Dynamic behavior of hepatitis C virus quasispecies in patients undergoing orthotopic liver transplantation.

We have studied the distribution of viral sequences from the 5' noncoding region and from a fragment of the E2/NS2 region of the hepatitis C virus (HCV) genome in samples obtained before and after liver transplantation in two patients with HCV cirrhosis. The population of viral sequences in both regions were established by sequencing cloned PCR products. In both cases, the complexity of the viral quasispecies decreased after transplantation, although the consensus nucleotide and amino acid sequences remained unchanged. It is suggested that both positive and negative selection and random sampling events contribute substantially in shaping the genetic composition of HCV quasispecies and that recurrence of HCV infection may occur under equilibrium conditions.     

Human immunodeficiency virus seroconversion and evolution of the hepatitis C virus quasispecies

When chronic hepatitis C virus (HCV) infections are complicated by acquisition of human immunodeficiency virus (HIV), liver disease appears to accelerate and serum levels of HCV RNA may rise. We hypothesized that HIV might affect the HCV quasispecies by decreasing both complexity (if HIV-induced immunosuppression lessens pressure for selecting HCV substitutions) and the ratio of nonsynonymous (d(N)) to synonymous (d(S)) substitutions, because d(N) may be lower (if there is less selective pressure). To test this hypothesis, we studied the evolution of HCV sequences in 10 persons with chronic HCV infection who seroconverted to HIV and, over the next 3 years, had slow or rapid progression of HIV-associated disease. From each subject, four serum specimens were selected with reference to HIV seroconversion: (i) more than 2 years prior, (ii) less than 2 years prior, (iii) less than 2 years after, and (iv) more than 2 years after. The HCV quasispecies in these specimens was characterized by generating clones containing 1 kb of cDNA that spanned the E1 gene and the E2 hypervariable region 1 (HVR1), followed by analysis of clonal frequencies (via electrophoretic migration) and nucleotide sequences. We examined 1,320 cDNA clones (33 per time point) and 287 sequences (median of 7 per time point). We observed a trend toward lower d(N)/d(S) after HIV seroconversion in 7 of 10 subjects and lower d(N)/d(S) in those with rapid HIV disease progression. However, the magnitude of these differences was small. These results are consistent with the hypothesis that HIV infection alters the HCV quasispecies, but the number of subjects and observation time may be too low to characterize the full effect.     

Structural basis for broad neutralization of hepatitis C virus quasispecies

Monoclonal antibodies directed against hepatitis C virus (HCV) E2 protein can neutralize cell-cultured HCV and pseudoparticles expressing envelopes derived from multiple HCV subtypes. For example, based on antibody blocking experiments and alanine scanning mutagenesis, it was proposed that the AR3B monoclonal antibody recognized a discontinuous conformational epitope comprised of amino acid residues 396–424, 436–447, and 523–540 of HCV E2 envelope protein. Intriguingly, one of these segments (436–447) overlapped with hypervariable region 3 (HVR3), a domain that exhibited significant intrahost and interhost genetic diversity. To reconcile these observations, amino-acid sequence variability was examined and homology-based structural modelling of E2 based on tick-borne encephalitis virus (TBEV) E protein was performed based on 413 HCV sequences derived from 18 subjects with chronic hepatitis C. Here we report that despite a high degree of amino-acid sequence variability, the three-dimensional structure of E2 is remarkably conserved, suggesting broad recognition of structural determinants rather than specific residues. Regions 396–424 and 523–540 were largely exposed and in close spatial proximity at the surface of E2. In contrast, region 436–447, which overlaps with HVR3, was >35 Å away, and estimates of buried surface were inconsistent with HVR3 being part of the AR3B binding interface. High-throughput structural analysis of HCV quasispecies could facilitate the development of novel vaccines that target conserved structural features of HCV envelope and elicit neutralizing antibody responses that are less vulnerable to viral escape.     

Tracking hepatitis C virus quasispecies major and minor variants in symptomatic and asymptomatic liver transplant recipients.

