乙型肝炎病毒全基因序列的测定

测定７ 例慢性 Ｈ Ｂ Ｖ 携带者 Ｈ Ｂ Ｖ 基因组全序列,经同源性比较,确定基因型。２ 例基因型为 Ｂ 型,余均为 Ｃ 型;血清型ａｄｒ ４ 例,ａｄｗ ３ 例。各序列间 Ｘ 基因差异最大。未见 Ａ１８９６ 、 Ｔ１７６２ Ａ１７６４等重要位点的变异。结合已有的２ 株 Ｈ Ｂ Ｖ 中国流行株全基因序列,初步建立以中国流行株序列为基础的 Ｈ Ｂ Ｖ 标准序列,该标准序列与国外标准序列仅有２２ 个位点的差异     

乙型肝炎病毒全基因序列的测定

Seven cases of chronic hepatitis B virus carriers were included for sequencing of whole gene sequences of hepatitis B virus (HBV). The serotype of 4 strains of HBV were adr, and 3 strains were adw. Two strains were classified to genotype B, and the other 5 strains to genotype C. No significant mutations, such as A¹⁸⁹⁶, T¹⁷⁶²A¹⁷⁶⁴ were present. With other reported 2 complete sequences of HBV strains prevailing in mainland China, 7 strains of HBV whole sequences of genotype C from mainland China were analyzed to produce the consensus sequence of HBV of China. Comparing of the consensus sequence with that from Genbank, there were only 22 sites with different nucleotides.     

乙肝病毒表面抗原和抗体双阳性者中病毒S区基因序列分析

为了解乙肝病毒 (HBV)表面抗原和抗体双阳性患者中病毒的基因型及其HVBS区是否有变异。用放射免疫试剂检测HBsAg阳性样品中的抗 HBs抗体 ,用聚合酶链反应法检测双阳性样品中的HBVDNA ,然后对阳性样品进行克隆和基因序列分析 ,并将所得序列与HBV不同基因型的代表株进行比较分析。结果显示 389例HBsAg阳性样品中有 10例为抗HBs抗体阳性 ;该 10例双阳性样品中有 5例为HBVDNA阳性 ;序列分析显示该 5株HBV均为B基因型 ,其中 4株为adw亚型 ,1株为adr亚型 ;其中有 2株在S区的“a”决定簇的氨基酸发生了变异     

Point mutation in the S gene of hepatitis B virus for a d/y or w/r subtypic change in two blood donors carrying a surface antigen of compound subtype adyr or adwr

Genomes of hepatitis B virus (HBV) were cloned from the plasma of a blood donor who carried subviral particles of three distinct subtypes in the following proportions: adr, 25%; ayr, 63%; and adyr, 12%. HBV DNA clones were classified into two groups based on a difference at only one nucleotide in the S gene. Two clones had A as nucleotide 365 that formed part of the codon for lysine as amino acid residue 122 and produced a surface antigen of subtype adr in transfected NIH 3T3 cells. The remaining four clones had G determining the codon for arginine and produced a surface antigen of subtype ayr in transfected cells. Similarly, HBV genomes were cloned from the plasma of an individual who carried subviral particles of subtypes adr (71%) and adwr (29%). Two clones had T and A as nucleotides 476 and 479, respectively. The other seven clones had C and G as the respective nucleotides. Based on a comparison with previously reported HBV genomes of various subtypes, the mutation of nucleotide 479, forming part of the codon for lysine or arginine as amino acid residue 160, was deduced to determine the w or r subtype, respectively. When NIH 3T3 cells were transfected separately with the genome of subtype adw or adr, derived from plasma containing a surface antigen of subtype adwr, and then cocultured, they produced subviral particles of either subtype adw or adr. When cells were transfected with the genomes of subtypes adw and adr simultaneously, however, subviral particles were produced that possessed w and r determinants on the selfsame particles. These results attributed the d/y or w/r subtypic change to a point mutation in the S gene and favored coinfection of hepatocytes with an HBV genome and its mutant as the mechanism of compound subtypes.     

