Sequence analysis of the hepatitis C virus genome recovered from serum, liver, and peripheral blood mononuclear cells of infected chimpanzees.

Of 13 different strains of hepatitis C virus (HCV) in the inoculum used, only 1 persisted in human lymphocyte cell lines infected in vitro (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325-3329, 1996). To determine whether that particular strain (designated H1-2) has a tropism for lymphocytes in vivo, we sequenced hypervariable region 1 (HVR1) of the genome of HCV recovered from the sera, livers, and peripheral blood mononuclear cells (PBMC) of chimpanzees infected with plasma H77, the same inoculum used for the in vitro studies. In the PBMC collected from two chimpanzees during the early phase of infection, H1-2 was detected as the only or predominant HVR1 sequence. H1-2 was also detected in PBMC obtained during persistent infection from a chimpanzee that had been treated with immunosuppressants. From the livers of these chimpanzees, two to six different strains were recovered but H1-2 was not detected. Thus, H1-2 appeared to have an affinity for lymphocytes not only in vitro but also in vivo. In samples collected from a chimpanzee after 6 years of infection, however, such tissue compartmentalization of the HCV genome was not observed; a single strain became predominant in the serum, liver, and PBMC. An HCV strain capable of replicating in both the liver and PBMC probably emerged during in vivo replication and persisted.     

Sequence evolution of the hypervariable region in the putative envelope region E2/NS1 of hepatitis C virus is correlated with specific humoral immune responses.

Sequence evolution of the hypervariable region 1 (HVR1) in the N terminus of E2/NS1 of hepatitis C virus (HCV) was studied retrospectively in six chimpanzees inoculated with the same genotype 1b strain, containing a unique predominant HVR1 sequence. Immediately after inoculation, all animals contained the same HVR predominant sequence. Two animals developed an acute self-limiting infection. Anti-HVR1 immunoglobulin G (IgG) was produced 40 to 60 days after inoculation and rapidly disappeared after normalization of transaminases. Another chimpanzee, previously infected with human immunodeficiency virus type 1, showed a delayed response to HVR1 epitopes after superinfection with HCV. No sequence variation of HVR1 was observed in these two animals during the transient viremia in the acute phase. Three other chimpanzees developed a chronic HCV infection. During follow up, sequence evolution occurred in two animals and their anti-HVR1 response remained at varying but detectable levels. The first mutations occurred immediately after the production of anti-HVR1 during the acute phase. However, IgM anti-HVR1 was not detectable. Remarkably, HVR1 sequences remained conserved for more than 6 years in another chronically infected animal. This correlated with the complete absence of detectable anti-HVR1 during this period. Seven years after inoculation, anti-HVR1 IgG was produced and coincided with an HVR1 alteration. These results strongly suggest the involvement of neutralizing anti-HVR antibodies in sequence evolution of HVR1 through immune selection.     

Follow-Up Study of Hypervariable Region Sequences of the Hepatitis C Virus (HCV) Genome in an Infant with Delayed Anti-HCV Antibody Responses

An infant born prematurely and infected with hepatitis C virus (HCV) one month after birth was followed for 4.5 years. The patient did not produce detectable anti-HCV antibodies until two years after the onset of hepatitis. Before seroconversion, a single clone of HCV, as determined by quasispecies of the hypervariable region (HVR) of the HCV genome, was almost exclusively found in the serum. After seroconversion, however, another distinct lineage of HCV clones replaced it within half a year. As HCV infection persisted further in the presence of anti-HCV antibodies, many derivatives of both sequence lineages emerged to exhibit the typical quasispecies feature of HVR sequences. Neither seroconversion nor the changes in HVR sequences influenced the serum aminotransferase titers.     

Towards a solution for hepatitis C virus hypervariability: mimotopes of the hypervariable region 1 can induce antibodies cross-reacting with a large number of viral variants.

The hypervariable region 1 (HVR1) of the putative envelope protein E2 of hepatitis C virus (HCV) is the most variable antigenic fragment in the whole viral genome and is mainly responsible for the large inter-and intra-individual heterogeneity of the infecting virus. It contains a principal neutralization epitope and has been proposed as the major player in the mechanism of escape from host immune response. Since anti-HVR1 antibodies are the only species shown to possess protective activity up to date, developing an effective prevention therapy is a very difficult task. We have approached the problem of HVR1 variability by deriving a consensus profile from >200 HVR1 sequences from different viral isolates and used it as a template to generate a vast repertoire of synthetic HVR1 surrogates displayed on M13 bacteriophage. This library was affinity selected using many different sera from infected patients. Phages were identified which react very frequently with patients' sera and bind serum antibodies that cross-react with a large panel of HVR1 peptides derived from natural HCV variants. When injected into experimental animals, the 'mimotopes' with the highest cross-reactivity induced antibodies which recognized the same panel of natural HVR1 variants. In these mimotopes we identified a sequence pattern responsible for the observed cross-reactivity. These data may hold the key for future development of a prophylactic vaccine against HCV.     