To evaluate the possibility that distinct viral quasispecies play a role in the pathogenesis of progressive hepatitis C virus (HCV) infection, we performed a detailed evaluation of HCV quasispecies before and after liver transplantation in five patients infected with HCV genotype 1, three of whom developed severe recurrent hepatitis C and two of whom developed asymptomatic posttransplant infections with high-titered viremia. HCV quasispecies were characterized by using a combination of nucleotide sequencing plus heteroduplex tracking assay of the second envelope gene hypervariable region (HVR). An average of 30 HVR clones were analyzed per specimen; an average of five specimens were analyzed per patient over a 6- to 24-month study period. The complexity of HCV quasispecies in pretransplant serum varied, ranging from one to nine genetically distinct variants for the five patients. However, in all five cases, relatively homogenous quasispecies variants emerged after liver transplantation. In the three patients who developed recurrent hepatitis, quasispecies major variants present in pretransplant serum were efficiently propagated immediately after liver transplantation and were propagated throughout the course of acute and chronic hepatitis. In contrast, in the two asymptomatic cases, we observed rapid depletion of pretransplant quasispecies major variants from posttransplant serum, followed by emergence of new quasispecies variants by posttransplant day 30. Genetic analysis suggested that in these cases, the new quasispecies variants were derived from minor variants present at relatively low clonal frequency (less than 5% of HVR clones) within the pretransplant quasispecies populations. These data demonstrate that quasispecies tracking patterns are associated with the rapidity and severity of HCV-associated liver disease after liver transplantation. Further characterization of HCV quasispecies in animal model systems is warranted.     

Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge

A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.     

丙型肝炎病毒准种及其临床意义研究进展

丙型肝炎病毒（HCV）易发生变异,在宿主体内主要以准种形式存在。HCV准种能够参与病毒逃避宿主的免疫监控及抗病毒治疗的耐药性,导致病毒在宿主体内持续存在,形成慢性感染。该文就HCV准种的产生、研究方法、基因组各区段内准种的研究及其临床意义作一综述。     

Impact of highly active antiretroviral therapy and immunologic status on hepatitis C virus quasispecies diversity in human immunodeficiency virus/hepatitis C virus-coinfected patients

This study analyzes the effect of highly active antiretroviral therapy (HAART), and thus immunologic status, on hepatitis C virus (HCV) load and quasispecies diversity in patients coinfected with the human immunodeficiency virus (HIV) and HCV. Three cohorts of coinfected patients were analyzed retrospectively over a period of 7 to 10 months: group A was antiretroviral drug na?ve at baseline and then on HAART for the remainder of the study, group B did not receive antiretroviral therapy at any point, and group C was on HAART for the entire study. HCV quasispecies diversity was analyzed by sequencing hypervariable region 1. In a longitudinal analysis, there was no significant change from baseline in any immunologic, virologic, or quasispecies parameter in any of the three groups. However, in comparison to groups A and B, group C had significantly higher CD4+- and CD8+-cell counts, a trend toward a higher HCV load, and significantly increased number of HCV clones, entropy, genetic distance, and ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site (Ka/Ks). In addition, CD4+-cell count was positively correlated with HCV load, genetic distance, and Ka. Interestingly, patients infected with HCV genotype 2 or 3 had a significantly higher CD4+-cell count, HCV load, genetic distance, and Ka/Ks than those infected with genotype 1. These results suggest that there is no immediate effect of HAART on HCV but that, with prolonged HAART, immune restoration results in an increase in HCV load and quasispecies diversity.     

Analysis of hepatitis C virus proteins based on amino acid sequence data and literature data

To analyze the interrelationships between the amino acid sequences of the proteins of hepatitis C virus and the functional characteristics of different variants of this virus, a database of protein functional mapping of hepatitis C virus was developed. The database contains amino acid sequences (both full-size and fragmentary) retrieved from accessible databases and experimental data published in literature. The database also contains the results of comparison and treatment of primary data, including alignments and functional regions. On the basis of these data, variable and conservative regions of envelope proteins of hepatitis C virus were revealed. Antigenic and functional maps of structural and nonstructural proteins of the virus were constructed. The most variable region of the envelope protein E2 (HVR1) was analysed. It is assumed that the conservatism of some amino acid positions of HVR1 is related to the functions of this region.     

