丙型肝炎病毒基因分型及其临床意义

为了探讨丙型肝炎病毒（ＨＣＶ）感染的基因型及其临床意义，本文对沈阳地区６１例ＨＣＶ－ＲＮＡ阳性血清的聚合酶链反应（ＰＣＲ）产物，用限制性片段长度多态性（ＲＦＬＰ）分析进行基因分型，结果为：ＨＣＶⅡ型感染占７０．５％（４３／６１），ＨＣＶⅢ型感染占２７．９％（１７／６１），ＨＣＶⅡ／Ⅲ型混合感染占１．６％（１／６１）。初步证明沈阳地区ＨＣＶ感染以Ⅱ型为主。对９１例肝癌病人中ＨＣＶ－ＲＮＡ阳性的２７例血清基因分型，Ⅱ型占７４％（２０／２７），其余为Ⅲ型。可见ＨＣＶⅡ型感染与肝癌发生有密切关系。     

山东省流行性出血热病人与宿主动物血清学分型及其意义的研究

目的为了查明流行性出血热（ＥＨＦ）病人血清型与宿主动物血清型关系，探讨血清学演变及其意义。方法采用ＩＦＡ、ＨＩ和ＲＰＨＩ法分型。结果在４５８例病人中，存在家鼠型、野鼠型和未定型病人，分别占９１．４８％、７．２１％和１．３１％。同时检测鼠肺，其中家鼠ＥＨＦ病毒（ＥＨＦＶ）抗原阳性率为６．５８％（１６／２４３），病毒抗原型均为家鼠型。野鼠４份阴性；在鼠血清中，家鼠血清ＥＨＦ抗体阳性率为１０．７１％（４４／４１１），血清型为家鼠型，野鼠阳性率为４．１７％（３／４８），血清型为野鼠型。结论证实当地是以家鼠型为主的家、野鼠型混合型疫区，ＥＨＦＶ抗原型和血清型别均与宿主动物种类相一致。     

HCV RNA链扩增反应（PCR）检测及其基因分型方法的建立

我们用选自ＨＣＶ５’ＮＣＲ和Ｃ区引物建立了检测血清中ＨＣＶＲＮＡ及其基因型的ＲＴ／ＰＣＲ方法，可以敏感而特异地检出血清中ＨＣＶＲＮＡ及其４种基因型。５０例抗ＨＣＶ阳性献血员ＨＣＶＲＮＡ检出率为８４％（４２／５０），４２例ＨＣＶＲＮＡ阳性者中ＨＣＶⅡ型占５９．５％（２５／４２），Ｉ型和混合型（包括Ⅰ／Ⅱ、Ⅱ／Ⅲ、Ⅰ／Ⅲ和Ⅰ／Ⅱ／Ⅲ各２例）分别占１１．９％和１９％，另有４例Ｃ区分型结果阴性，为未定型，占９．５％，没有发现Ⅳ型。上述结果说明我国献血员中存在无症状ＨＣＶ携带者，ＨＣＶ基因型则以Ⅱ型为主，Ⅰ型次之，Ⅲ型较少。     

重型肝炎患者丙型肝炎病毒抗体的存在状态及其意义

采用合成肽酶免疫分析法（ｓｐＥＬＩＳＡ）和美国Ｏｒｔｈｏ公司第２代ＥＬＩＳＡ（２ｎｄＥＬＩｓＡ）对１０３例重型肝炎患者血清丙型肝炎病毒抗体（抗ＨＣＶ）作筛选，阳性血清用重组免疫印迹法（４·ＲＩＢＡ）作鉴定，并用逆转录聚合酶链反应检测ＨＣＶＲＮＡ，以重新评价重型肝炎患者血清抗ＨＣＶ存在状态及其意义。发现ｓｐＥＬＩＳＡ和２ｎｄＥＬＩＳＡ检测抗ＨＣＶ的阳性率分别为２２．３３％（２３／１０３）和１９．１０％（１７／８９），阳性血清经４·ＲＩＢＡ鉴定仅５９．０９％（１３／２２）阳性，ＨＣＶＲＮＡ检出率亦仅为６０．００％（９／１５）。动态观察，约１／３抗ＨＣＶ阳性患者抗ＨＣＶ持续２～４周消失。血清反复冻融可使阳性检测吸光值（Ａ４９２）下降，阴性检测Ａ４９２值升高。血清抗ＨＣＶ阳性和（或）ＨＣＶＲＮＡ阳性患者均合并乙型肝炎病毒（ＨＢＶ）感染，但合并ＨＣＶＲＮＡ阳性患者预后较差。结果提示单独ＨＣＶ感染不是本地区重型肝炎发生的主要原因，部分患者抗ＨＣＶ存在可能属既往感染或系“被动输入”所致，也可能为假阳性，检测ＨＣＶＲＮＡ对于确切了解重肝患者ＨＣＶ感染状况和判断预后有一定的意义。     

