Marked sequence diversity in the putative envelope proteins of hepatitis C viruses.

The nucleotide sequences of cDNAs (414 base pairs) encoding parts of putative envelope proteins (gp35 and gp70) of 40 isolates of hepatitis C virus (HCV-J) derived from 30 independent plasma or liver specimens from Japanese patients (13 with chronic hepatitis, 14 with hepatocellular carcinoma and 3 hemophiliacs who had received imported clotting factors), were analyzed using the polymerase chain reaction. Approximately 29-38% of the nucleotide sequences of the HCV-J isolates examined differed from those of isolates from the United States (HCV-US). Furthermore, 12-24% and 8-17% sequence diversities were found within the isolates of HCV-J and HCV-US, respectively. The diversities of the amino acid sequences were the same or greater than those of the nucleotide sequences. We confirmed that two hypervariable regions (HVR1 and HVR2) were present in this amplified region, as described in our previous report (Hijikata et al., 1991a) and we found that the HVR1 regions of HCV-J and HCV-US were 27 and 21 amino acids in length, respectively, and began from the N-terminal amino acid of gp70. HVR2 was found in HCV-J, but not in HCV-US isolates, in which the corresponding region of the genome was conserved. During the analysis, plural HCV genomes were found in 6 of 30 specimens. These plural HCV genomes in a single specimen were concluded to be derived from the same HCV ancestor, because of their relative low sequence diversities (about 10% in their nucleotide sequences).(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic characterization of hypervariable region 1 in patients chronically infected with hepatitis C virus genotype 2.

The hypervariable region 1 (HVR1) has been most reliably identified in the genome of HCV genotype 1 isolates and thought to possibly play a role in immune evasion and development of chronic infection. There are few studies, however, of other HCV genotypes to determine if they also have such a hypervariable region present, and it is unclear whether or not there is any genotype-dependent difference in the genetic characteristics of HVR1. We determined the nucleotide sequence of 5' end of E2/NS1 region of the HCV genome spanning HVR1 of multiple genotype 1 and 2 HCV isolates and carried out a detailed genetic analysis. Similarity plots identified two hypervariable regions within the genotype 2 sequences, a larger one corresponding to HVR1 as well as a smaller 27-nucleotide region of hypervariability. The synonymous substitutions per synonymous site (ds) was greater than nonsynonymous substitutions per nonsynonymous site (dn) within genotype 1 group whereas dn and ds were similar in the genotype 2 group. Analysis of amino acid sequences revealed several conserved sites across genotype 1 and 2 (amino acid numbers 2,6, 20 and 26) and overall similar hydropathic profiles were found within two genotypes. Still, despite the hypervariability, the HVR1 showed a genotype-specific phylogenetic clustering. Thus, HVR1 appears to be conserved between genotypes in keeping with it having an important survival function. Genotype 2 appears to have a greater rate of nonsynonymous substitutions within HVR1, suggesting a greater positive evolutionary pressure.     

不同基因型HCV膜区（E2）基因克隆及亲、疏水性分析

目的 研究丙型肝炎病毒（HCV）不同基因型膜区序列变异及其变异规律。方法 克隆不同基因型HCV的膜区基因,序列测定,核苷酸和氨基酸序列比较和分析。结果 不同基因型膜区基因相似性不同,从同一血清样本获得的不同克隆间,其同源性核苷酸大于99．2％,氨基酸大于99．9％。相同亚型核苷酸和氨基酸同源性大于80％。同型基因核苷酸和氨基酸同源性分别为（63．9－75．0）％和（64．9－72．8）％。异型基因同源性核苷酸为（59．4－67．0）％,氨基酸为（56．5－66．3）％。氨基酸序列变化在某些部位有一定保守性。不同基因HCV高变区（HVR）的亲水性和疏水性变化小于其下游3′端区域。结论 HCV基因变化可能有一定规律性。     

Superinfection by homotypic virus in hepatitis C virus carriers: Studies on patients with post-transfusion hepatitis

