对其非结构蛋白ＮＳＳ区扩增产物进行了杂交基因分型。结果表明，这组病人中抗－ＨＣＶ阳性率为４１％，肾移植术后再透析者达５６．５２％，且与透析时间、输血次数、受血量成正相关；国产抗－ＨＣＶ试剂同美国Ｏｒｔｈｏ公司试剂比较阳性符合率达９１．４３％；抗－ＨＣＶ阳性患者中有３１．４３％（１１／３２）血清ＨＣＶＲＮＡＮＳ５阳性；透析患者中，ＨＣＶ基因型各型均有，以混合型为主，占６３．６４％。     

TLMV5‘非编码区基因的克隆与序列分析

目的 调查ＴＴＶ阴性的非甲￣庚型肝炎患者中ＴＴＶ－ｌｉｋｅ ｍｉｎｉ ｖｉｒｕｓ（ＴＬＭＶ）感染情况,对ＴＬＭＶ５’非编码区（５’ＮＣＲ）部分基因进行分子克隆与序列分析。方法 采用巢式ＰＣＲ技术检测５３例ＴＴＶ阴性的非甲－庚肝炎患者血清ＴＬＭＶ ＤＮＡ,对ＰＣＲ产物进行克隆、测序和分析。结果 ５３例病例中ＴＬＭＶ ＤＮＡ阳性３７例（６９．８％）,对其中８株ＴＬＭＶ基因克隆测序,并与Ｔａｋａｈａｓｈ     

丙型肝炎病毒基因分型及其与干扰素治疗应答的关系

目的为了解山西省丙型肝炎病毒的基因型和基因型对干扰素疗效的预示价值。方法用ＨＣＶ５’ＮＣ区酶切分型方法对９４例丙型肝炎病人进行基因分型，并观察其中４５例患者对干扰素α１ｂ治疗的应答。结果显示ＨＣＶⅠ组（Ⅰ、Ⅱ型）感染８０例（８５１％），ＨＣＶⅡ组（Ⅲ、Ⅳ型）感染１２例（１２８％），ＨＣＶⅠ／Ⅱ组混合感染２例（２１％）。在接受干扰素治疗的病例中，ＨＣＶⅠ组感染（３５例）的应答率为３７１％，持续应答率为１７１％，而Ⅱ组感染（１０例）的应答率为８０％，持续应答率为６０％，两组相比，有显著性差异（Ｐ＜００５，Ｐ＜００２５）。结论表明山西省以ＨＣＶⅠ组感染为主，干扰素对ＨＣＶⅡ组感染的疗效优于ＨＣＶⅠ组感染，ＨＣＶ基因型有预测干扰素疗效的意义。     

乙型肝炎病毒C基因启动子及前C变异的动态研究

目的明确乙型肝炎病毒（ＨＢＶ）Ｃ基因启动子（ＢＣＰ）与前Ｃ变异株在慢性乙型肝炎患者中的动态消长,及与ＨＢｅＡｇ血清学状态的关系。方法对３２例慢性乙型肝炎患者的１０５份系列血清进行错配聚合酶链反应（ＰＣＲ）－限制性片段长度多态性分析,并对其中１５例患者系列血清标本的ＰＣＲ产物进行直接测序。结果ＢＣＰ和Ａ８３变异株感染率均明显增高（６２．５％＞４６．９％;３１．３％＞１２．５％）;ＢＰＣ变异株更多地（１４／１６）表现为优势积累,且先于ＨＢｅＡｇ血清阴转;ＢＣＰ野株／变异株比例变化影响ＨＢｅＡｇ血清学的稳定性。结论两种类型的变异株在炎症活动中均具有选择优势,最终造成ＨＢｅＡｇ（－）的ＨＢＶ感染;可能与ＨＢＶ感染之持续有关。     

丙型肝炎病毒非结构基因5b(NS 5b)区一级结构的变异

目的　分析中国丙型肝炎病毒非结构基因５ ｂ 区核苷酸序列变异。方法　以逆转录套式聚合酶链反应（ＲＴｎｅｓｔｅｄＰＣＲ） 从４９ 例中国病人的血清中获得互补的ＤＮＡ片段,产物克隆后测序。结果　３３ 株系基因Ⅱ１ ｂ 型,１６ 株系Ⅲ２ ａ 型,中国ＨＣＶ株型与日本ＨＣＶ株型同属１ 个基因亚型,但在一些核苷酸保守位点上有一定的差异,对３３ 株Ⅱ１ ｂ 型和１６ 株Ⅲ２ ａ 型间的同源性进行比较,发现１６ 株Ⅲ２ ａ 型的同源性低于３３ 株Ⅱ１ ｂ 型的同源性。首次在ＨＣＶＮＳ５ ｂ 区发现１ 个新的３ 个核苷酸的缺失突变及１ 个移码突变。结论　同一基因亚型之内不同ＨＣＶ株的核苷酸序列可能具有一定的地理分布特征,每个地区有一定的流行株。     

