Molecular and bioinformatic evidence of hepatitis C virus evolution in brain

BACKGROUND: Neurocognitive deficits in patients with hepatitis C virus (HCV) infection prompted a search for HCV in brain. RESULTS: HCV was present in the brains of 7 (54%) of 13 patients with viremia, as determined by 5' UTR and E1 (envelope 1) gene analysis. Brain HCV RNA consensus sequences differed from those in plasma and liver in 4 (57%) of 7 patients. The quality of HCV RNA from postmortem brain and liver was assessed and demonstrated to be suitable for sequence analysis. Quasispecies analysis revealed that several mutations present in clones from >1 brain region were absent in clones from liver and plasma. Brain-specific mutations defined several families of related sequences. The patterns of brain-specific mutations in these families were consistent with the evolution of HCV RNA from a common ancestor. Single-nucleotide-polymorphism analysis confirmed that a prominent brain-specific mutation constituted approximately 10% of HCV RNA in cerebellum and medulla but that this mutation was undetectable in the liver and plasma of the same patient. CONCLUSIONS: This study introduces novel methods for assessing RNA from postmortem samples. It increases the reported cases of HCV in the brain, provides the first E1 sequences from the brain, and contributes to the growing evidence that HCV replicates and evolves within the brain.     

丙型肝炎病毒感染自然过程中的准种变化

目的观察丙型肝炎病毒(HCV)持续感染者与自然阴转者外周血HCV准种构成的变化规律。方法应用基因扩增、分子克隆和测序的方法,对未接受过治疗的4例HCV持续感染者与4例自然阴转者前后间隔10年血清中HCV高变区1(HVR1)基因片段进行了序列分析及遗传进化关系比较。结果与持续感染者相比,自然阴转者外周血HCVHVR1区准种群体组内平均遗传距离、熵值较小。4例持续感染者中有3例10年前后血清HCVHVR1准种群体组内与组间遗传距离有明显差异。8例感染者中有7例血清HCV准种KA/KS值大于1。结论在丙型肝炎的自然病程中HCV准种遗传复杂度、变异度大小可能与丙型肝炎的转归相关;HCV准种构成可能发生改变。     

Multigene tracking of quasispecies in viral persistence and clearance of hepatitis C virus

AIM: To investigate the evaluation of hepatitis C virus (HCV) quasispecies in the envelope region and its relationship with the outcome of acute hepatitis C. METHODS: HCV quasispecies were characterized in specimens collected every 2-6 mo from a cohort of acutely HCV-infected subjects. We evaluated two individuals who spontaneously cleared viremia and three individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2, including hypervariable region 1 (HVR1). To assess the quasispecies complexity and to detect variants for sequencing, 33 cloned cDNAs representing each specimen were assessed by a combined method of analysis of a single-stranded conformational polymorphism and heteroduplex analysis. The rates of both synonymous and nonsynonymous substitutions for the E1, HVR1 and E2 regions outside HVR1 were analyzed. RESULTS: Serum samples collected from chronic phase of infection had higher quasispecies complexity than those collected from acute phase of infection in all individuals examined. The genetic diversity (genetic distance) within HVR1 was consistently higher than that in the complete E1(0.0322±0.0068 vs-0.0020±0.0014, P<0.05) and E2 regions outside HVR1 (0.0322±0.0068 vs 0.0017±0.0011, P<0.05) in individuals with persistent viremia, but did not change markedly over time in those with clearance of viremia. For individuals with persistent viremia, the rate of nonsynonymous substitutions within the HVR1 region (2.76×10-3±1.51×10-3) predominated and gradually increased, as compared with that in the E1 and E2 regions outside HVR1 (0.23×10-3±0.15×10-3, 0.50×10-3±0.10×10-3). By contrast, the rates of both nonsynonymous and synonymous substitutions for the E1 and E2 regions including HVR1 were consistently lower in individuals with clearance of viremia. CONCLUSION: HCV persistence is associated with a complexity quasispecies and positive selection of HVR1 by the host immune system.     