Genotype and subtype analyses of hepatitis B virus (HBV) and possible co-infection of HBV and hepatitis C virus (HCV) or hepatitis D virus (HDV) in blood donors, patients with chronic liver disease and patients on hemodialysis in Surabaya, Indonesia

Four subtypes (adw, adr, ayw, and ayr ) and eight genotypes (A to H) of the hepatitis B virus (HBV) have been identified. They appear to be associated with particular geographic distribution, ethnicity, and possibly clinical outcomes. In this study, hepatitis B surface antigen (HBsAg) subtyping and HBV genotyping were carried out on sera obtained from HBsAg-positive HBV carriers, including healthy blood donors; patients with acute hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; and patients on hemodialysis all located in Surabaya, Indonesia. We report here that all HBV isolates tested in Surabaya belonged to genotype B, with more than 90% of them being classified into subtype adw. Our results also revealed that prevalence of hepatitis C virus (HCV) co-infection among HBV carriers in Surabaya was approximately 10% for healthy blood donors and patients with chronic liver disease, and approximately 60% for patients on maintenance hemodialysis. Interestingly, HBsAg titers were lower in HBV carriers with HCV co-infection than in those without HCV co-infection. We also found that prevalence of hepatitis D virus (HDV) co-infection was < 0.5% among HBV carriers in Surabaya.     

The complete nucleotide sequences of the cloned hepatitis B virus DNA; subtype adr and adw

The complete nucleotide sequences of two different subtypes (adr and adw) of hepatitis B virus (HBV) DNA cloned in E. coli were determined. The sequence of the viral genome of the adr clone was 3188 nucleotides long, and that of the adw clone was 3200 nucleotides long. The adr and adw clones differed from the reported cloned ayw HBV DNA (3182 nucleotides long) in 11.2% and 10.0% of nucleotides, respectively. Heterogeneity of the HBV genome in the clones with the same subtype was observed.     

Genotype-specific genomic markers associated with primary hepatomas, based on complete genomic sequencing of hepatitis B virus

We aimed to identify genomic markers in hepatitis B virus (HBV) that are associated with hepatocellular carcinoma (HCC) development by comparing the complete genomic sequences of HBVs among patients with HCC and those without. One hundred patients with HBV-related HCC and 100 age-matched HBV-infected non-HCC patients (controls) were studied. HBV DNA from serum was directly sequenced to study the whole viral genome. Data mining and rule learning were employed to develop diagnostic algorithms. An independent cohort of 132 cases (43 HCC and 89 non-HCC) was used to validate the accuracy of these algorithms. Among the 100 cases of HCC, 37 had genotype B (all subgenotype Ba) and 63 had genotype C (16 subgenotype Ce and 47 subgenotype Cs) HBV infection. In the control group, 51 had genotype B and 49 had genotype C (10 subgenotype Ce and 39 subgenotype Cs) HBV infection. Genomic algorithms associated with HCC were derived based on genotype/subgenotype-specific mutations. In genotype B HBV, mutations C1165T, A1762T and G1764A, T2712C/A/G, and A/T2525C were associated with HCC. HCC-related mutations T31C, T53C, and A1499G were associated with HBV subgenotype Ce, and mutations G1613A, G1899A, T2170C/G, and T2441C were associated with HBV subgenotype Cs. Amino acid changes caused by these mutations were found in the X, envelope, and precore/core regions in association with HBV genotype B, Ce, and Cs, respectively. In conclusion, infections with different genotypes of HBV (B, Ce, and Cs) carry different genomic markers for HCC at different parts of the HBV genome. Different HBV genotypes may have different virologic mechanisms of hepatocarcinogenesis.     

The first large-scale nucleic acid amplification testing (NAT) of donated blood using multiplex reagent for simultaneous detection of HBV, HCV, and HIV-1 and significance of NAT for HBV.