佛山市庚型肝炎病毒检测和部分基因序列分析

目的了解佛山庚型肝炎病毒（ＨＧＶ）感染状况，分析ＨＧＶ非结构基因（ＮＳ）３区部分核苷酸序列。方法采用逆转录聚合酶链反应检测血清ＨＧＶＲＮＡ，对一例肝炎患者的ＨＧＶ（ＨＧＶＣ－ＦＳ）ＮＳ３区８１８ｂｐ片段作克隆及序列分析。结果８０例非甲－戊型肝炎患者和１０５例静脉吸毒者ＨＧＶＲＮＡ检出率分别为６．３％（５／８０）和２３．８％（２５／１０５），ＨＧＶＣ－ＦＳＮＳ３区片段核苷酸序列与ＨＧＶ－Ｕ４４４０２、Ｕ４５９６６、Ｕ３６３８０及ＨＧＶＣ９６４相同区段同源性为８５．５％、８５．６％、８８．０％、８９．２％。结论佛山存在ＨＧＶ感染，静脉吸毒者感染率较高，ＨＧＶ可能不是非甲－戊型肝炎主要致病因素。ＨＧＶＣ－ＦＳ与ＨＧＶＣ９６４同源性最高。     

不同人群血清单项丙型肝炎病毒核心抗体存在状态

为调查不同人群血清单项丙型肝炎病毒核心抗体（抗－ＨＣＶｃ）存在状态，应用合成肽酶免疫分析筛选抗－ＨＣＶｃ，经重组免疫印迹和中和抑制试验鉴定。发现孕妇、供血员、慢性乙型肝炎、非甲非乙型肝炎、透析患者、原因不明肝硬化和输血后肝炎单项抗－ＨＣＶｃ阳性率分别为０．８％（１１／１４３６）、２．８％（１５／５４１）、３．１％（１５／４８４）、１３．６％（９／６６）、１１．５％（１０／８７）、２８．６％（４／１４）和３０％（３／１０）。单项抗－ＨＣＶ阳性的孕妇和供血员血清仅２６．７％～２７．３％检出ＨＣＶＲＮＡ，透析和各种肝病患者检出率高达５５．６％～１００％。８例单项抗－ＨＣＶｃ阳性肝炎患者病理检查显示不同程度的肝损害。结果提示：不同人群单项抗－ＨＣＶｃ阳性的意义有差别，检测ＨＣＶＲＮＡ对区别抗－ＨＣＶｃ存在属既往或现症感染十分重要。     

竞争性逆转录聚合酶链反应定量检测对HCV RNA

为了解感染丙型肝炎病素（ＨＣＶ）后的（ＨＣＶＲＮＡ）含量与丙型肝炎病情轻重，预后，治疗反应的关系，采用竞争性逆转录聚合酶链反应（ＣＲＴ－ＰＣＲ）的方法，对９例感染了ＨＣＶ患者的血清进行了ＨＣＶＲＮＡ的定量检测。结果：６．６×１０３拷贝１毫升占８８．９％，６．６×１０４拷贝／毫升占１１．１％。结果提示：急慢性丙型肝炎及无症状ＨＣＶ感染者体内均含有较高的病毒滴度，且滴度低预后好，抗病毒疗效好。     

上海地区丙型肝炎病毒基因分型的研究

目的 研究上海地区不同人群中丙型肝炎病毒基因型的分布和频率。方法 应用新改进的第二代谱线探针分析（Ｌｉｎｅ Ｐｒｏｂｅ Ａｓｓａｙ,ＬｉＰＡ）试验盒,对１０９份血清的ＨＣＶ ＲＮＡ逆转录－巢式ＰＣＲ阳性分离物进行了基因分型。结果 ８１份丙型肝炎患者ＨＣＶ分离物中７１份（８６．７％）为１ｂ型,４份（４．９％）为２ｃ型,２份（２．５％）为３ｂ份,１份（１．２％）为６ａ型,２份（２．５％）为混合型９２／     