Human Monoclonal Antibody HCV1 Effectively Prevents and Treats HCV Infection in Chimpanzees

Hepatitis C virus (HCV) infection is a leading cause of liver transplantation and there is an urgent need to develop therapies to reduce rates of HCV infection of transplanted livers. Approved therapeutics for HCV are poorly tolerated and are of limited efficacy in this patient population. Human monoclonal antibody HCV1 recognizes a highly-conserved linear epitope of the HCV E2 envelope glycoprotein (amino acids 412–423) and neutralizes a broad range of HCV genotypes. In a chimpanzee model, a single dose of 250 mg/kg HCV1 delivered 30 minutes prior to infusion with genotype 1a H77 HCV provided complete protection from HCV infection, whereas a dose of 50 mg/kg HCV1 did not protect. In addition, an acutely-infected chimpanzee given 250 mg/kg HCV1 42 days following exposure to virus had a rapid reduction in viral load to below the limit of detection before rebounding 14 days later. The emergent virus displayed an E2 mutation (N415K/D) conferring resistance to HCV1 neutralization. Finally, three chronically HCV-infected chimpanzees were treated with a single dose of 40 mg/kg HCV1 and viral load was reduced to below the limit of detection for 21 days in one chimpanzee with rebounding virus displaying a resistance mutation (N417S). The other two chimpanzees had 0.5–1.0 log₁₀ reductions in viral load without evidence of viral resistance to HCV1. In vitro testing using HCV pseudovirus (HCVpp) demonstrated that the sera from the poorly-responding chimpanzees inhibited the ability of HCV1 to neutralize HCVpp. Measurement of antibody responses in the chronically-infected chimpanzees implicated endogenous antibody to E2 and interference with HCV1 neutralization although other factors may also be responsible. These data suggest that human monoclonal antibody HCV1 may be an effective therapeutic for the prevention of graft infection in HCV-infected patients undergoing liver transplantation.     

Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses.

We developed an in vitro assay for antibodies to hepatitis C virus (HCV) that bind to virions and prevent initiation of the replication cycle in susceptible cells in vitro. These antibodies therefore appear to be capable of neutralizing the virus. Using this assay and a standard inoculum of HCV of known infectivity, we have measured the antibody in serial serum samples obtained from the same chronically infected patient over 14 years following onset of his hepatitis. Such antibody was found in sera collected within 5 years of onset of hepatitis but not in later sera. In double immunoprecipitation experiments with anti-human immunoglobulin, the same sera that contained neutralizing antibody were found to contain antibody that bound to HCV to form antigen-antibody complexes immunoprecipitable with anti-human globulin. Similarly, plasma collected from this patient in 1990, 13 years after onset of hepatitis, and which contained HCV that had diverged genetically from the 1977 strain, did not contain antibody capable of neutralizing either the 1977 or the 1990 strain of HCV. However, plasma collected a year later (1991, 14 years after onset of hepatitis) contained neutralizing antibody to the 1990, but not the 1977, strain of HCV. These results suggest that HCV does induce antivirion antibody, as measured by blocking of initiation of the replication cycle of virus in cells and by the formation of immunoprecipitable antigen-antibody complexes but that these antibodies are isolate specific and change over time. Thus, these antivirion antibodies function as neutralizing antibodies and are probably in vitro correlates of the attempt of the host to contain the emergence of neutralization-resistant variants of HCV over time.     

Genetic drift in hypervariable region 1 of the viral genome in persistent hepatitis C virus infection.