The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) of voluntarily donated blood after serological pre-screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. From February 1, 2000 to April 30, 2001, specimens from 6,805,010 units of serologically negative donation were screened in minipools of 50 samples within 24 hr after blood donation by NAT using multiplex HBV/HCV/HIV-1 reagent for blood transfusion including short shelf-life platelets. Among them, 112 HBV DNA-positives, 25 HCV RNA positives and 4 HIV-1 RNA positives were screened out and we could prevent transfusion of these NAT positive units. Subtypes/genotypes of HBV DNA, adr/C, adw/A, adw/B, adw/C, ayr/C and ayw/D were found and adr/C was predominant. A total of 61.6 % of them (69/112) were negative by overnight EIA. Sixth three of HBV NAT-positive samples carried virus loads less than 10(4) copies/mL and 92.1 % of them (58/63) were negative by overnight EIA. The virus growth curves of HBV in 6 cases obtained by retrospective and prospective follow-up study showed exponential straight lines in the early stage of serological window periods and the log times of HBV growth (10 fold increase) in serological window period were between 4.6 and 7.6 days. NAT screening with highly sensitive reagents in pool of specimens is useful to exclude blood units with low level of HBV and HBV mutants from blood transfusion.     

利用基因芯片分析拉米夫定治疗过程中HBV DNA的基因变异

依据乙型肝炎病毒(Hepatitis B virus;HBV)聚合酶基因序列研制HBV基因芯片,此芯片可分析HBV的7个基因型、4种血清型和HBV聚合酶基因rtV173、rtL180、rtM204和rtV207位点的突变。利用此芯片对A、B两组共计45例拉米夫定治疗12个月的患者进行服药前和服药后3、6、9、12个月的动态检测,其中C基因型39例,且血清型均为adr;B基因型6例,其血清型均为adw。在完成全程检测的38例患者中,17例ALT升高的A组出现1例拉米夫定耐药变异株,而21例ALT正常的B组出现4例变异株,且所有变异株均为rtM204 V／rtL180M,其中2例野生株和变异株共存。rtM204V变异最早在服药6个月时出现,随后出现rtL180M变异。10份PCR产物测序分析表明,芯片检测结果与测序结果基本一致,仅在rtL173位点出现1例差异。进一步分析HBV DNA变异与HBV DNA含量、ALT水平和HBeAg血清转换率的相关性,初步结果表明变异株的出现与治疗过程中的DNA反弹呈正相关,而与起始HBVDNA水平、ALT值无关联。HBV基因芯片可初步用于HBV DNA检测,可能是临床追踪评价抗病毒治疗效果的较好方法之一。     

Genetic Diversity of the Hepatitis B Virus Strains in Cuba: Absence of West-African Genotypes despite the Transatlantic Slave Trade

Cuba is an HBsAg low-prevalence country with a high coverage of anti-hepatitis B vaccine. Its population is essentially the result of the population mix of Spanish descendants and former African slaves. Information about genetic characteristics of hepatitis B virus (HBV) strains circulating in the country is scarce. The HBV genotypes/subgenotypes, serotypes, mixed infections, and S gene mutations of 172 Cuban HBsAg and HBV-DNA positive patients were determined by direct sequencing and phylogenetic analysis. Phylogenetic analysis of HBV S gene sequences showed a predominance of genotype A (92.4%), subgenotype A2 (84.9%) and A1 (7.6%). Genotype D (7.0%) and subgenotype C1 (0.6%) were also detected but typical (sub)genotypes of contemporary West-Africa (E, A3) were conspicuously absent. All genotype A, D, and C strains exhibited sequence characteristics of the adw2, ayw2, and adrq serotypes, respectively. Thirty-three (19.1%) patients showed single, double, or multiple point mutations inside the Major Hydrophilic domain associated with vaccine escape; eighteen (10.5%) patients had mutations in the T-cell epitope (amino acids 28-51), and there were another 111 point mutations downstream of the S gene. One patient had an HBV A1/A2 mixed infection. This first genetic study of Cuban HBV viruses revealed only strains that were interspersed with strains from particularly Europe, America, and Asia. The absence of genotype E supports previous hypotheses about an only recent introduction of this genotype into the general population in Africa. The presence of well-known vaccine escape (3.5%) and viral resistance mutants (2.9%) warrants strain surveillance to guide vaccination and treatment strategies.     