Although heterotypic superinfection and mixed infections of hepatitis C virus (HCV) may be possible for hepatitis flares in chronic hepatitis C, the possibility of homotypic HCV superinfection in HCV carriers with post-transfusion hepatitis has not been explored. Six HCV carriers with post-transfusion non-A, non-B hepatitis found in a prospective study of post-transfusion hepatitis were included. Serum samples before transfusion and during hepatitis were selected to determine genotypes of HCV and nucleotide sequences of the hypervariable region (HVR). The genotypes identified before and after transfusion were concordant in all. There were four with type 1b and one each with type 2a and type 2b. Amplified nucleotide sequences of the HVR before transfusion and during hepatitis were compared in four patients, and a, >95% homology was observed in three, suggesting persistence of original viruses. In contrast, only a 51% homogeneity was seen in a given patient, suggesting a homotypic HCV superinfection. Phylogenotic tree analysis validated further these findings. This study implies that HCV carriers can be reinfected by homotypic HCV, and this may contribute to hepatitis flares in chronic hepatitis C. These findings also confirm a weak or inadequate protective immunity in HCV infection and justify protection from reinfection of HCV of patients with chronic hepatitis C. © 1996 Wiley-Liss, Inc.     

Molecular characterization of hepatitis C virus genotype 2a from the entire sequences of four isolates

Genotype 2a hepatitis C virus (HCV) has different characteristics from genotype 1b, such as responsiveness to interferon therapy. Such type-specific characteristics appear to be due to differences in the HCV genome sequence. The complete sequences of genotype 2a HCV genome isolated from four patients with chronic hepatitis C were determined, and nucleotide and deduced amino acid sequences were compared within genotype 2a, as well as between genotype 2a and 1b. Whereas the amino acid sequence similarity of the core region was highest within genotype 1b, the NS3 and NS4B regions of exhibited greater similarity than the core region in genotype 2a. The serine protease and helicase motifs in the NS3 region were well conserved in genotype 2a to the same degree as in genotype 1b. However, the putative secondary structure of 2a isolates was significantly different from that of the 1b isolates. Analysis of amino acid similarity between genotypes 2a and 1b revealed the lowest degree of similarity in the E1 region, followed by the NS2 and NS5A region. Sequences of genotype 2a in the interferon-sensitivity determining region (ISDR) located in the NS5A region had a deletion of four amino acids compared with that of genotype 1b. When the ISDR of the genotype 2a was aligned for maximal similarity, it exhibited similarity of only 52.5-55.0% when compared with that of HCV-J, which belongs to genotype 1b. These findings for the entire sequences of genotype 2a isolates will contribute to virological studies of HCV.     

Antibody responses against B-cell epitopes of the hypervariable region 1 of hepatitis C virus in self-limiting and chronic human hepatitis C followed-up using consensus peptides.

A rare collection of serum samples from patients with hepatitis C virus (HCV) infection followed up from the onset of clinical symptoms was acquired. RNA corresponding to the hypervariable region 1 (HVR1) of E2 protein of HCV isolated from nine patients was reverse-transcribed, amplified, sequenced, and HVR1 amino acid sequences were deduced. These sequences and a selection of HVR1 amino acid sequences of matching HCV genotypes from protein and translated DNA sequence databanks were used to create the HVR1 amino acid consensus. The degenerated peptides mimicking N- and C-termini of the consensus were synthesized. Most (76%) of 17 patients followed up for the period from 1 week to a minimum of 7 months from the onset of acute symptoms developed antibodies reacting with peptides representing N- and/or C- termini of HVR1. Antibody recognition of the consensus HVR1 peptides indicates that the variability of HVR1 sequence on the protein level is limited with certain conserved structure(s) being untouched. A tendency was observed for a slower development of anti-HVR1 antibody response in patients developing chronic HCV, as compared to those with self-limiting HCV infection.     

Genotype- and Subtype-Independent Full-Genome Sequencing Assay for Hepatitis C Virus

Hepatitis C virus (HCV) exhibits a high genetic diversity and is classified into 6 genotypes, which are further divided into 66 subtypes. Current sequencing strategies require prior knowledge of the HCV genotype and subtype for efficient amplification, making it difficult to sequence samples with a rare or unknown genotype and/or subtype. Here, we describe a subtype-independent full-genome sequencing assay based on a random amplification strategy coupled with next-generation sequencing. HCV genomes from 17 patient samples with both common subtypes (1a, 1b, 2a, 2b, and 3a) and rare subtypes (2c, 2j, 3i, 4a, 4d, 5a, 6a, 6e, and 6j) were successfully sequenced. On average, 3.7 million reads were generated per sample, with 15% showing HCV specificity. The assembled consensus sequences covered 99.3% to 100% of the HCV coding region, and the average coverage was 6,070 reads/position. The accuracy of the generated consensus sequence was estimated to be >99% based on results from in vitro HCV replicon amplification, with the same extrapolated amount of input RNA molecules as that for the patient samples. Taken together, the HCV genomes from 17 patient samples were successfully sequenced, including samples with subtypes that have limited sequence information. This method has the potential to sequence any HCV patient sample, independent of genotype or subtype. It may be especially useful in confounding cases, like those with rare subtypes, intergenotypic recombination, or multiple genotype infections, and may allow greater insight into HCV evolution, its genetic diversity, and drug resistance development.     