The hypervariable region 1 (HVR1) of the putative second envelope glycoprotein (gp70) of hepatitis C virus (HCV) contains a sequence-specific immunological B-cell epitope that induces the production of antibodies restricted to the specific viral isolate, and anti-HVR1 antibodies are involved in the genetic drift of HVR1 driven by immunoselection (N. Kato, H. Sekiya, Y. Ootsuyama, T. Nakazawa, M. Hijikata, S. Ohkoshi, and K. Shimotohno, J. Virol. 67:3923-3930, 1993). We further investigated the sequence variability of the HCV genomic region that entirely encodes the envelope proteins (gp35 and gp70); these sequences were derived from virus isolated during the acute and chronic phases of hepatitis in one patient, and we found that HVR1 was a major site for genetic mutations in HCV after the onset of hepatitis. We carried out epitope-mapping experiments using the HVR1 sequence derived from the acute phase of hepatitis and identified two overlapping epitopes which are each composed of 11 amino acids (positions 394 to 404 and 397 to 407). The presence of two epitopes within HVR1 suggested that epitope shift happened during the course of hepatitis. Four of six amino acid substitutions detected in HVR1 were located within the two epitopes. We further examined the reactivities of anti-HVR1 antibodies to the substituted amino acid sequences within the two epitopes. HVR1 variants in both epitopes within the HVR1 escaped from anti-HVR1 antibodies that were preexisting in the patient's serum.     

Occurrence of antibodies reactive with more than one variant of the putative envelope glycoprotein (gp70) hypervariable region 1 in viremic hepatitis C virus-infected patients.

The hepatitis C virus (HCV) is a frequent cause of chronic liver disease. A mechanism proposed as being responsible for virus persistence is evasion of the host immune response through a high mutation rate in crucial regions of the viral genome. We have sequenced the hypervariable region 1 (HVR1) of the virus isolated from three serum samples, collected during 18 months of follow-up, from an asymptomatic HCV-infected patient. A synthetic peptide of 27 amino acids, corresponding to the HVR1 sequence found to be predominant in both the second and third samples, was used as the antigen for detection of antibodies by enzyme-linked immunosorbent assay (ELISA). We observed reactivity against this HVR1 sequence in the first serum sample before the appearance of the viral isolate in the bloodstream; the reactivity increased in the second and third samples while the cognate viral sequence became predominant. Moreover, our results show that antibodies from all three samples recognize a region mapping at the carboxyl-terminal part of the HVR1 and are cross-reactive with the HVR1 sequence previously found in the same patient. The presence of anti-HVR1 antibodies was investigated in a further 142 HCV patients: 121 viremic and 21 nonviremic. Two synthetic peptides were used, the first corresponding to the sequence derived from the patient described above and the second one synthesized according to the sequence of the HCV BK strain. A high frequency of positive reactions against both HVR1 variants was detected in the samples from the viremic individuals. Finally, antibodies cross-reactive with both variants were shown to be present by competitive ELISA in 6 of 10 viremic patients. The potential negative implications of this observation for the host are discussed.     

Hypervariable region 1 sequence stability during hepatitis C virus replication in chimpanzees

The putative envelope 2 (E2) gene of hepatitis C virus (HCV) contains a highly variable region referred to as hypervariable region 1 (HVR1). We hypothesized that this genetic variability is driven by immune selection pressure, rather than representing the accumulation of random mutations in a region with relatively little functional constraint. To test this hypothesis, we examined the E2 sequence of a human inoculum that was passaged through eight chimpanzees, which appear to have a replicative rate (opportunity for chance mutation) similar to that of humans. Acute-phase plasma samples from a human (the inoculum) and six of eight serially infected chimpanzees were studied. For each, 33 cloned cDNAs were examined by a combined heteroduplex-single-stranded conformational polymorphism assay to assess quasispecies complexity and optimize selection of clones with unique gel shift patterns (clonotypes) for sequencing. The sequence diversity of HCV was significantly lower in the chimpanzees than in the humans, and during eight serial passages there was no change in the sequence of the majority clonotype from each animal examined. Similarly, the rates of protein sequence altering (nonsynonymous) substitution were lower in the chimpanzees than in the humans. These findings demonstrate that nonsynonymous mutations indicate selection pressure rather than being an incidental result of HCV replication.     