Research on Hepatitis B virus Genotypes and Subgenotypes among Bai Nationality in Dali, Yunnan Province

To investigate the distribution of hepatitis B virus （HBV） genotypes and subgenotypes among the Bai nationality in Dali, a total of 100 serum samples from patients with chronic HBV-infection were collected for the detection of HBV genotypes and subgenotypes by genotype-specific primers and restriction fragment length polymorphism （RLFP）, respectively. Among the 100 samples, the proportions of genotype B, C and mixed genotype （B＋C） were 41%, 25% and 34%, respectively. All the genotype B strains belonged to subgenotype Ba In genotype C, 84% were Subgenotype Cs and 12% were subgenotype Ce. The distribution of genotypes B, C and B＋C showed no significant difference between male and female patients （P=0.182） and among the age groups of patients （P=0.812）. The rates of HBeAg/HBeAg positivity were no significantly different among genotypes B, genotype C and mixed genotype （B＋C） （P=0.077/P=0.663）. In Dali, genotypes B, B＋C and C existed among Bai nationality with chronic HBV-infection, and genotype B was the major genotype. Subgenotypes Ba and Cs were the predominant strains in patients with HBV genotype B/C infection. The most prominent characteristic was the higher prevalent rate of mixed genotype （B＋C） in patients.     

Research on hepatitis B virus genotypes and subgenotypes among Bai nationality in Dali, Yunnan Province

To investigate the distribution of hepatitis B virus (HBV) genotypes and subgenotypes among the Bai nationality in Dali, a total of 100 serum samples from patients with chronic HBV-infection were collected for the detection of HBV genotypes and subgenotypes by genotype-specific primers and restriction fragment length polymorphism (RLFP), respectively. Among the 100 samples, the proportions of genotype B, C and mixed genotype (B C) were 41%, 25% and 34%, respectively. All the genotype B strains belonged to subgenotype Ba. In genotype C, 84% were Subgenotype Cs and 12% were subgenotype Ce. The distribution of genotypes B, C and B C showed no significant difference between male and female patients (P=0.182) and among the age groups of patients (P=0.812). The rates of HBeAg/HBeAg positivity were no significantly different among genotypes B, genotype C and mixed genotype (B C) (P=0.077/P=0.663). In Dali, genotypes B, B C and C existed among Bai nationality with chronic HBV-infection, and genotype B was the major genotype. Subgenotypes Ba and Cs were the predominant strains in patients with HBV genotype B/C infection. The most prominent characteristic was the higher prevalent rate of mixed genotype (B C) in patients.     

Comparison and significance of specific and non-specific cellular immunity in patients with chronic hepatitis B caused by infection with genotypes B or C of hepatitis B virus

The present study was designed to investigate possible relationships between the genotypes of hepa-titis B virus (HBV) and the HBV-specific cytotoxic T lymphocyte (CTL) responses. HBV genotypes, HBV specific CTL HBV DNA and other markers of HBV infection were determined in 138 patients with chronic hepatitis B. The results showed that the patients infected with genotype C (n=62) had a significantly lower HBV-specific CTL response than those who were infected with HBV genotype B (P<0.01). HBV DNA titer was higher in patients infected with HBV genotype C than in those infected with HBV geno-type B (P<0.01). Both alanine aminotransferase (ALT) and total bilirubin (TBIL) were higher in HBV genotype C infected patients than in those infected with genotype B (P<0.01 and <0.05, respectively). These results suggest that compared with CHB patients infected with HBV genotype B, the higher HBV DNA level and more severe liver damages in the patients infected with genotype C of HBV may be as-sociated with genotype C of the virus.     

Hepatitis B virus of genotype B with or without recombination with genotype C over the precore region plus the core gene

