Sequence analysis of the core gene of 14 hepatitis C virus genotypes.

We previously sequenced the 5' noncoding region of 44 isolates of hepatitis C virus (HCV), as well as the envelope 1 (E1) gene of 51 HCV isolates, and provided evidence for the existence of at least 6 major genetic groups consisting of at least 12 minor genotypes of HCV (i.e., genotypes I/1a, II/1b, III/2a, IV/2b, 2c, V/3a, 4a-4d, 5a, and 6a). We now report the complete nucleotide sequence of the putative core (C) gene of 52 HCV isolates that represent all of these 12 genotypes as well as two additional genotypes provisionally designated 4e and 4f that we identified in this study. The phylogenetic analysis of the C gene sequences was in agreement with that of the E1 gene sequences. A major division in the genetic distance was observed between HCV isolates of genotype 2 and those of the other genotypes in analysis of both the E1 and C genes. The C gene sequences of 9 genotypes have not been reported previously (i.e., genotypes 2c, 4a-4f, 5a, and 6a). Our analysis indicates that the C gene-based methods currently used to determine the HCV genotype, such as PCR with genotype-specific primers, should be revised in light of these data. We found that the predicted C gene was exactly 573 nt long in all 52 HCV isolates, with an N-terminal start codon and no in-frame stop codons. The nucleotide and predicted amino acid identities of the C gene sequences were in the range of 79.4-99.0% and 85.3-100%, respectively. Furthermore, we mapped universally conserved, as well as genotype-specific, nucleotide and deduced amino acid sequences of the C gene. The predicted C proteins of the different HCV genotypes shared the following features: (i) high content of proline residues, (ii) high content of arginine and lysine residues located primarily in three domains with 10 such residues invariant at positions 39-62, (iii) a cluster of 5 conserved tryptophan residues, (iv) two nuclear localization signals and a DNA-binding motif, (v) a potential phosphorylation site with a serine-proline motif, and (vi) three conserved hydrophilic domains that have been shown by others to contain immunogenic epitopes. Thus, we have extended analysis of the predicted C protein of HCV to all of the recognized genotypes, confirmed the existence of highly conserved regions of this important structural protein, and demonstrated that the genetic relatedness of HCV isolates is equivalent when analyzing the most conserved (i.e., C) and the most variable (i.e., E1) genes of the HCV genome.     

Sequence analysis of the 5'' noncoding region of hepatitis C virus.

We have determined the nucleotide sequence of the 5' noncoding (NC) region of the hepatitis C virus (HCV) genome in 44 isolates from around the world. We have identified several HCV isolates with significantly greater sequence heterogeneity than reported previously within the 5' NC region. The most distantly related isolates were only 90.1% identical. Nucleotide insertions were seen in three isolates. Analysis of the nucleotide sequence from 44 HCV isolates in this study combined with that of 37 isolates reported in the literature reveals that the 5' NC region of HCV consists of highly conserved domains interspersed with variable domains. The consensus sequence was identical to the prototype HCV sequence. Nucleotide variations were found in 45 (16%) of the 282 nucleotide positions analyzed and were primarily located in three domains of significant heterogeneity (positions -239 to -222, -167 to -118, and -100 to -72). Conversely, there were three highly conserved domains consisting of 18, 22, and 63 completely invariant nucleotides (positions -263 to -246, -199 to -178, and -65 to -3, respectively). Two nucleotide domains within the 5' NC region, conserved among all HCV isolates studied to date, shared statistically significant similarity with pestivirus 5' NC sequences, providing further evidence for a close evolutionary relationship between these two groups of viruses. Additional analysis revealed the presence of short open reading frames in all HCV isolates. Our sequence analysis of the 5' NC region of the HCV genome provides additional information about conserved elements within this region and suggests a possible functional role for the region in viral replication or gene expression. These data also have implications for selection of optimal primer sequences for the detection of HCV RNA by the PCR assay.     

Genomic structure of the human prototype strain H of hepatitis C virus: comparison with American and Japanese isolates.