Hepatitis C virus NS3/4A quasispecies diversity in acute hepatitis C infection in HIV‐1 co‐infected patients

The growing number of cases of acute hepatitis C (AHC) infections among human immunodeficiency virus type 1 (HIV‐1)‐positive men who have sex with men (MSM) in the last 10 years has promoted the search for predictors of AHC clearance as well as for epidemiological networks of viral transmission. We characterized the diversity and catalytic efficiency of HCV NS3/4A protease quasispecies in AHC patients coinfected with HIV‐1. Plasma samples obtained at HCV diagnosis from 18 MSM HIV‐coinfected patients with AHC were studied. Five HCV monoinfected patient samples with AHC were also investigated. An average of 39 clones from each sample was analysed. The catalytic efficiency of the dominant quasispecies (i.e. the most abundant) from each quasispecies was also assayed for mitochondrial antiviral signalling protein (MAVS) cleavage. Phylogenetic analysis identified two clusters of patients with highly related viruses, suggesting a common source of HCV infection. None of the 18 MSM HIV‐coinfected patients spontaneously cleared HCV, although 78% of the treated patients achieved a sustained virological response after early treatment with pegylated interferon (pegIFN) plus ribavirin (RBV). The synonymous‐nonsynonymous (ds/dn) mutation ratio, a marker of selective pressure, was higher in AHC compared to 26 HIV‐1‐infected men with genotype 1a chronic hepatitis C (CHC) (P < 0.0001). NS3/4A proteases from AHC patients also exhibited higher catalytic efficiency compared to CHC patients (P < 0.0001). No differences were found when ds/dn mutation ratios and NS3/4A protease catalytic efficiencies from AHC HIV‐coinfected patients were compared with AHC monoinfected patients. The presence of epidemiological networks of HCV transmission was confirmed among HIV‐1‐positive MSM. In addition, substantial genetic diversity was demonstrated in AHC. NS3/4A protease efficiency cleaving MAVS may be associated with virus transmission and response to pegIFN/RBV treatment.     

Mutations within the E2 and NS5A protein in patients infected with hepatitis C virus type 3a and correlation with treatment response

Defined regions of hepatitis C virus (HCV) envelope 2 (E2), PePHD, and nonstructural 5A (NS5A) protein (PKR-binding domain) have been shown to interact with interferon alfa (IFN-alpha)-inducible double-stranded RNA-activated protein kinase (PKR) in vitro, suggesting a possible mechanism of HCV to evade antiviral effects of IFN-alpha. The clinical correlation between amino acid mutations within the E2 PePHD or the NS5A PKR-binding domain and response to antiviral treatment in HCV-3a-infected patients is unknown. Thirty-three patients infected with HCV-3a isolates were treated with IFN-alpha with or without ribavirin. The carboxyterminal half of E2 and of the NS5A gene were sequenced. Sixteen patients achieved a sustained virological response (SR), 6 patients an end-of-treatment response with relapse thereafter (ETR), and 11 patients were nonresponders (NR). Within the PePHD of the E2 protein 0.5 (range, 0-2) mutations were observed in SR patients, whereas the number of mutations in ETR or NR patients was 0.2 (0-1). Quasispecies analyses showed almost no heterogeneity. The mean number of mutations within the PKR-binding domain of the NS5A protein was 1.6 (range, 0-4) in SR patients, 1 (0-2) in ETR patients, and 1.6 (0-3) in NR patients. Patients with higher numbers of mutations within the E2 or NS5A region showed a trend towards lower pretreatment viremia. Phylogenetic and conformational analyses of E2 or NS5A sequences allowed no differentiation between sensitive and resistant isolates. However, mutations within the E2 PePHD in SR patients were frequent, and hydrophobic mutations within the hydrophilic area of PePHD at codon 668 and 669 were exclusively observed in sustained virological responders.     

Genomic variability of within‐host hepatitis C variants in acute infection

Interactions between the host immune system and the viral variants determine persistence of hepatitis C virus (HCV) infection after the acute phase of infection. This study describes the genetic variability of within‐host HCV viral variants in acute infection and correlates it with host‐ and virus‐related traits and infection outcome. Next generation sequence data (Illumina, MiSeq platform) of viral genomes from 116 incident acute infections (within 180 days of infection) were analysed to determine all the single nucleotide polymorphism (SNP) frequencies above a threshold of 0.1%. The variability of the SNPs for the full open reading frame of the genome as well as for each protein coding region were compared using mean standardized Shannon entropy (SE) values calculated separately for synonymous and nonsynonymous mutations. The envelope glycoproteins regions (E1 and E2) had the highest SE values (indicating greater variability) followed by the NS5B region. Nonsynonymous mutations rather than synonymous mutations were the main contributors to genomic variability in acute infection. The mean difference of Shannon entropy was also compared between subjects after categorizing the samples according to host and virus‐related traits. Host IFNL3 allele CC polymorphism at rs12979860 (vs others) and viral genotype 1a (vs 3a) were associated with higher genomic variability across the viral open reading frame. Time since infection, host gender or continent of origin was not associated with the viral genomic variability. Viral genomic variability did not predict spontaneous clearance.     