The entire nucleotide sequences of 70 hepatitis B virus (HBV) isolates of genotype B (HBV/B), including 38 newly determined and 32 retrieved from the international DNA database (DDBJ/EMBL/GenBank), were compared phylogenetically. Two subgroups of HBV/B were identified based on sequence divergence in the precore region plus the core gene, one with the recombination with genotype C and the other without it. The analysis over the entire genome of HBV/B by the SimPlot program located the recombination with genotype C in the precore region plus the core gene spanning nucleotide positions from 1740 to 1838 to 2443 to 2485. Within this genomic area, HBV/B strains with the recombination had higher nucleotide and amino acid homology to genotype C than those without the recombination (96.9 versus 91.1% in nucleotides and 97.0 versus 92.9% in amino acids). There were 29 HBV/B strains without the recombination, and they were all recovered from carriers in Japan. The remaining 41 HBV/B isolates having the recombination with genotype C were from carriers in China (12 strains), Hong Kong (3 strains), Indonesia (4 strains), Japan (3 strains), Taiwan (4 strains), Thailand (3 strains), and Vietnam (12 strains). Due to the frequency of the distribution of HBV/B without the recombination (29 of 32 isolates, or 91%) and the fact that it was exclusive to Japan, it was provisionally classified into the Bj (j standing for Japan) subgroup, and HBV/B with the recombination was classified into the Ba (a for Asia) subgroup. Virological differences between HBV/Bj and HBV/Ba may be reflected in the severity of clinical disease in the patients infected with HBV of genotype B, which seems to be under strong geographic influences in Asia.     

内江地区慢性乙型肝炎患者的基因型及拉米夫定联合阿德福韦酯抗病毒治疗效果

目的探讨内江地区慢性乙型肝炎患者的基因型及拉米夫定联合阿德福韦酯抗病毒的治疗效果。方法将201例慢性乙型肝炎患者进行HBV基因型的测定,其中120例HBeAg(+)慢性乙型肝炎患者随机分为三组:A组、B组和C组,每组40例。A组给予拉米夫定(LAM)治疗;B组给予恩替卡韦(ETV)治疗;C组给予LAM联合阿德福韦酯(ADV)治疗,比较治疗情况。结果 201例慢性乙型肝炎患者中B型119例(59.2%),C型68例(33.8%),B/C混合型10例(5.0%),未知型4例(2.0%),各分型之间的性别、年龄差异无统计学意义(P0.05)。C型感染者HBeAg阳性率为86.8%,显著高于B型的51.3%(P0.05)。HBeAg(+)的3组患者治疗12、24和48周时,B组和C组患者的ALT复常率、HBV DNA阴转率及48周时的HBeAg血清转换率均显著高于A组(P0.05);C组患者与B组比较差异无统计学意义(P0.05)。治疗期间均未见不良反应发生。C组患者(包括B型19例、C型21例)中B型的HBV DNA阴转率及HBeAg血清转换率显著高于C型(P0.05)。结论地处西南方的内江地区慢性乙型肝炎患者主要以B型为主,C型次之,B型和C型共占93.0%,其他型别仅占较少部分。ETV方案或LAM联合ADV方案治疗HBeAg(+)慢性乙肝疗效优于LAM治疗。初始LAM联合ADV治疗基因B型HBeAg(+)慢性乙型肝炎疗效优于C型。     

Characterization of restriction endonuclease maps of hepatitis B viral DNAs

The HBV DNA isolated from Dane particles of 9 patients' plasma was cloned into the EcoRI or BamHI site of the pUC8 plasmids. Two plasmids with full length HBV DNA and four plasmids containing the HBV surface antigen gene were obtained. Based on our cloned HBV DNA and a comparison with 7 complete sequences and 5 restriction endonuclease patterns of HBV DNA published by others, we can recognize common restriction sites shared by different subtypes (adw, adr, ayw, and adyw): (1) a HincII site in the S gene, (2) a BamHI site in the X region, and (3) two BglII sites in the C gene. In addition adw has specific sites for HincII, BamHI, and PstI in the pre-S region. A unique XhoI site is present in the pre-S region in all subtypes except for adw.     

Serotypes and mating types of clinical strains ofCryptococcus neoformans isolated in Taiwan

Twenty-one strains ofCryptococcus neoformans isolated from patients in Taiwan were characterized for serotypes and mating types. Slide agglutination test was performed with 8 factor-specific sera (Iatron Company, Japan) to determine the serotypes. Wheat bran agar (WBA) and malt extract agar (MEA, Wickerham) media were used for the mating tests. Twenty of the isolates were of serotype A, and one was serotype B. Except for 2 strains of serotype A, all of the serotype A strains mated withFilobasidiella neoformans var.neoformans, mating type a. The only serotype B strain mated withF. neoformans var.bacillispora mating type a in MEA medium. These data revealed the low prevalence (1/21; 4.8%) ofC. neoformans var.gattii in Taiwan, a subtropically located island.     