Genomic RNA from the human prototype strain H of the hepatitis C virus (HCV-H) has been molecularly cloned and sequenced. The HCV-H sequence reported consists of 9416 nucleotides including the 5' and 3' untranslated regions. HCV-H shows 96% amino acid identity with the American isolate HCV-1 but only 84.9% with the Japanese isolates HCV-J and HCV-BK. In addition to the hypervariable region (region V) previously identified in the putative E2 domain, three other variable domains were identified: region V1 (putative E1), region V2 (putative E2), and region V3 (putative NS5). These regions appear rather conserved (86-100%) among the American isolates (HCV-1 and HC-J1) or among various Japanese isolates (HCV-J, HCV-BK, HCV-JH, and HC-J4) but show striking heterogeneity when the two subgroups are compared (42-87.5% amino acid difference). A structural similarity between the 5'-terminal hairpin structure of HCV and of poliovirus was observed. This study further suggests the existence of at least two genomic subtypes of HCV and confirms a distant relationship between HCV and pestiviruses.     

Genetic organization and diversity of the hepatitis C virus.

The nucleotide sequence of the RNA genome of the human hepatitis C virus (HCV) has been determined from overlapping cDNA clones. The sequence (9379 nucleotides) has a single large open reading frame that could encode a viral polyprotein precursor of 3011 amino acids. While there as little overall amino acid and nucleotide sequence homology with other viruses, the 5' HCV nucleotide sequence upstream of this large open reading frame has substantial similarity to the 5' termini of pestiviral genomes. The polyprotein also has significant sequence similarity to helicases encoded by animal pestiviruses, plant potyviruses, and human flaviviruses, and it contains sequence motifs widely conserved among viral replicases and trypsin-like proteases. A basic, presumed nucleocapsid domain is located at the N terminus upstream of a region containing numerous potential N-linked glycosylation sites. These HCV domains are located in the same relative position as observed in the pestiviruses and flaviviruses and the hydrophobic profiles of all three viral polyproteins are similar. These combined data indicate that HCV is an unusual virus that is most related to the pestiviruses. Significant genome diversity is apparent within the putative 5' structural gene region of different HCV isolates, suggesting the presence of closely related but distinct viral genotypes.     

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     

Predicted secondary structure of the hepatitis G virus and GB virus-A 5'' untranslated regions consistent with an internal ribosome entry site

We describe comparative sequence analysis of 20 isolates of the recently discovered hepatitis G virus (HGV) and propose a model of the RNA secondary structure at the 3' end of the 5' untranslated region (UTR) of this virus. A single AUG starting at nucleotide position 552, which was in-frame and continuous with the putative polyprotein, was conserved in all 20 isolates and appeared to be the most likely site for the initiation of polyprotein synthesis. This consensus AUG was 14 amino acid residues upstream of a sequence with identity to the envelope protein E1 of hepatitis C virus (HCV), but the actual function of this N-terminal peptide of HGV is still not certain. None of the isolates encoded a sequence with similarity to the nucleocapsid protein of any known virus. The RNA secondary structure of the fragment under study was determined using thermodynamic modelling and validated using the results of comparative sequence analysis. Further support for the model was gained from the prediction of significant sequence and structural homology in the RNA secondary structure of the complete 5'-UTR of GB virus-A (GBV-A). A possible mechanism for translation initiation in HGV and GBV-A was suggested by the identification of features in common with the internal ribosome entry sites (IRESs) of HCV and picornaviruses.     

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。     

Hepatitis C virus variants from Vietnam are classifiable into the seventh, eighth, and ninth major genetic groups.

Thirty-four (41%) of 83 hepatitis C virus (HCV) isolates from commercial blood donors in Vietnam were not classifiable into genotype I/1a, II/1b, III/2a, IV/2b, or V/3a; for 15 of them, the sequence was determined for 1.6 kb in the 5'-terminal region and 1.1 kb in the 3'-terminal region. Comparison of the 15 Vietnamese isolates among themselves and with reported full or partial HCV genomic sequences indicated that they were classifiable into four major groups (groups 6-9) divided into six genotypes (6a, 7a, 7b, 8a, 8b, and 9a). Vietnamese HCV isolates of genotypes 7a, 7b, 8a, 8b, and 9a were significantly different from those classified into groups 4, 5, and 6 based on divergence within partial sequences; those of genotype 6a were homologous to a Hong Kong isolate (HK2) of genotype 6a. Phylogenetic trees based on the envelope 1 (E1) gene (576 bp) of 55 isolates and a part of the nonstructural 5 (NS5) region (1093 bp) of 43 isolates revealed at least nine major groups, three of which (groups 7, 8, and 9) were identified only in Vietnamese blood donors. With a prospect that many more HCV isolates with significant sequence divergence will be reported from all over the world, the domain of the HCV genome to be compared and criteria for grouping/typing and genotyping/subtyping will have to be determined, so that they may be correlated with virological, epidemiological, and clinical characteristics.     