Genotyping hepatitis C virus by heteroduplex mobility analysis using temperature gradient capillary electrophoresis

The genotype of the infecting hepatitis C virus (HCV) helps determine the patient's prognosis and the duration of treatment. Heteroduplex mobility analysis (HMA) is a rapid, inexpensive method for genotyping of HCV that does not require sequencing. We developed an HMA that uses temperature gradient capillary electrophoresis (TGCE) to differentiate HCV genotypes. A 56-bp region of the HCV 5' untranslated region (UTR) that was conserved within a genotype yet whose sequence differed between genotypes was amplified for HMA-TGCE analysis. HCV amplicons of types 1, 2a, 2b, 3a, 4, and 6a were hybridized in pairs and analyzed by TGCE. Amplicons hybridized to the same subtype yielded one homoduplex peak, while hybridization of different subtypes resulted in heteroduplexes and generated multiple TGCE peaks. Heteroduplexes contain thermodynamically unstable nucleotide mismatches that reduced their TGCE mobilities compared to those of homoduplexes. Three HCV subtypes (subtypes 1a, 3a, and 4) generated unique peak patterns when they were combined with each genotype analyzed and were chosen as the reference genotypes. A blinded study with 200 HCV-infected samples was 97% accurate compared to genotyping by 5' UTR sequence analysis. The majority of discordant results were unexpected sequence variants; however, five of nine sequence variants were correctly genotyped. The assay also detected and correctly genotyped mixed HCV infections. Compared to conventional HMA, TGCE improves the resolution, with better separation of heteroduplexes and homoduplexes. All common HCV genotypes can be detected and differentiated by this HMA-TGCE assay.     

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.     

Use of sequence analysis of the NS5B region for routine genotyping of hepatitis C virus with reference to C/E1 and 5' untranslated region sequences

Nucleotide sequence analysis of the NS5B region was performed to identify genotypes of 8,479 hepatitis C virus (HCV) RNA-positive patient samples collected in the Canadian province of Quebec. Genotypes could be determined for 97.3% of patients. Genotypes 1 to 6 were found in 59.4, 9.0, 25.7, 3.6, 0.6, and 1.8% of patients, respectively. Two isolates did not classify within the six genotypes. The subtype 1 distribution was 76.7% 1a, 22.6% 1b, and 0.7% others, while the subtype 2 distribution was 31.8% 2a, 47.6% 2b, 10.9% 2c, 4.1% 2i, and 5.6% others. Subtype 3a accounted for 99.1% of genotype 3 strains, while all genotype 5 samples were of subtype 5a. The subtype 4 distribution was 39.2% 4a, 15.4% 4k, 11.6% 4d, 10.2% 4r, and 23.6% others. The subtype 6 distribution was 40.4% 6e, 20.5% 6a, and 39.1% others. The 5' untranslated region (5'UTR) sequences of subtype 6e were indistinguishable from those of genotype 1. All samples that did not classify within the established subtypes were also sequenced in C/E1 and 5'UTR. C/E1 phylogenetic reconstructions were analogous to those of NS5B. The sequences identified in this study allowed the provisional assignments of subtypes 1j, 1k, 2m, 2r, 3i, 4q, 6q, 6r, and 6s. Sixty-four (0.8%) isolates classifying within genotypes 1 to 6 could not be assigned to one of the recognized subtypes. Our results show that genotyping of HCV by nucleotide sequence analysis of NS5B is efficient, allows the accurate discrimination of subtypes, and is an effective tool for studying the molecular epidemiology of HCV.     

Greater diversity of hepatitis C virus genotypes found in Hong Kong than in mainland China.