丙型肝炎病毒高变区1中介导感染的关键氨基酸残基鉴定

丙型肝炎病毒( HCV)包膜E2蛋白氨基端的高变区1(HVR1)由27个氨基酸组成,是HCV蛋白中变异频率最高的肽段.HVR1含中和抗体表位,同时对HCV细胞侵入起重要作用,其结构与功能的关系目前尚不清楚.本研究对H77株包膜蛋白基因中的HVR1进行了一系列缺失突变,然后将突变体表达质粒与假病毒包装质粒共转染人胚肾(H...     

Compartmentalization of hepatitis C virus quasispecies in blood mononuclear cells of patients with mixed cryoglobulinemic syndrome

The aim of this study was to investigate the quasispecies heterogeneity of hepatitis C virus (HCV) in the plasma, cryoprecipitate, and peripheral lymphocytes of chronically infected HCV patients with mixed cryoglobulinemia (MC). We studied 360 clones from 10 HCV-positive patients with MC and 8 age-, gender- and HCV genotype-matched subjects with chronic HCV infection but without MC. A partial nucleotide sequence encompassing the E1/E2 region, including hypervariable region 1 (HVR1), was amplified and cloned from plasma, cryoprecipitates, and peripheral blood mononuclear cells (PBMC), and the genetic diversity and complexity and synonymous and nonsynonymous substitution rates were determined. Heterogeneous selection pressure at codon sites was evaluated. Compartmentalization was estimated by phylogenetic and phenetic (Mantel's test) approaches. The patients with MC had 3.3 times lower nonsynonymous substitution rates (1.7 versus 5.7 substitutions/100 sites). Among the subjects with HCV genotype 1, the MC patients had significantly less complexity than the controls, whereas the diversity and complexity were similar in the genotype 2 patients and controls. Site-specific selection analysis confirmed the low frequency of MC patients showing positive selection. There was a significant correlation between positive selection and the infecting HCV genotype. The quasispecies were less heterogeneous in PBMC than in plasma. Significant compartmentalization of HCV quasispecies was observed in the PBMC of four of nine subjects (three with MC) and seven of nine cryoprecipitates. In one subject with MC, we detected a 5-amino-acid insertion at codons 385 to 389 of HVR1. Our results suggest reduced quasispecies heterogeneity in MC patients that is related to a low selection pressure which is probably due to an impaired immune response, the HCV genotype, and/or the duration of the infection. The frequent HCV quasispecies compartmentalization in patients' PBMC suggests a possible pathogenetic significance.     

Within-host dynamics of the hepatitis C virus quasispecies population in HIV-1/HCV coinfected patients

HIV/HCV coinfected individuals under highly active antiretroviral therapy (HAART) represent an interesting model for the investigation of the role played by the immune system in driving the evolution of the HCV quasispecies. We prospectively studied the intra-host evolution of the HCV heterogeneity in 8 coinfected subjects, selected from a cohort of 32 patients initiating HAART: 5 immunological responders (group A) and 3 immunological non-responders (group B), and in two HCV singly infected controls not assuming drugs (group C). For all these subjects at least two serial samples obtained at the first observation (before HAART) and more than 1 year later, underwent clonal sequence analysis of partial E1/E2 sequences, encompassing the whole HVR1. Evolutionary rates, dated phylogenies and population dynamics were co-estimated by using a Bayesian Markov Chain Monte Carlo approach, and site specific selection pressures were estimated by maximum likelihood-based methods. The intra-host evolutionary rates of HCV quasispecies was 10 times higher in subjects treated with HAART than in controls without immunodeficiency (1.9 and 2.3 × 10(-3) sub/site/month in group A and B and 0.29 × 10(-3) sub/site/month in group C individuals). The within-host Bayesian Skyline plot analysis showed an exponential growth of the quasispecies populations in immunological responders, coinciding with a peak in CD4 cell counts. On the contrary, quasispecies population remained constant in group B and in group C controls. A significant positive selection pressure was detected in a half of the patients under HAART and in none of the group C controls. Several sites under significant positive selection were described, mainly included in the HVR1. Our data indicate that different forces, in addition to the selection pressure, drive an exceptionally fast evolution of HCV during HAART immune restoration. We hypothesize that an important role is played by the enlargement of the viral replicative space.