长春地区人与猪戊型肝炎病毒分子流行病学及部分基因序列分析

目的分析长春地区猪和人群流行的戊型肝炎病毒的系统进化关系。方法参照文献设计引物,对长春地区猪和人群流行的8株HEV(其中3株来源于人,5株来源于猪)进行RT-PCR,并将RT-PCR产物克隆到p MD18-T载体,筛选阳性重组质粒测序,测序结果采用Clustal X v.1.8进行多重比对分析,并与基因1~4型HEV代表株的核苷酸及氨基酸进行同源性比较,绘制遗传进化树。结果本研究获得的8株HEV核苷酸同源性在91.2%~99.1%,推导氨基酸同源性在97.4%~100%之间;基因1~4型内各株序列间同源性分别为:87.9%~100%,100%,85.9%~96.6%,84.8%~100%。结论研究表明获得的长春各株病毒均属基因4型,由进化关系看长春地区猪HEV与人HEV可能由同一毒株进化而来。     

Hepatitis C in human immunodeficiency virus-coinfected patients: Increased variability in the hypervariable envelope coding domain

Patients coinfected with the hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) were studied with regard to nucleotide sequence variability in the E2/NS1 first hypervariable region of the HCV genome. The nucleotide variability within individual patients was compared to patients infected only with HCV. The proportion of predicted synonymous and nonsynonymous amino acid changes, and the relationship to putative high-antigenicity sites, were evaluated in the hypervariable envelope domain. Ninety-one clones from 10 patients with HCV/HIV coinfection were sequenced, following polymerase chain reaction (PCR) amplification of the hypervariable region. The control HCV group included 53 clones from 7 patients. Sequence analysis encompassed the region coding for amino acids 384 to 414. Consensus sequences from each patient were used as the internal standard for nonsynonymous amino acid codon variability. Cumulative proportional comparison at each amino acid site revealed increased variability in HCV RNA from patients with HCV/HIV coinfection versus HCV alone (P < .05). The greatest variability was observed at amino acids 386, 397, 400, 402, 405, 407, and 414, with >l0 percent clonal variation at these sites. Jameson-Wolf plots were used to predict putative high-antigenicity domains. Nonsynonymous clonal variation resulted in alteration of putative antigenic sites within the hypervariable region. All clones had at least one high-probability site. Clones with unique predicted antigenic domains were observed more frequently in HIV/HCV coinfected patients, and, independent of viral titer, were consistent with increased sequence variability. These data suggest an accumulation of envelope variants in the HCV/HIV coinfected patients, which could be related to ineffective viral clearance, and may help explain prior reports of interferon (IFN) resistance in this patient group. (Hepatology 1996 Apr;23(4):688-94)     

Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infections.

E2/nonstructural protein 1, the putative envelope glycoprotein (gp72) of HCV, possesses an N-terminal hypervariable (E2 HV) domain from amino acids 384 to 414 of unknown significance. The high degree of amino acid sequence variation in the E2 HV domain appears to be comparable to that observed in the human immunodeficiency virus type 1 gp120 V3 domain. This observation and the observation that the HCV E2 HV domain lacks conserved secondary structure imply that, like the V3 loop of human immunodeficiency virus 1 gp120, the N-terminal E2 region may encode protective epitopes that are subject to immune selection. Antibody-epitope binding studies revealed five isolate-specific linear epitopes located in the E2 HV region. These results suggest that the E2 HV domain is a target for the human immune response and that, in addition to the three major groups of HCV, defined by nucleotide and amino acid sequence identity among HCV isolates, E2 HV-specific subgroups also exist. Analysis of the partial or complete E2 sequences of two individuals indicated that E2 HV variants can either coexist simultaneously in a single individual or that a particular variant may predominate during different episodes of disease. In the latter situation, we found one individual who developed antibodies to a subregion of the E2 HV domain (amino acids 396-407) specific to a variant that was predominant during one major episode of hepatitis but who lacked detectable antibodies to the corresponding region of a second variant that was predominant during a later episode of disease. The data suggest that the variability in the E2 HV domain may result from immune selection. The findings of this report could impact vaccine strategies and drug therapy programs designed to control and eliminate HCV.     