丙型肝炎病毒E1、E2 蛋白的表达及初步用于丙型肝炎患儿血清学诊断的临床研究

在昆虫细胞中表达丙型肝炎病毒(HCV)包膜糖蛋白E1和E2,将该蛋白应用于丙型肝炎患儿血清抗体的检测。用PCR方法自HCV H株扩增出包膜蛋白E基因,构建重组杆状病毒,在昆虫细胞中稳定表达包膜糖蛋白E1和E2,并用此蛋白与56例丙型肝炎患儿血清反应。在56份HCV感染者血清中,有7例E1抗体阳性,其中2例检出E2抗体。在16例PCR检测HCV RNA阳性而抗HCV阴性的血清中,有5例与表达的E1蛋白反应。HCV E蛋白基因表达的E1蛋白可用于HCV患儿的血清学诊断。     

慢性肝炎患者血清中丙型肝炎病毒核心区和包膜1区基因变异研究

目的 探讨丙型肝炎病毒（HCV）核心（C）区和包膜1（E1）区变异与其感染慢性化的关系。方法 10例HCV慢性感染者和2例急性感染者血标本,采用逆转录－聚合酶链反应（RT－PCR）扩增HCV的C区羧基端、E1及E2区氨基端片段（1kb),扩增产物进行克隆,以单链构象多态性（SSCP）和异质性双体（HD）分析对每份血清的30个克隆的C／E1区准种（quasispecies)进行筛选,挑选每例标本HCV的优势株与劣势株序列进行测定,并分析推导的氨基酸序列。结果 各例患者血清中HCV的C／E1区扩增片段形成的SSCP条带间差异明显,而HD与同源双体之间差距不明显。HCV急性感染者和慢性感染者血清病毒C区无发生变异,后者E1区氨基酸替换率为1．32％,但功能性氨基酸无改变。结论 HCV的E1区序列变异以准种形式存在,但与免疫逃避可能无关。     

Complexity and diversity of hepatitis C virus RNA in african americans and whites: analysis of the envelope-coding domain

African Americans infected with hepatitis C virus (HCV) are less responsive than whites to interferon-based therapy. HCV quasi species have been implicated. Quasi-species complexity and diversity were evaluated in matched African American and white individuals. Complexity and diversity in the first hypervariable region were similar in the 2 groups. Both the frequency of nonsynonymous amino acid substitutions and the mean ratio of nonsynonymous mutations to synonymous mutations were greater in clones derived from white patients. Racial differences in amino acid usage at otherwise conserved positions were observed. Differences in amino acid representation at key positions are suggestive of immunologic and functional selection along racial lines.     

Analysis of nevirapine resistance mutations in cloned HIV type 1 variants from HIV-infected Ugandan infants using a single-step amplification-sequencing method (AmpliSeq)

We analyzed the genetic linkage of nevirapine (NVP) resistance mutations and the genetic complexity of HIV-1 variants in Ugandan infants who were HIV infected despite single dose (SD) prophylaxis. Plasma samples were obtained from six HIV-infected infants who had two or more NVP resistance mutations detected by population sequencing (ViroSeq). ViroSeq PCR products were cloned and transformed, and a single-step amplification-sequencing reaction (AmpliSeq) was used to analyze NVP resistance mutations in cloned HIV-1 variants directly from bacterial colonies. Fifty clones were analyzed for each infant sample. This analysis revealed numerous NVP resistance mutations not detected by population sequencing, genetically linked NVP resistance mutations, and a high degree of genetic complexity at codons that influence NVP susceptibility.     

Sequence evolution of putative cytotoxic T cell epitopes in NS3 region of hepatitis C virus