上海等地区慢性丙型肝炎患者中HCV基因型的分布

目的：探讨上海等地区丙型肝为患者ＨＣＶ基因型的分布，方法：以一种新的ＨＤＶ基因分型方法－线探针试验（ＩＮＮＯ－ＬｉＰＡ）对来源于上海等地区９６例慢性丙型肝炎患者进行ＨＣＶ基因分型，结果：显示９６例患者中以１ｂ型为主（占７７．６％）其次为２ａ／２ｃ（占８．６２％）并发现１ａ４ａ和４ｅ基因亚型，８２例感染单一型（１ｂ）基他１４例为混合感染，有一例慢性丙型肝炎患者感染５个亚型，对其中３９例病人应用干扰素     

丙型肝炎病毒基因分型研究

采用逆转录－多聚酶链反应（ＲＴ／ＰＣＲ）法检测了泰安地区４６例输血后丙型肝炎（ＰＴＨ－Ｃ）患者的血清ＨＣＶ－ＲＮＡ〉对其中３４例生进物ＰＣＲ产物用限制性长度多生分析泊（ＲＦＬＰ）进行了ＨＣＶ基因分型。结果ＨＣＶ－Ⅱ型检出率为７３．５％，ＨＣＶ－Ⅲ型检出率为１１．８％；ＨＣＶⅡ／ＨＣＶⅢ型混合感染率为１４．７％，未发现Ⅰ、Ⅳ和Ⅴ型感染。     

Serotyping and genotyping of hepatitis C virus in Taiwanese patients with type C chronic liver disease and uraemic patients on maintenance haemodialysis

BACKGROUND: To evaluate a recombinant immunoblot hepatitis C virus (HCV) serotyping assay, which determines HCV serotypes 1, 2, and 3 by detecting type-specific antibodies to core-and NS-4-derived peptides. METHODS: Immunoreactivity of type-specific antibodies among 173 chronic hepatitis C patients and 43 haemodialysis patients in Taiwan was examined and the serotyping results were compared with genotyping by Okamoto's method. Serial specimens from 29 patients undergoing interferon-alpha therapy were also evaluated. RESULTS: Of the 205 specimens for which genotyping data were available, 51.2% were of serotype 1, 31.7% of serotype 2, 1.0% of serotype 3, 2.4% of either serotype 1 or 3, and the remaining 13.7% were untypable. The serotypable rate was significantly lower in haemodialysis patients than in chronic hepatitis C patients (70.0% vs 94.9%; P < 0.001). Serotyping of genotype 2b specimens was significantly more dependent on core peptide bands than other genotypes. Using genotyping as the reference, the overall sensitivity, specificity and concordance of the recombinant immunoblot HCV serotyping assay were 86.3%, 97.2% and 83.9%, respectively. However, the serotyping assay had significantly lower sensitivity (69.2%), specificity (77.8%) and concordance (53.8%) for genotype 2b specimens. Of nine HCV complete responders, one lost type-specific antibodies 6 months after the cessation of interferon-alpha treatment. CONCLUSIONS: These results suggest that, except for less than optimal performance with immunocompromised or genotype 2b patients, the HCV serotyping assay is a practical and useful method for HCV typing in the clinical setting in Taiwan.     

HCV血清分型方法的建立及其评价

目的建立HCV血清分型方法,评价其在慢性丙型肝炎(丙肝)抗体阳性标本中的分型率及血清型与基因型的相关性。方法克隆表达HCVCore、非结构蛋白(non-structural protein,NS)4两个区段的型别特异性表位嵌合抗原,并应用酶联免疫竞争抑制法建立血清分型方法。分别应用酶联免疫吸附试验和聚合酶链反应法检测200例慢性丙肝患者的血清抗体和HCVRNA,并用建立的方法进行血清分型。同时采用该方法检测90例乙型肝炎患者、11例戊型肝炎患者和16例其他肝病患者的血清,评价其特异性。结果在200例慢性丙肝患者的血清标本中,基因1b型128例,2a型72例,型别特异性抗体阳性157例,分型率为78.50%,与基因型一致率为98.09%。而在另外117例乙型肝炎、戊型肝炎和其他肝病患者的血清中,均未检测出HCV型别特异性抗体。结论建立的HCV血清分型方法具有较高的分型率和特异性,可用于HCV抗体的血清学分型和预测干扰素疗效。     

Serotyping of hepatitis C virus in chronic type C hepatitis in Taiwan: Correlation with genotypes