Mutations in the putative HCV-E2 CD81 binding regions and correlation with cell surface CD81 expression

The hepatitis C virus (HCV) envelope (E)2 protein interacts with the cellular receptor CD81 leading to modulation of B and T cell function. Recently, a higher binding affinity of subtype 1a in comparison with 1b derived E2 proteins for CD81 in vitro was described. The importance of mutations within the putative CD81 binding regions of different HCV geno-/subtypes in correlation with CD81 expression is unknown. In the present study, CD81 expression on blood lymphocytes of patients with chronic hepatitis C infected with different HCV geno-/subtypes were analysed by fluorescence activated cell sorter analyses. In addition, the putative CD81 binding regions on the E2 gene comprising the hypervariable region (HVR)2 were analysed by direct sequencing. CD81 expression on CD8(+) T-lymphocytes from patients infected with subtype 1a (n = 6) was significantly higher in comparison with subtype 1b (n = 12) and 3 (n = 5) infected patients before and during antiviral therapy (P = 0.006; P = 0.021, respectively). Sequencing of the putative CD81 binding regions in the E2 protein comprising the HVR2 (codon 474-495 and 522-552 according to the HCV-1a prototype HCV-H) showed a highly conserved motif within HVR2 for subtype 1a isolates and an overall low number of mutations within the putative CD81 binding regions, whereas numerous mutations were detected for subtype 1b isolates (12.0 vs 23.6%). HCV-3 isolates showed an intermediate number of mutations within the putative binding sites (19.2%; P = 0.022). In conclusion, the highly conserved sequence within HVR2 and putative CD81 binding sites of subtype 1a isolates previously associated with a high CD81 binding affinity in vitro is correlated with high CD81 expression on CD8(+) T-lymphocytes in vivo.     

Molecular epidemiology of dengue virus serotype 2 in the Taiwan 2002 outbreak with envelope gene and nonstructural protein 1 gene analysis

The genetic relationships among dengue virus serotype 2 (DEN-2) isolates from the Taiwan 2002 epidemic were studied by sequence analysis of the envelope (E) and nonstructural protein 1 (NS1) genes. A 0-0.4% divergence among 10 isolates revealed an epidemic strain in the outbreak. Phylogenetic study demonstrated that the 2002 Taiwan isolates were of the Cosmopolitan genotype, which is different from the Asian 1 and Asian 2 genotypes of Taiwan DEN-2 isolates from 1981 to 1998 and the American/Asian genotype of 2005 Taiwan isolates. Although grouping results from both E and NS1 gene sequence analyses were the same, the usage of the NS1 gene as a sequence analysis target has not been validated for the lower bootstrap support values of branches in the phylogenetic tree. Our result showing the same genotype changes in Taiwan and Philippines isolates suggests strain transfer of DEN-2 to nearby countries resulting in the same trend of genotype change.     

Envelope gene of the Friend spleen focus-forming virus: deletion and insertions in 3'' gp70/p15E-encoding region have resulted in unique features in the primary structure of its protein product.

A nucleotide sequence was determined for the envelope (env) gene of the polycythemia-inducing strain of the acute leukemia-inducing Friend spleen focus-forming virus (SFFV) and from this the amino acid sequence of its gene product, gp52, was deduced. All major elements of the gene were found to be related to genes of other retroviruses that code for functional glycoproteins. Although the carboxyl terminus of gp52 is encoded by sequences highly related to sequences in its putative parent, ecotropic Friend murine leukemia virus, the majority of the protein (69%), including the amino terminus, is encoded by dualtropic virus-like sequences. Nucleotide sequence comparisons suggest that the nonecotropic region may be more closely related to the 5' substitution in dualtropic mink cell focus-inducing viruses that it is to the 5' end of xenotropic virus env genes. A large deletion and two unique insertions have been located in the env gene of polycythemia-inducing SFFV and may account for some of the unusual structural characteristics, aberrant processing, and pathogenic properties of gp52. As a consequence of the deletion, amino-terminal gp70 and carboxyl-terminal p15E-encoding sequences are juxtaposed and it appears that translation from the p15E region, 3' to the deletion, continues in the standard reading frame used by other retroviruses. Insertions of six base pairs and one base pair at the very 3' end of the gp52-encoding region results in a SFFV-unique amino acid sequence and a premature termination codon.     