2010年舟山某医院拉米夫定无良好应答乙肝患者耐药监测分析

目的观察2010年舟山某医院拉米夫定单药治疗无良好应答的乙肝患者其基因型和P区耐药位点突变的关系。方法随机选取舟山某医院就诊的124例拉米夫定单药治疗无良好应答的本地乙肝患者,男64例,女60例,平均年龄(46.92±15.16)岁,用实时荧光定量PCR检测其基因型和HBV-DNA,并用毛细管电泳测序技术对P区进行突变位点检测。结果 124例患者35例(28.23%)B型和89例(71.77%)C基因型。C型HBV-DNA为(5.83±0.91)log10 copies/mL,高于B型的(5.07±1.13)log10 copies/mL,差异具有统计学意义(t=3.55,P<0.01)。拉米夫定在B基因型中的耐药率为45.71%(16/35),低于C基因型的65.17%(58/89),差异具有统计学意义(2χ=3.951,P<0.05)。拉米夫定耐药株在B基因型中rtM204 I/V/S位点突变率为68.75%(11/16),高于C基因型的37.93%(22/58),差异具有统计学意义(2χ=4.821,P<0.05)。结论对于本地区拉米夫定治疗无良好应答的C基因乙肝患者,应加强拉米夫定的耐药位点筛查,而拉米夫定治疗无良好应答的B基因乙肝患者应加强rtM204 I/V/S位点检测。     

Plant growth promotion and Penicillium citrinum

Background

Lamivudine is an oral nucleoside analogue widely used for the treatment of chronic hepatitis B. The main limitation of lamivudine use is the selection of resistant mutations that increases with time of utilization. Hepatitis B virus (HBV) isolates have been classified into eight genotypes (A to H) with distinct geographical distributions. HBV genotypes may also influence pathogenic properties and therapeutic features. Here, we analyzed the HBV genotype distribution and the nature and frequency of lamivudine resistant mutations among 36 patients submitted to lamivudine treatment for 12 to 84 months.

Results

Half of the patients were homosexual men. Only 4/36 (11%) patients were HBV DNA negative. As expected for a Brazilian group, genotypes A (24/32 positive individuals, 75%), D (3/32, 9.3%) and F (1/32, 3%) were present. One sample was from genotype C, which is a genotype rarely found in Brazil. Three samples were from genotype G, which had not been previously detected in Brazil. Lamivudine resistance mutations were identified in 20/32 (62%) HBV DNA positive samples. Mean HBV loads of patients with and without lamivudine resistance mutations were not very different (2.7 × 10⁷ and 6.9 × 10⁷ copies/mL, respectively). Fifteen patients showed the L180M/M204V lamivudine resistant double mutation. The triple mutant rt173V/180M/204V, which acts as a vaccine escape mutant, was found in two individuals. The three isolates of genotype G were entirely sequenced. All three showed the double mutation L180M/M204V and displayed a large genetic divergence when compared with other full-length genotype G isolates.

Conclusion

A high (55%) proportion of patients submitted to long term lamivudine therapy displayed resistant mutations, with elevated viral load. The potential of transmission of such HBV mutants should be monitored. The identification of genotypes C and G, rarely detected in South America, seems to indicate a genotype distribution different to that observed in non treated patients. Disparities in routes of transmission (genotype G seems to be linked to homosexual behavior) and in pathogenic properties (genotype C is very aggressive) among HBV genotypes may explain the presence of rare genotypes in the present work.     

Characteristics of pre-S2 region of hepatitis B virus

The nucleotide sequence of our cloned HBV DNA (subtype adw) has been determined. When the 165-nucleotide sequence of the pre-S2 region was compared with 7 other published sequences (subtypes adw, adr, ayw, and adyw), we found 38 nucleotide substitutions among different subtypes and 4 (adr) or 6 (adw and ayw) substitutions within the same subtype. Analysis of the predicted amino acid sequence from the known nucleotide sequences indicates that: there are 20 amino acid substitutions, the longest conserved amino acid sequence is located between amino acids 23 to 34, and the 54th amino acid is identical within the same subtype but varies in different subtypes.