To evaluate the usefulness of a new serologic assay to group hepatitis C virus (HCV), genotypes identified by this serotyping method were compared to those identified by a polymerase chain reaction (PCR) assay with type-specific primers in 71 Taiwanese patients with chronic type C hepatitis. The group-specific antibodies against different HCV genotypes were detected by using an enzyme-linked immunosorbent assay (ELISA) based on group-specific recombinant peptides (C14-1 and C14-2) within the NS4 region. Among 71 patients positive for current second-generation HCV antibodies, HCV RNA was detected in 55 patients by PCR with primers from the 5′ untranslating region, and in 52 by genotype-specific PCR. In 49 (89%) of 55 viremic patients, the results of serotyping by ELISA showed complete agreement with those determined by PCR genotyping, and none of the patients showed a group opposite to that of HCV genotype. The positive rate of group-specific antibodies (69/71;97%) was even better than that of the PCR (55/71;78%). We conclude that this new serotyping assay is highly sensitive and specific for the determination of HCV genotypes, and will be useful in future epidemiologic studies, as well for clinical application.     

Hepatitis C Virus Genotyping versus Serotyping in Egyptian Patients

Background: The RNA genome of hepatitis C virus (HCV) displays extensive sequence variation. In this study, serotyping and genotyping techniques were applied to assess this variability by comparing the performance of the serotyping assay with a panel of well-characterized HCV strains isolated from chronic active hepatitis (CAH) patients. Patients and Methods: 60 serum samples from CAH patients were analyzed. All isolates were genotyped by a line probe assay and the results of genotyping and serotyping were evaluated. Results: The overall sensitivity of the serotyping and genotyping techniques was 81.16% with a concordance of 73.3%. Type 4 was detected in 73.3% of cases and it was highly heterogeneous. Conclusion: Type 4 HCV is the most prevalent type in Egyptian CAH patients and there is a high concordance between the results of serotyping and genotyping techniques. Received: January 18, 2000 · Revision accepted: December 3, 2000     

HCV serotypes in Brazilian patients.

OBJECTIVE: To investigate the prevalence of the different types of hepatitis C virus (HCV) in a population of chronic HCV carriers using the Murex HCV serotyping 1-6 assay. METHODS: All serum samples from these patients had a positive nested PCR HCV reaction. The sera were submitted to ELISA, modified, for the identification of antibodies against HCV serotypes 1, 2, 3, 4, 5 and 6 (Murex HCV serotyping 1-6 assay). RESULTS: The viral serotype was identified in 166 (75.8%) of the 219 patients, 108 (65.11%) males and 58 (34.9%) females. Patient age ranged from 12 to 73 years, with a mean of 41.1 years. The form of acquisition of the disease most frequently reported was blood transfusion. The results showed a predominance of type 1 (70.0%), followed by type 3 (22.3%) and type 2 (4.2%). CONCLUSION: Samples presenting low and very close optical density readings may lead to discrepant diagnoses concerning HCV serotypes and should be confirmed by genotyping. The serotyping can be useful in clinical practice and can be of help in establishing the prognosis of the disease, also favoring epidemiologic studies independently of the technology required for genotyping tests.     

丙型肝炎病毒血清学分型的研究

本文用EIA法对辽宁省部分地区的105例抗-HCV阳性者(包括各种丙型肝炎患者和无症状丙型肝炎病毒感染者)进行抗-HCV型特异性抗体检测,结果显示血清学方法的分型率为72.9％(76/105),其中血清1型44人(57.9％),血清2型29人(38.2％),1/2混合型占3.9％(3/76)。提示在辽宁省部分地区的抗-HCV阳性人群中HCV血清1、2型均存在,但以血清1型为主。     

急性丙型肝炎患者病毒株部分基因克隆及其序列分析

目的探讨HCV感染在肝炎发生中的病原学作用。方法对89例急性肝炎进行HCV标志物分析,并从HCVRNA阳性的5例非甲非乙型急性肝炎患者血清中提取HCVRNA,经随机引物逆转录合成cDNA,先以型别特异的PCR法分型,再用巢式PCR扩增部分核心基因区序列,将产物连接p-GEM-T载体,在大肠杆菌中表达后测序分析。结果非甲非乙型急性肝炎中HAV、HBV和HCV阳性分别占47.2%、28.1%和15.7%,HAV和HBV双重感染占14.6%,非甲、非乙和非丙肝炎占9%;HCV分型显示Ⅱ型、Ⅲ型和Ⅱ/Ⅲ混合型分别占85.8%、7.1%和7.1%;Ⅱ型血清用于序列分析,扩增序列424bp与原设计完全一致。急性肝炎株间核苷酸同源性在98.1%～99.5%。氨基酸同源性在97.6%～99.2%;前者的同源性在Ⅰ型为91.9%,在Ⅱ型为94.3%～95.6%;后者与Ⅰ型、Ⅱ型的同源性在92.3%～95.8%。结论HCV为引起非甲非乙型急性肝炎的主要病毒之一,以Ⅱ型为主,应引起临床重视。     