Sequence analysis of the hepatitis C virus (HCV) core gene suggests the core protein as an appropriate target for HCV vaccine strategies

Summary. Hepatitis C virus (HCV) is a major health problem with a prevalence of 1% in the United States population, and a significant percentage of infected patients progress to chronic liver disease and cirrhosis. Interferon therapy has demonstrated that the immune system can be modulated to alter the acute course of the disease, but long-term treatments remain elusive. Prevention of hepatitis C infection is therefore an important strategy to mitigate the impact of this disease. Initial attempts at vaccination have focused on recombinant envelope vaccines, which have shown an ability to protect against very low titre challenges of HCV in chimps. The need for vaccines capable of protecting against higher titre challenges has led to the search for alternative vaccine strategies. The most highly conserved structural protein in the HCV genome is the core protein, and vaccine strategies targeting the core protein have been proposed to increase vaccine efficacy. The variability of HCV core sequences and genotypes in the Ann Arbor patient population are not known, and the present study was undertaken to assess the theoretical feasibility of developing a HCV core vaccine by excluding promiscuous core (C) gene variability as a mechanism of vaccine failure. Results of nucleotide and deduced amino acid sequence analysis from 13 of 14 patients studied reveal a 93% nucleotide and 96.4% amino acid core sequence homology in the C gene regions studied. Genotype analysis revealed four of 14 to be type 1a and nine of 14 to be type 1b with one infection not being sufficiently characterized to determine genotype. These results demonstrate a sufficiently high degree of conservation of HCV core sequences in our patient population to permit design of a vaccine directed against core protein.     

Nucleotide sequence and mutation rate of the H strain of hepatitis C virus.

Patient H is an American patient who was infected with hepatitis C virus (HCV) in 1977. The patient became chronically infected and has remained so for the past 13 years. In this study, we compared the nucleotide and predicted amino acid sequences of the HCV genome obtained from plasma collected in 1977 with that collected in 1990. We find that the two HCV isolates differ at 123 of the 4923 (2.50%) nucleotides sequenced. We estimate that the mutation rate of the H strain of HCV is approximately 1.92 x 10(-3) base substitutions per genome site per year. The nucleotide changes were exclusively base substitutions and were unevenly distributed throughout the genome. A relatively high rate of change was observed in the region of the HCV genome that corresponds to the non-structural protein 1 gene region of flaviviruses, where 44 of 960 (4.6%) nucleotides were different. Within this region there was a 39-nucleotide domain in which 28.2% of the nucleotides differed between the two isolates. In contrast, relatively few nucleotide substitutions were observed in the 5' noncoding region, where only 2 of 276 (0.7%) nucleotides were different. Our results suggest that the mutation rate of the HCV genome is similar to that of other RNA viruses and that genes appear to be evolving at different rates within the virus genome.     

Polymorphism of the TT virus and its frequency in Polish blood donors

OBJECTIVE: To determine, in Polish blood donors, the frequency of TT virus (TTV) using different primers and the sequence diversity of TTV genotypes. MATERIALS AND METHODS: Two-hundred blood donors were studied. TTV DNA was detected by the polymerase chain reaction (PCR) using primers for the coding (ORF1) and non-coding (NC) regions. Twenty isolates were genotyped by sequencing the ORF1 fragment. RESULTS: TTV DNA was detected in 78% of donors using NC primers and in 10% using ORF1 primers. The frequency of TTV DNA detection by NC primers was observed to increase with donor age, whereas the frequency of detection by ORF primers did not differ between various age-groups. The nucleotide sequence homology of Polish TTV isolates ranged from 59 to 99%. Three genotypes (1b, 2b and 2c) were identified. CONCLUSIONS: The frequency of TTV detection depends on the primers used for the PCR. Using the NC primers the virus is detected in the majority of donors, whereas the ORF1 primers strongly underestimate the prevalence of TTV. The frequency of TTV DNA increases with age. Polish TTV isolates are highly polymorphic and are classified as 1b, 2b and 2c.