AIM: Quasispecies of hepatitis C virus (HCV) are the foundation for rapid sequence evolution of HCV to evade immune surveillance of hosts. The consensus sequence evolution of a segment of HCV NS3 region, which encompasses putative cytotoxic T cell epitopes, was evaluated. METHODS: Three male patients, infected with HCV through multiple transfusions, were identified from clinical symptoms and monitored by aminotransferase for 60 months. Blood samples taken at months 0, 32, and 60 were used for viral RNA extraction. A segment of HCV NS3 region was amplified from the RNA extraction by RT-PCR and subjected to subcloning and sequencing. HLA types of these three patients were determined using complement-dependent microlymphocytotoxic assay. CTL epitopes were predicted using MHC binding motifs. RESULTS: No patient had clinical symptoms or elevation of aspartate/alanine aminotransferase. Two patients showed positive HCV PCR results at all 3 time points. The other one showed a positive HCV PCR result only at month 0. A reported HLA-A2-restricted CTL epitope had no alteration in the HLA-A2-negative carrier over 60 months. In the HLA-A2-positive individuals, all the sequences from 0 month 0 showed an amber mutation on the initial codon of the epitope. Most changes of consensus sequences in the same patient occurred on predicted cytotoxic T cell epitopes. CONCLUSION: Amber mutation and changes of consensus sequence in HCV NS3 region may be related to viral immune escape.     

At least 12 genotypes of hepatitis C virus predicted by sequence analysis of the putative E1 gene of isolates collected worldwide.

In a previous study we sequenced the 5' noncoding (NC) region of 44 isolates of hepatitis C virus (HCV) and identified heterogeneous domains that provided evidence for additional genetic groups of HCV not previously recognized. In this study we have determined the complete nucleotide sequence of the putative envelope 1 (E1) gene in 51 HCV isolates from around the world and found that they could be grouped into at least 12 distinct genotypes. The E1 gene sequence of 8 of these genotypes has not been reported previously. Although the genetic relatedness of HCV isolates determined by the previous analysis of the 5' NC region predicted the relationships observed in the E1 gene, analysis of the 5' NC sequence alone did not accurately predict all HCV genotypes. The nucleotide and amino acid sequence identities of the E1 gene among HCV isolates of the same genotype were in the range of 88.0-99.1% and 89.1-98.4%, respectively, whereas those of HCV isolates of different genotypes were in the range of 53.5-78.6% and 49.0-82.8%, respectively. The latter differences are similar to those found when comparing the envelope gene sequences of the various serotypes of the related flaviviruses as well as other RNA viruses. We found that some genotypes of HCV were widely distributed around the world, whereas others were identified only in discreet geographical regions. Four genotypes were identified exclusively in Africa and comprised the majority of HCV isolates on that continent. The E1 gene was exactly 576 nucleotides in length in all 51 HCV isolates with no in-frame stop codons. Analysis of the predicted E1 protein identified several conserved domains that may be important for maintaining its biological function: (i) eight invariant cysteine residues, (ii) three potential N-linked glycosylation sites, (iii) a domain of nine amino acids (GHRMAWDMM), and (iv) an amino acid doublet (GV) near the putative cleavage site at the C terminus of the protein. In conclusion, the discovery of at least 12 genotypes of HCV has important implications for HCV diagnosis and vaccine development.     

Influence of specific CD4+ T cells and antibodies on evolution of hypervariable region 1 during acute HCV infection

BACKGROUND/AIMS: Several studies suggest that the evolutionary rate of HVR1 sequence in acute HCV hepatitis derives from the action of a continuous immune-driven positive selection. However, these studies have not been performed examining the relationship between HVR1 evolution and the development of specific immunity to autologous HVR1 sequences. METHODS: We performed a longitudinal analysis of HVR1 sequences and specific antibodies and CD4+ T cells in ten HCV acutely infected patients with different clinical outcomes (recovery versus persistence). RESULTS: We showed that although both recovered and chronically evolving individuals developed IFN-gamma+ T cells specific for Core and NS sequences, HVR1-specific CD4+ T cells were detected only in patients clearing the virus. On the contrary, all patients displayed anti-HVR1 antibodies that recognized sequences exclusively carried by autologous viruses. Measurements of genetic diversity and the number of non-synonymous per synonymous substitutions within HVR1 sequences before and after antibody appearance showed an increase of these parameters only in concomitance with the appearance of anti-HVR1 antibodies. CONCLUSIONS: The evidence that anti-HVR1 antibodies favor HVR1 variant selection suggests that viral complexity in chronically infected patients could represent a virus adaptive strategy to escape the continuous selective process mediated by anti-HVR1 antibodies.     

Hepatitis cRNA quasispecies complexity in patients with alcoholic liver disease.