Hepatitis C Serotypes in Nonalcoholic and Alcoholic Patients

Genotyping of the hepatitis C virus (HCV) RNAcan be performed by a variety of methods followingpolymerase chain reaction amplification of a stable RNAportion of the genome. The gold standard isamplification of the RNA from the NS5 region, followed bydirect sequencing and homology comparison. This methodis extremely labor intensive. In this study, we comparedan immunoblot serotyping technique (HCV SIA) to a reversehybridization line-probe assay (LiPA)for genotype classification among non-alcoholic HCVinfected patients. We then compared and contrasted theresponse in this cohort to a population of alcoholic patients with HCV infection. To validate theserotype assay, sera from 110 patients with chronic HCVinfection was utilized. Serotyping (Chiron SIA) andgenotyping by the LiPA (Line Probe Assay, Innogenetics) reverse-hybridization technique was performed.Additionally, both methods were compared tosequence-derived genotyping in 26 patients based on PCRamplification of the NS5 region. After the validationphase, sera from 105 alcoholic patients wasgenotypically classified by the serologic method. Thenonalcoholic and alcoholic groups were then comparedwith regard to serotype, demographics, and frequency ofuntypable test results. Among typable pairs, the overallconcordance rate between serotyping and LiPA-basedgenotyping was 93.75% . Patients with genotype 1 byreverse hybridization demonstrated a 95.8% concordance with serotype. Untypable samples were presentfor both techniques, but since they occurred indifferent patients, the techniques were complementary.Alcoholic patients were significantly more likely to be infected with untypable serotypes than thosewithout a pattern of alcohol abuse. These patients werealso more likely to be HCV RNA negative than sera fromtypable patients. Serotype 1 was associated with high HCV RNA titer and poor interferontreatment response among both nonalcoholic and alcoholicpatients. An immunoblot method for the evaluation ofgenotype classification was rapid and easily performed compared to sequence-based genotyping. Therewas a high degree of concordance compared toreverse-hybridization and sequence-based genotypecharacterization methods. Failure to detect HCV RNA inthe serum is associated with a higher likelihood ofclassification failure. This problem was particularlyprevalent in the alcoholic population. HCV RNA titersand treatment outcomes were strongly associated with serotype classification results, demonstratingclinical utility of this assay technique.     

Serotyping and Genotyping of Hepatitis C Virus in Belgium

Background: Given that both pathogenicity and the response to treatment are possibly associated with hepatitis C virus (HCV) serotype, it appeared sensible to establish the prevalence of the different HCV types in Belgium. Materials and Methods: The HCV serotypes were determined in 68 HCV-RNA and anti-HCV-positive samples taken from Belgian patients and compared with the results of the genotyping assay. Possible associations with age and sex were investigated. Results: Antibodies were identified in 55 (80.9%) of the 68 samples, with serotype 1 (58.8%) and serotype 3 (19.1%) showing the highest prevalence. 17 samples contained several serotypes with serotype 1 being detected in 82.4% of cases. Nine of the 11 samples undetermined by serotyping could be determined by genotyping. There was no significant difference in the distribution of HCV types with respect to gender. Compared with genotype 3 (p < 0.01) and genotypes 2 and 4 (p = 0.05), genotype 1 was detected among older patients. Conclusion: Our data showed a 96.0% correlation between the serotyping and genotyping assays. Genotypes 1 and 3 are the most prevalent types among Belgian patients. The data suggest that genotype 1 spread earlier than genotypes 2, 3 and 4. This corroborates previous European studies. Received: January 3, 2002 · Revision accepted: December 2, 2002 Liesbet De Cock (corresponding author)     

有偿供血员,丙型肝炎及肝癌患者HCV基因分型

为了解本地区ＨＣＶ基因亚型的分布情况，作者对采自６６例有偿供血员、８７例输血后丙型肝炎、１５例散发性丙型肝炎和９例原发性肝癌患者的共１７７份ＨＣＶＲＮＡ阳性的血清标本，进行了ＨＣＶ基因亚型分析。结果发现除２例输血后丙肝患者系ＨＣＶ－Ⅲ感染外，其余被检者均为ＨＣＶ－Ⅱ感染。此结果表明ＨＣＶ－Ⅱ是本地区的ＨＣＶ伏势株