Immunity in Chimpanzees Chronically Infected with Hepatitis C Virus: Role of Minor Quasispecies in Reinfection

We have previously reported that chimpanzees chronically infected with hepatitis C virus (HCV) could be reinfected, even with the original infecting strain. In this study we tested the hypothesis that this might reflect the presence of minor quasispecies to which there was little or no immunity. To evaluate this hypothesis, we sequenced multiple clones taken at intervals after primary infection and rechallenge from four chronically infected chimpanzees. The inoculum used in these studies (HCV-H, genotype 1a) revealed 17 separate variants among 46 clones sequenced. Following challenge, each of the four challenged animals showed marked alterations of their quasispecies distribution. The new variants, which appeared 1 to 6 weeks after challenge, were either identical to or closely resembled variants present in the challenge inoculum. These results, paralleled by an increase in viremia in some of the challenged animals, suggest that quasispecies in the challenge inoculum were responsible for signs of reinfection and that there was little immunity. However, the newly emerged quasispecies completely took over infection in only one animal. In the remaining three chimpanzees the prechallenge quasispecies were able to persist. The natural evolution of infection within chimpanzees resulted in variants able to compete with the inoculum variants. Whether through reexposure or the natural progression of infection, newly emerged quasispecies are likely to play a role in the pathogenesis of chronic HCV infection.Hepatitis C virus (HCV) is estimated to chronically infect about 400 million people worldwide. More than half of these develop chronic active hepatitis, cirrhosis, or hepatocellular carcinoma. The HCV genome consists of a single-stranded RNA molecule approximately 10 kb long which contains a single open reading frame encoding approximately 3,000 amino acids (1, 5). There are at least six genotypes of HCV, and within a given patient the genomes are distributed among quasispecies which show sequence variation, particularly in the variable regions of the genome (4, 9). Hypervariable region 1 (HVR1) is a 27-amino-acid segment in the amino terminus of the second envelope protein which has been identified as the most variable region of the viral genome (11, 20). Sequential changes have been observed during the course of chronic HCV infections in chimpanzees and in humans (4, 11, 12). It has been postulated that these reflect immune system selection of neutralizing epitopes encoded by HVR1 (18, 19) and that persistent infection depends on the ability of the virus to continually evade the effects of neutralizing antibody (7, 10, 15, 17, 20). Due to its variability, HVR1 has been used extensively as an indicator of viral evolution.We have previously reported that chronically infected chimpanzees could seemingly be reinfected, even with the original infecting strain (13). In a recent report a similar phenomenon was observed in patients with posttransfusion hepatitis (6). We postulated that this might reflect the presence of minor quasispecies in the inoculum to which there was little or no immunity (13). Here we test this hypothesis by sequencing multiple clones of HVR1 derived at intervals after initial infection and after rechallenge.     

Differential amplification of hypervariable region 1 of hepatitis C virus by partially mismatched primers

The quasispecies nature of hepatitis C virus (HCV) has been well documented over its whole genome and the most variable domain is located at the 5' end of the second envelope region, the so-called hypervariable region 1 (HVR1). HVR1 has therefore been extensively used as the target for characterizing HCV quasispecies profiles. In this study, we reported our finding that partially mismatched primers preferentially amplify different HVR1 sequences in a heterogeneous virus population. This finding suggests a possible mechanism of bias during the amplification of HVR1 sequences and may be responsible for some conflicting data regarding evolutionary or clinical implications of HCV quasispecies.     

Epitope mapping of antibodies directed against hypervariable region 1 in acute self-limiting and chronic infections due to hepatitis C virus.

Epitopes of hypervariable region 1 (HVR1) were mapped by enzyme-linked immunosorbent assay using follow-up sera of patients, all of whom were infected with the same isolate of hepatitis C virus (HCV). Our results suggest that (i) an early appearance (up to month 13 postinfection) of antibodies directed to the N terminus of HVR1 is associated with acute self-limiting infections of HCV and (ii) isolate-independent antibodies which are mainly directed to the C terminus of HVR1 seem to persist in chronically infected patients. The relevance of HVR1-specific antibodies for neutralization was evaluated by characterization of a rabbit serum.     

Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes

Hepatitis C(HCV) genome is highly variable,particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene.The variability of HCV genome has been a major obstacle for de-veloping HCV vaccines.Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes,we synthesized an minigene of HCV-derived multi-epitope peptide an-tigen(CMEP) ,which contains 9 B-cell HVR1 mimotopes in E2,2 conserved CTL epitopes in C,1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3.This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP.The immunogenic properties of CEMP were characterized by HCV infected patients' sera,and found that the reactivity frequency reached 75%.The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%.Meanwhile,we constructed an HCV DNA vaccine candidate,plasmid pVAX1.0-st-CMEP carrying the recombinant gene(st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene.Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody,which was of the same cross reactivity as the fusion protein GST-CMEP.Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes,and would be of the value as a candidate for the development of HCV vaccines.     

Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes

Hepatitis C (HCV) genome is highly variable, particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene. The variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes, we synthesized an minigene of HCV-derived multi-epitope peptide antigen (CMEP), which contains 9 B-cell HVR1 mimotopes in E2, 2 conserved CTL epitopes in C, 1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3. This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP. The immunogenic properties of CEMP were characterized by HCV infected patients’ sera, and found that the reactivity frequency reached 75%. The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%. Meanwhile, we constructed an HCV DNA vaccine candidate, plasmid pVAX1.0-st-CMEP carrying the recombinant gene (st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene. Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody, which was of the same cross reactivity as the fusion protein GST-CMEP. Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes, and would be of the value as a candidate for the development of HCV vaccines.     

四份中国HCV阳性血清中包膜蛋白E1/E2准种基因的序列分析及新型插入突变的发现

为分析四份中国丙型肝炎病毒（HCV）阳性血清中包膜蛋白E1/E2基因的准种特征。本研究对从4份中国HCV阳性血清（1b亚型：274、366、383;2a亚型：283）中提取的HCV核酸,采用逆转录-聚合酶反应扩增编码全长E1/E2蛋白（191～764aa）的基因片段,随机挑取多个克隆测序。根据E1/E2基因核苷酸的序列与其他相关序列（来自于GenBank）构建亲缘性关系进化树,进行核苷酸与氨基酸同源性分析并对重要的基因位点进行分析。共获得阳性克隆序列43个（274株10个,283株12个,366株13个,383株8个）,发现高变区HVR1、HVR2的基因异质性高,而其他抗体中和表位及跨膜区I、II及N末端糖基化位点相对保守。并首次发现在HCV 2a亚型（283血清）中多个准种序列存在1279nt（E1区,313aa）处单碱基插入优势基因突变,导致HCV包膜蛋白编码突变与中断（E2区,398aa）。本研究对中国HCV代表株包膜蛋白E1/E2编码基因的准种多样性及一种新型插入突变进行了描述,可为进一步研究HCV免疫逃避与慢性化机制提供重要信息。关键词：丙型肝炎病毒;包膜蛋白;序列分析;准种;插入突变     

Variability or conservation of hepatitis C virus hypervariable region 1? Implications for immune responses

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is highly heterogeneous in its primary sequence and is responsible for significant inter- and intra-individual variation of the infecting virus, which may represent an important pathogenetic mechanism leading to immune escape and persistent infection. A binding site for neutralizing antibodies (Ab) has also been allegedly identified in this region. Prospective studies of serological responses to synthetic oligopeptides derived from naturally-occurring HVR1 sequences showed promiscuous recognition of HVR1 variants in most patients via binding to C-terminal amino acid residues with conserved physicochemical properties. Monoclonal antibodies generated by immunization of mice with peptides derived from natural HVR1 sequences were shown to recognize several HVR1 variants in line with evidence gathered from studies using human sera. In addition, selected mAbs were able to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded sequences, and were shown to specifically capture circulating and recombinant HCV particles, suggesting that HVR1 is expressed on intact virus particles and therefore potentially able to interact with cellular receptor(s). These findings suggest that it is possible to induce a broadly reactive clonal immune response to multiple HCV variants and that this mechanism could be used in principle to induce protective immunity for a large repertoire of HCV variants.     

Monoclonal antibodies with broad specificity for hepatitis C virus hypervariable region 1 variants can recognize viral particles

The hypervariable region 1 (HVR1) of the E2 protein of hepatitis C virus (HCV) is a highly heterogeneous sequence that is promiscuously recognized by human sera via binding to amino acid residues with conserved physicochemical properties. We generated a panel of mAbs from mice immunized with HVR1 surrogate peptides (mimotopes) affinity-selected with sera from HCV-infected patients from a phage display library. A high number of specific clones was obtained after immunization with a pool of nine mimotopes, and the resulting mAbs were shown to recognize several 16- and 27-mer peptides derived from natural HVR1 sequences isolated from patients with acute and chronic HCV infection, suggesting that HVR1 mimotopes were efficient antigenic and immunogenic mimics of naturally occurring HCV variants. Moreover, most mAbs were shown to bind HVR1 in the context of a complete soluble form of the E2 glycoprotein, indicating recognition of correctly folded HVR1. In addition, a highly promiscuous mAb was able to specifically capture bona fide viral particles (circulating HCV RNA) as well as rHCV-like particles assembled in insect cells expressing structural viral polypeptides derived from an HCV 1a isolate. These findings demonstrate that it is possible to induce a broadly cross-reactive clonal Ab response to multiple HCV variants. In consideration of the potentially important role of HVR1 in virus binding to cellular receptor(s), such a mechanism could be exploited for induction of neutralizing Abs specific for a large repertoire of viral variants.     