Alcohol abuse is described as a major cofactor in the development of hepatitis C (HCV) associated liver disease and may play a role in the outcome of interferon-based treatment interventions. The association between HCV viral heterogeneity and alcohol has not been previously described. This study was designed to evaluate the quasispecies nature of the HCV population in patients with compensated and decompensated alcoholic liver disease, to test the hypothesis that alcoholics have greater complexity than matched nonalcoholic controls. A nonisotopic heteroduplex complexity assay (HCA) was first validated by comparison with results of quasispecies complexity determined by subcloning and sequencing of amplicon products from the E2/NS1 hypervariable coding region (HVR). Subsequently, this methodology was applied to comparison of 20 compensated (Child's-Pugh A) and decompensated (Child's-Pugh B/C) alcoholic and 20 nonalcoholic controls. The complexity of the HVR, as well as the 5' Untranslated (5'UT) and the NS5b coding domains were evaluated. The HCA methodology provided a reasonable semiquantitative measure of HCV RNA quasispecies variability compared with subclone sequence homology comparison. Overall, alcoholic patients had greater quasispecies complexity (2.65 bands) than nonalcoholic controls (1.6 bands; P =.01). Subset analysis revealed that compensated alcoholic patients had a mean of 3.1 homo/heteroduplex bands per sample whereas Child's-Pugh B/C alcoholics showed intermediate complexity. A similar quasispecies complexity difference was seen in the 5'UTR, but not in the NS5b coding domain. Quasispecies complexity was not associated with viral titer, complementary DNA concentration, or genotype. The differences in quasispecies complexity may help explain reports of poor interferon responsiveness in alcoholic patients.     

Is investigation of hepatitis C virus NS5A gene heterogeneity a tool for predicting long‐lasting response to interferon therapy in patients with HCV‐1b chronic hepatitis?

Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) may repress the interferon (IFN)-induced protein kinase R (PKR). High variability of different regions in the carboxy-terminal half of NS5A implicated in the interaction with PKR (particularly the interferon sensitivity determining region (ISDR)) may be a predictor of response to IFN in patients infected with genotype 1b of HCV. We examined pretreatment serum samples from 17 HCV-1b infected patients included in the same schedule of IFN therapy. Seven patients were a rare series of sustained responders (SR) with a post-treatment follow-up of 5-7 years, while ten were nonresponders (NR). The carboxy-terminal half of the NS5A gene was amplified and directly sequenced in all 17 cases. In addition, the entire NS5A gene and the part of the HCV E2 gene coding for the hypervariable region 1 (HVR1) were amplified, cloned and sequenced in six cases (three NR and three SR). No difference in number and distribution of amino acid mutations was observed between isolates from SR and NR in any portion of the protein, including the ISDR region. Analysis of full length NS5A confirmed no difference between the two groups. The NS5A gene sequence was different among the six cases cloned although it appeared to be conserved in each individual patient independently of the quasispecies complexity evaluated through HVR1 examination. These data indicate that pretreatment analysis of theNS5A genomic variability has no value in predicting long-lasting response to IFN therapy in HCV-1b-infected patients, and that the HCV NS5A gene has high quasispecies homology.     

Early evolution of hepatitis C virus (HCV) quasispecies after liver transplant for HCV-related disease

BACKGROUND: End-stage liver disease as a result of chronic hepatitis C virus (HCV) infection is the main indication for liver transplant (LT), but allografts are systematically infected with HCV soon after transplant. Viral quasispecies are poorly described during the early posttransplant period. METHODS: For 17 patients who received an LT for HCV disease, plasma viral quasispecies evolution was determined by sequence analysis of hypervariable region 1 of the E2 envelope gene before transplant (BT), after 7 days (D7), and after 1 month (M1). T helper (Th)1/Th2 cytokine levels were determined concomitantly. RESULTS: HCV quasispecies showed a significant decrease in amino acid diversity at D7 and M1, compared with BT (P<.05). A correlation was observed between low plasma tumor necrosis factor-alpha levels at D7 and decreased quasispecies amino acid complexity at the same date. Nucleic acid diversity was lower for genotype 1 than for genotype 3 infection (P<.05). The complexity and diversity of amino acids were lower in patients with hepatocellular carcinoma (HCC) BT than in those without HCC (P<.05). Conserved amino acid residues within quasispecies were shared by the whole cohort before and after LT. CONCLUSION: Viral structural and/or host immunological features could favor the emergence of fitter HCV strains after LT.     