A hepatitis C virus (HCV) genotyping PCR assay based on type-specific primers was expanded to include genotype 6a as well as genotypes 1a, 1b, 2a, and 3a. The nucleotide sequences of a 194-bp fragment in the center of the HCV core gene showed that the homologies between genotype 6a and genotypes 1a, 1b, 2a, 2b, 3a, and 5 were 81.2, 82.1, 73.8, 77.3, 81.4, and 78.9%, respectively. A high degree of homology (99.6%) was seen in the amplified core region among eight clinically unrelated genotype 6a isolates. Although the Hong Kong Chinese patients had predominantly genotype 1b (70%), it was noteworthy that genotype 6a was the second most common genotype (14%). Four other HCV genotypes--1a, 1b, 2a, and 2b--were also present. In contrast, HCV infection by mainland China was confined to genotypes 1b and 2a. Thus, we found a greater diversity of HCV genotypes in Hong Kong than in mainland China.     

Nested restriction site-specific PCR to detect and type hepatitis C virus (HCV): a rapid method to distinguish HCV subtype 1b from other genotypes

Genotypic differentiation of hepatitis C virus (HCV) has become an integral part of clinical management and epidemiologic studies of hepatitis C infections. Thus, it is extremely important in areas such as the Czech Republic, where current instrumentation and kits for assessing HCV infection are too costly for widespread use. We describe a new and relatively inexpensive method called nested restriction site-specific PCR (RSS-PCR) that generates a "fingerprint" pattern to represent an HCV genotype without the use of restriction endonucleases and that specifically differentiates HCV genotype 1b from the other HCV genotypes. The RSS-PCR method was applied directly to serum samples from patients with hepatitis C from the Czech Republic and from patients with known HCV genotypes from the United States. The method was validated by comparison of the subtype determined by RSS-PCR to the subtype determined from analysis of the 5' noncoding region (NC) or the nonstructural protein gene (NS5b) nucleotide sequence of HCV in these clinical samples. From 75 Czech samples containing HCV RNA, three distinct RSS-PCR patterns were observed; 54 were predicted to contain subtype 1b, 19 were predicted to contain subtype 1a, and 2 were predicted to contain subtype 3a. Among 54 samples predicted to contain HCV genotype 1b, all were confirmed by their 5' NC or NS5b sequences to be subtype 1b. Thus, both the sensitivity and specificity of the RSS-PCR test for the differentiation of HCV subtype 1b from the others were 100%. While the assay described here was designed to specifically differentiate HCV subtype 1b from the other HCV genotypes, the RSS-PCR method can be modified to differentiate any HCV genotype or subtype of interest. Its simplicity and speed may provide new opportunities to study the epidemiology of HCV infections and the relationship between HCV genotypes and clinical outcome by more laboratories throughout the world.     

Evaluation of a new hepatitis C virus sequencing assay as a routine method for genotyping

The determination of HCV genotypes, subtypes and isolates has been helpful in understanding the evolution and the epidemiology of the virus, and is an important factor in the pre-treatment evaluation. A new simpler and automated sequencing based system has been developed recently, the Visible Genetics TruGene Hepatitis C Assay. The aim of the study was to compare this new genotyping assay with reverse hybridization based Innogenetics INNO-LiPA HCV II assay that is used most commonly. Eighty-eight HCV-RNA positive patients were enrolled and divided in four groups: 26 hemodialysed patients, 30 untreated patients with chronic HCV hepatitis, 12 IFN non-responder patients with chronic HCV hepatitis, 20 asymptomatic HCV positive subjects. The 5'-UTR region was amplified by RT-PCR and the nucleotide sequences determined by the TruGene assay. In parallel, the amplicons were also tested by INNO-LiPA. Concordant results were obtained in 80 out of 88 cases (90.9%). The new assay allowed to genotype 2 samples not typed by LiPA as 1b and 2a/c. The new system also allowed the subtyping of 3 untypable samples, classified as genotype 1 by INNO-LiPA, as genotype 1b (1 sample) and, as genotype 4 (2 samples). The difference between these genotype 4 isolates and the closest genotype 1 isolate was 6 nucleotides. One LiPA genotype 1a sample was typed as 1b and 2 genotype 1b samples were all typed as 1a by the sequence analysis. In conclusion, the new assay is a sensitive and rapid method that is suitable for accurate large-scale genotyping.     