Long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis C virus

Two chimpanzees (Ch1535 and Ch1536) became infected with hepatitis C virus (HCV) following intrahepatic inoculation with RNA transcribed from a full-length cDNA clone of the virus. Both animals were persistently infected and have been followed for 60 weeks. They showed similar responses to infection, with transient liver enzyme elevations and liver inflammatory responses, which peaked at weeks 17 (Ch1535) and 12 (Ch1536) postinoculation (p.i.). Antibody responses to structural and nonstructural proteins were first detected at weeks 13 (Ch1535) and 10 (Ch1536) p.i. Serum RNA titers increased steadily during the first 10 to 13 weeks but decreased sharply in both animals following antibody and inflammatory responses. Despite direct evidence of humoral immune responses to multiple viral antigens, including hypervariable region 1 (HVR1), both animals remained chronically infected. Detailed sequence analysis of serum HCV RNA revealed no change in the majority HVR1 sequence in Ch1535 and a single-amino-acid mutation in Ch1536, with very little clonal variation in either animal. Full-length genome analysis at week 60 revealed several amino acid substitutions localized to antigens E1, E2, p7, NS3, and NS5. Of these, 55.6 and 40% were present as the majority sequence in serum RNA isolated at week 26 p.i. (Ch1535) and week 22 p.i. (Ch1536), respectively, and could represent immune escape mutations. Mutations accumulated at a rate of 1.57 x 10(-3) and 1.48 x 10(-3) nucleotide substitutions/site/year for Ch1535 and Ch1536, respectively. Taken together, these data indicate that establishment of a persistent HCV infection in these chimpanzees is not due to changes in HVR1; however, the possibility remains that mutations arising in other parts of the genome contributed to this persistence.     

Conservation of the conformation and positive charges of hepatitis C virus E2 envelope glycoprotein hypervariable region 1 points to a role in cell attachment

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease. The HCV polyprotein contains a hypervariable region (HVR1) located at the N terminus of the second envelope glycoprotein E2. The strong variability of this 27-amino-acid region is due to its apparent tolerance of amino acid substitutions together with strong selection pressures exerted by anti-HCV immune responses. No specific function has so far been attributed to HVR1. However, its presence at the surface of the viral particle suggests that it might be involved in viral entry. This would imply that HVR1 is not randomly variable. We sequenced 460 HVR1 clones isolated at various times from six HCV-infected patients receiving alpha interferon therapy (which exerts strong pressure towards quasispecies genetic evolution) and analyzed their amino acid sequences together with those of 1,382 nonredundant HVR1 sequences collected from the EMBL database. We found that (i) despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of HVR1 were highly conserved, and (ii) HVR1 is a globally basic stretch, with the basic residues located at specific sequence positions. This conservation of positively charged residues indicates that HVR1 is involved in interactions with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). As with many other viruses, possible interaction with GAGs probably plays a role in host cell recognition and attachment.     

Evolutionary rate and genetic drift of hepatitis C virus are not correlated with the host immune response: studies of infected donor-recipient clusters

Six donor-recipient clusters of hepatitis C virus (HCV)-infected individuals were studied. For five clusters the period of infection of the donor could be estimated, and for all six clusters the time of infection of the recipients from the donor via blood transfusion was also precisely known. Detailed phylogenetic analyses were carried out to investigate the genomic evolution of the viral quasispecies within infected individuals in each cluster. The molecular clock analysis showed that HCV quasispecies within a patient are evolving at the same rate and that donors that have been infected for longer time tend to have a lower evolutionary rate. Phylogenetic analysis based on the split decomposition method revealed different evolutionary patterns in different donor-recipient clusters. Reactivity of antibody against the first hypervariable region (HVR1) of HCV in donor and recipient sera was evaluated and correlated to the calculated evolutionary rate. Results indicate that anti-HVR1 reactivity was related more to the overall level of humoral immune response of the host than to the HVR1 sequence itself, suggesting that the particular sequence of the HVR1 peptides is not the determinant of reactivity. Moreover, no correlation was found between the evolutionary rate or the heterogeneity of the viral quasispecies in the patients and the strength of the immune response to HVR1 epitopes. Rather, the results seem to imply that genetic drift is less dependent on immune pressure than on the rate of evolution and that the genetic drift of HCV is independent of the host immune pressure.