In vitro selection of a neutralization-resistant hepatitis C virus escape mutant

Effective immunization against hepatitis C virus (HCV) infections is likely to require the induction of both robust T and B cell immunity. Although neutralizing antibodies may play an important role in control of infection, there is little understanding of the structure of the HCV envelope glycoproteins and how they interact with such antibodies. An additional challenge for vaccine design is the genetic diversity of HCV and the rapid evolution of viral quasispecies that escape antibody-mediated neutralization. We used a cell culture-infectious, chimeric HCV with the structural proteins of genotype 1a virus to identify envelope residues contributing to the epitope recognized by a broadly neutralizing, murine monoclonal antibody, AP33. By repetitive rounds of neutralization followed by amplification, we selected a population of viral escape mutants that resist stringent neutralization with AP33 and no longer bind the antibody. Two amino acid substitutions, widely separated in the linear sequence of the E2 envelope protein (N415Y and E655G), were identified by sequencing of cloned cDNA and shown by reverse genetics analysis to contribute jointly to the AP33 resistance phenotype. The N415Y mutation substantially lowered virus fitness, most likely because of a defect in viral entry, but did not reduce binding of soluble CD81 to immobilized HCV-pseudotyped retrovirus particles. The in vitro selection of an HCV escape mutant recapitulates the ongoing evolution of antigenic variants that contributes to viral persistence in humans and reveals information concerning the conformational structure of the AP33 epitope, its role in viral replication, and constraints on its molecular evolution.     

Evolution of hepatitis C viral quasispecies and hepatic injury in perinatally infected children followed prospectively

Perinatal infection with hepatitis C virus (HCV) is characterized by a wide range of alanine aminotransferase (ALT) levels. The mechanisms responsible for this variability are unknown. We examined whether the evolution of the HCV quasispecies was associated with different ALT profiles in perinatally infected children. Sequences within HCV envelope 1 and 2 genes, inclusive of the hypervariable region 1, the viral load, and the nascent humoral immunity were analyzed in serial serum samples from 12 perinatally infected children prospectively followed for a median of 53 months. These patients were selected to represent two different ALT patterns during the first year of life: 6 had high levels (maximum values ranging from 4.2 to 30 times the normal upper limit), and 6 had normal or slightly elevated levels (< 2 times the normal upper limit). Two patterns of viral evolution were identified according to the ALT profiles. Biochemical evidence of hepatic injury was invariably associated with a mono- or oligoclonal viral population, whereas mild or no liver damage correlated with the early emergence of a heterogeneous viral quasispecies. Consistent with selective immune pressure, amino acid changes occurred almost exclusively within the hypervariable region 1 and were temporally associated with antibody seroconversion; at this time, the difference in genetic diversity between the two groups was highly significant (P = 0.002). The two patterns of viral evolution persisted over time and did not correlate with viral load or genotype. Our study demonstrates that, in perinatally infected children, the evolution of HCV quasispecies correlates with hepatic injury. The sequences reported in this paper have been deposited in the GenBank database (accession nos. DQ 504441-DQ 507112).     

Analysis of hepatitis C virus quasispecies transmission and evolution in patients infected through blood transfusion

BACKGROUND & AIMS: Studies on hepatitis C virus (HCV) quasispecies dynamics in the natural course of infection are rare owing to difficulties in obtaining samples from the early phase of infection. METHODS: We studied 15 patients from the Transfusion-Transmitted Viruses Study who seroconverted to anti-HCV after receiving infected blood. Follow-up serum samples were collected every 2-3 weeks for 6 months, at 10 months, and at 11-16 years. Viral quasispecies in the second envelope hypervariable region 1 (E2/HVR1) and 5' untranslated region (5'UTR) were analyzed with single-strand conformation polymorphism (SSCP) and heteroduplex mobility assay (HMA). RESULTS: Seven patients cleared infection within 7-24 weeks (mean, 14.0 wk) and 3 patients eventually became anti-HCV negative. In 6 patients with resolving hepatitis the SSCP band pattern remained stable, whereas in one patient minor changes appeared before clearance. In contrast, in all 8 patients progressing to chronicity, major changes in the E2/HVR1 quasispecies developed at 8-22 weeks (mean, 13.1 wk). Shannon entropy and medium mobility shift values derived from HMA gels remained stable in patients with resolving hepatitis but changed in those who developed chronic infection. Only 2 patients showed minor changes in 5'UTR. A decrease in E2/HVR1 complexity at the time of transmission (bottleneck) was found in 5 patients altogether. CONCLUSIONS: Changes in E2/HVR1 quasispecies 8-22 weeks after infection, likely caused by mounting immune pressure, were predictive of ensuing chronic infection, whereas stability was associated with resolution. Our study also showed that composition of HCV quasispecies may be preserved during transmission from host to host.