Hepatitis C viral genotype influences the clinical outcome of patients with acute posttransfusion hepatitis C

Most patients with an acute infection of hepatitis C virus (HCV) will develop chronic hepatitis, and only about 15-20% of the cases will resolve spontaneously. The mechanism for the different outcomes in patients with acute HCV infection remains unclear. HCV genotype has been recognized as an important factor affecting the clinical course and outcome of chronic hepatitis C patients. In order to evaluate the role of HCV genotype in the clinical course and outcome of acute posttransfusion hepatitis C, 67 patients with acute posttransfusion hepatitis C from a prospective study of posttransfusion non-A, non-B hepatitis were enrolled. Thirty-nine patients (58.2%) were HCV genotype 1b. Among the 67 patients with acute posttransfusion hepatitis C, 53 (79.1%) progressed to chronic hepatitis. Significantly more patients with genotype 1b than non-1b genotypes developed chronic hepatitis (89.7% vs. 64.3%; P = 0.019). There was no significant difference in gender, mean age, amount of transfused blood, hepatitis symptoms, jaundice, incubation period, peak serum alanine transaminase, or serum HCV RNA titer between patients with HCV genotype 1b and non-1b infections. Patients who developed chronic hepatitis had a significantly greater incidence of genotype 1b infection (66.0% vs. 28.6%; P = 0.013) and a longer incubation period (7.3 weeks vs. 5.4 weeks; P = 0.052) than patients whose infection was resolved. Patients with a genotype 1b infection that resolved itself spontaneously all had an incubation period of less than 6 weeks. Multivariate logistic regression analysis revealed that genotype 1b and an incubation period > or = 6 weeks were significant predictive factors for the development of chronic hepatitis. Therefore, the HCV genotype can influence the outcome of patients with acute HCV infection.     

丙型肝炎病毒基因型分布及HLA-Cw基因多态性的关联性分析

目的 对邯郸不同HCV基因型的分布情况进行统计,结合HLA-Cw的基因多态性,分析HCV与HLA-Cw的关联性.方法 采用荧光PCR法进行HCV基因分型检测,计算不同HCV基因型的阳性率,从而对该地区的丙肝病毒感染者进行流行病学调查;采用病例-对照研究,对所有研究对象的基因组进行HLA基因分型,统计HCV感染者和健康对照者之间HLA-Cw基因频率的差异和关联度;对所有丙型肝炎病毒感染者,采用统计学方法分析HLA基因型别和血清中HCV-RNA的基因型别的关联性,从而寻找与不同HCV基因型感染相关的HLA-Cw基因型.结果 邯郸HCV 1b亚型在人群中的分布最多,其次为混合型1b/2型;HLA-Cw* 8与HCV的慢性感染有关,而其他7个基因位点经研究关联不大,进一步研究发现,HLA-Cw * 8基因与HCV 1b亚型的易感性密切相关.结论 HLA-Cw基因与HCV感染密切相关,可进行易感人群分析,为控制HCV的传播提供可参考的理论依据.     

Hepatitis C virus infection among dialysis patients in Tunisia: incidence and molecular evidence for nosocomial transmission

In order to study the incidence of hepatitis C virus (HCV) infection in Tunisian haemodialysis patients and detect its nosocomial transmission, 395 patients were enrolled in a prospective study (November 2001-2003). HCV serological and virological status was determined initially using, respectively a third generation ELISA and an RT-PCR qualitative assay. The genotype of the HCV isolates was determined by sequencing NS5B region. The issue of nosocomial transmission was addressed by sequencing the HVR-1 region of the E2 gene. About 20% of the patients had anti-HCV antibodies and HCV-RNA was detected in 73% of the anti-HCV positive patients. Two cases of de novo HCV infection were identified in two dialysis centers, during virological follow-up of patients susceptible to HCV infection. The incidence of de novo HCV infection was 0.5%. Determining the genotypes in the first center disclosed that all HCV-positive patients were infected with genotype 1b; sequencing of the HVR-1 region of the E2 gene provided strong evidence that the isolate from the newly infected patient and another infected dialysis patient were closely related, confirming nosocomial contamination. The investigation of the second center is pending. Besides, one patient with negative HCV serology had detectable HCV-RNA at the beginning of the study. This case had HCV genotype 1b, two other infected dialysis patients in the same unit had HCV genotypes 4k and 3a; thus precluding nosocomial transmission. Thanks to molecular and phylogenetic methods, one case of nosocomial HCV transmission in haemodialysis was confirmed. Epidemiological investigation suggested nosocomial transmission via the medical and/or nursing staff.     