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 virus genotyping: interrogation of the 5' untranslated region cannot accurately distinguish genotypes 1a and 1b

Although the 5' untranslated region (5' UTR) is the most conserved region of the hepatitis C virus (HCV) genome, it has been suggested that interrogation of this region is sufficient for determination of the HCV genotype. We compared two methods of determination of the HCV genotype: (i) direct sequencing of the DNA of the NS-5b region and (ii) reverse line probe assay (LiPA; INNO-LiPA HCV II; Innogenetics N.V.) of the 5' UTR. There was 100% concordance between the two methods for genotype but only 80% concordance for subtype. A significant percentage of genotype 1a isolates were misclassified by LiPA as genotype 1b. Sequence analysis revealed that the only consistent difference in the 5' UTR for these genotype 1a isolates misclassified as genotype 1b was a single nucleotide (A/G) at position -99 of the HCV genome. All isolates with discordant results analyzed had a G at this position, consistent with LiPA determination of these samples as subtype 1b. However, sequence analysis of 222 nucleotides in the NS-5b region clearly identified all of these isolates as subtype 1a. Population distribution data from the University of Pittsburgh Medical Center of over 200 samples analyzed by sequencing of the NS-5b region and over 1,000 samples analyzed by LiPA also indicated that INNO-LiPA HCV II cannot accurately differentiate HCV genotype 1a isolates from HCV genotype 1b isolates. We provide evidence that the A/G at position -99 represents a sequence polymorphism in the HCV genome that cannot differentiate subtype 1a from subtype 1b isolates. In conclusion, the 5' UTR is not heterogeneous enough for use in determination of the HCV subtype and cannot be used for differentiation of HCV genotypes 1a and 1b.     

The hypervariable region 1 protein of hepatitis C virus broadly reactive with sera of patients with chronic hepatitis C has a similar amino acid sequence with the consensus sequence.

Hypervariable region 1 (HVR1) proteins of hepatitis C virus (HCV) have been reported to react broadly with sera of patients with HCV infection. However, the variability of the broad reactivity of individual HVR1 proteins has not been elucidated. We assessed the reactivity of 25 different HVR1 proteins (genotype 1b) with sera of 81 patients with HCV infection (genotype 1b) by Western blot. HVR1 proteins reacted with 2-60 sera. The number of sera reactive with each HVR1 protein significantly correlated with the number of amino acid residues identical to the consensus sequence defined by Puntoriero et al. (G. Puntoriero, A. Lahm, S. Zucchelli, B. B. Ercole, R. Tafi, M. Penzzanera, M. U. Mondelli, R. Cortese, A. Tramontano, G. Galfre', and A. Nicosia. 1998. EMBO J. 17, 3521-3533. ) (r = 0.561, P < 0.005). The most widely reactive HVR1 protein, 12-22, had a sequence similar to the consensus sequence. The peptide with C-terminal 13-amino-acids sequence of HVR1 protein 12-22 (NH2-CSFTSLFTPGPSQK) was injected into rabbits as an immunogen. The rabbit immune sera reacted with 9 of 25 HVR1 proteins of genotype 1b including HVR1 protein 12-22 and with 3 of 12 proteins of genotype 2a. These results indicate that the HVR1 protein broadly reactive with patients' sera has a sequence similar to the consensus sequence, can induce broadly reactive sera, and could be one of the candidate immunogens in a prophylactic vaccine against HCV.     

Molecular epidemiology of hepatitis C virus genotype 4 isolates in Egypt and analysis of the variability of envelope proteins E1 and E2 in patients with chronic hepatitis

We analyzed hepatitis C virus (HCV) genotype 4 isolates circulating in the Alexandria District (Egypt) in terms of genetic divergence and the presence of different subtypes. Hypervariable region 1 (HVR1) and the NH2 region of the E2 protein were characterized, and the heterogeneity of subtype 4a isolates was evaluated by analyzing epitope frequencies, immunoproteasome prediction, and possible glycosylation patterns. The heterogeneity of the nucleotide sequences was greater than that found in previous studies, which reported only subtype 4a. Subtype 4a was most common (78% of cases), yet four new subtypes were found, with subtype 4m representing 11% of the cases and the other three subtypes representing another 11%. Substantial heterogeneity was also found when the intrasubtype 4a sequences were analyzed. Differences in the probability of glycosylation and in the positions of the different sites were also observed. The analysis of the predicted cytotoxic-T-lymphocyte epitopes showed differences in both the potential proteosome cleavage and the prediction score. The Egyptian isolates in our study also showed high variability in terms of the HVR1 neutralization epitope. Five of these isolates showed amino acid substitutions never previously observed (a total of six positions). Four of these residues (in four different isolates) were in positions involved in anchoring to the E2 glycoprotein core and in maintaining the HVR1 conformation. The results of this study indicate that HCV genotype 4 in Egypt is extremely variable, not only in terms of sequence, but also in terms of functional and immunological determinants. These data should be taken into account in planning the development of vaccine trials in Egypt.