Molecular evidence for nosocomial transmission of hepatitis C virus in a French hemodialysis unit.

A systematic virological follow-up of hemodialysis patients identified 11 cases of de novo hepatitis C virus (HCV) infection in the same unit that were not due to blood transfusion. There were three groups of infection, each occurring within a period of 3 months: four infections with genotype 1b, two infections with genotype 1b, and five infections, four with genotype 1a and one with genotype 5a. The possibility of patient-to-patient transmission was addressed by sequencing the first hypervariable region of the HCV genome in sera taken shortly after infection. Phylogenetic analysis indicated clustering of most of the cases of de novo infections. Sequence homologies identified potential contaminators among already infected patients. All patients who were infected with closely related HCV isolates were found to have been treated in the same area and during the same shift or on the previous one. These infections could have been due to occasional breaches of the usual hygiene measures. Strict adhesion to hygiene standards and routines, continuously supervised, remains the key rule in the management of dialysis patients. Nevertheless, the isolation of patients with HCV could reduce the risk of infection because occasional lapses of preventive hygiene measures or unpredictable accidents can always take place in a hemodialysis unit. This policy needs to be evaluated by large-scale prospective studies.     

Hepatitis C virus and its genotypes in patients suffering from chronic hepatitis C with or without a cryoglobulinemia-related syndrome

Recently, evidence has been presented for a possible association between hepatitis C virus (HCV) infection and essential mixed cryoglobulinemia (EMC). Eleven consecutive patients with EMC and two with cryoglobulinemia type I were examined for the presence of markers of HCV infection. Eleven of 13 patients (10 with EMC and 1 with type I cryoglobulinemia) had anti-HCV antibodies (as determined by a second generation anti-HCV assay) and HCV-RNA in plasma or serum. HCV-RNA was also detected in liver biopsies of five patients. Genotyping showed that HCV genotype 1 was found in 10 of 11 patients with HCV-RNA (9 genotype 1b and 1 genotype 1a) and only one patient had HCV genotype 2. However, a similar high prevalence of genotype 1b (100%) was found in a group of 14 consecutive patients with chronic hepatitis C, who had no clinical evidence of cryoglobulinemia. Concomitant infection was present in three patients with genotypes 2, 3 and 4, respectively. These findings stress the high prevalence of HCV infection in patients with EMC and further study shows that a difference in genotype prevalence was not found between HCV-related EMC and chronic hepatitis C without clinical manifestations of EMC. © 1994 Wiley-Liss, Inc.     

Characterization of hypervariable region in hepatitis C virus envelope protein during acute and chronic infection

Summary. Hepatitis C virus (HCV) causes persistent infection in most patients. To clarify the mechanisms underlying establishment of this persistent infection, nucleotide sequences of the E1/E2 region were characterized in 5 patients with acute and chronic HCV infection. We used direct DNA sequencing methods to identify the major sequence of HCV in each patient. Each HCV genome displayed a high frequency of nucleotide sequence variation in the hypervariable region (HVR) of E2. However, patient-specific conserved nucleotide sequences were identified in the E1/E2 region during the course of infection and conserved the higher-order protein structure.In the acute phase HCV infection, amino acid substitution in HVR-1 as the monthly rate of amino acids substitution per site (%) between each point exceeded 10.2%. In the chronic phase HCV infection, a significantly lower rate of amino acid substitution was observed in patients. The host immune responses to HVR-1 of each HCV isolates from all clinical courses were characterized using synthetic peptides and ELISA. One chronic patient serum (genotype 1b) did not react at all to its own HVR-1 peptides, however another patient (genotype 2b) reacted to all clinical course. These results indicated that HVR-1 might not always exhibit neutralizing epitopes of HCV infection. The sequence variation in HVR-1 may instead indicate the existence of various clones in acute phase infection and the adaption of these clones is thought to have caused persistent and chronic infection in each patient.