Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators

Background

Hepatitis C virus (HCV) infection is a major public health problem with more than 170 million cases of chronic infections worldwide. There is no protective vaccine currently available for HCV, therefore the development of novel strategy to prevent chronic infection is important. We reported earlier that a recombinant human antibody clone blocks viral NS3 helicase activity and inhibits replication of HCV 1b virus. This study was performed further to explore the mechanism of action of this recombinant antibody and to determine whether or not this antibody inhibits replication and infectivity of a highly efficient JFH1 HCV 2a virus clone.

Results

The antiviral effect of intracellular expressed antibody against the HCV 2a virus strain was examined using a full-length green fluorescence protein (GFP) labeled infectious cell culture system. For this purpose, a Huh-7.5 cell line stably expressing the NS3 helicase gene specific IgG1 antibody was prepared. Replication of full-length HCV-GFP chimera RNA and negative-strand RNA was strongly inhibited in Huh-7.5 cells stably expressing NS3 antibody but not in the cells expressing an unrelated control antibody. Huh-7.5 cells stably expressing NS3 helicase antibody effectively suppressed infectious virus production after natural infection and the level of HCV in the cell free supernatant remained undetectable after first passage. In contrast, Huh-7.5 cells stably expressing an control antibody against influenza virus had no effect on virus production and high-levels of infectious HCV were detected in culture supernatants over four rounds of infectivity assay. A recombinant adenovirus based expression system was used to demonstrate that Huh-7.5 replicon cell line expressing the intracellular antibody strongly inhibited the replication of HCV-GFP RNA.

Conclusion

Recombinant human anti-HCV NS3 antibody clone inhibits replication of HCV 2a virus and infectious virus production. Intracellular expression of this recombinant antibody offers a potential antiviral strategy to inhibit intracellular HCV replication and production.     

Hepatitis C virus genotype 3a with phylogenetically distinct origin is circulating in Pakistan

Background

Hepatitis C virus (HCV) is one of the leading causes of viral hepatitis worldwide and its genotype 3a is predominant in vast areas of Pakistan.

Findings

The present study reports the first full sequence of HCV 3a isolate PK-1 from Pakistan. This nucleotide sequence was compared with six other HCV genotype 3a full length sequences from different regions of the world by using statistical methods of phylogenetic analysis.

Conclusion

The nucleotide difference of these seven sequences shows that HCV genotype 3a of phylogenetically distinct origin is circulating in Pakistan.     

On Hepatitis C Virus Evolution: The Interaction between Virus and Host towards Treatment Outcome

Background

Hepatitis C is a disease spread throughout the world. Hepatitis C virus (HCV), the etiological agent of this disease, is a single-stranded positive RNA virus. Its genome encodes a single precursor protein that yields ten proteins after processing. NS5A, one of the non-structural viral proteins, is most associated with interferon-based therapy response, the approved treatment for hepatitis C in Brazil. HCV has a high mutation rate and therefore high variability, which may be important for evading the immune system and response to therapy. The aim of this study was to analyze the evolution of NS5A quasispecies before, during, and after treatment in patients infected with HCV genotype 3a who presented different therapy responses.

Methods

Viral RNA was extracted, cDNA was synthesized, the NS5A region was amplified and cloned, and 15 clones from each time-point were sequenced. The sequences were analyzed for evolutionary history, genetic diversity and selection.

Results

This analysis shows that the viral population that persists after treatment for most non-responder patients is present in before-treatment samples, suggesting it is adapted to evade treatment. In contrast, the population found in before treatment samples from most end-of-treatment responder patients either are selected out or appears in low frequency after relapse, therefore changing the population structure. The exceptions illustrate the uniqueness of the evolutionary process, and therefore the treatment resistance process, in each patient.

Conclusion

Although evolutionary behavior throughout treatment showed that each patient presented different population dynamics unrelated to therapy outcome, it seems that the viral population from non-responders that resists the treatment already had strains that could evade therapy before it started.     

Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures

Background

Hepatitis C virus (HCV) infection afflicts about 170 million individuals worldwide. However, the HCV life cycle is only partially understood because it has not been possible to infect normal human hepatocytes in culture. The current Huh-7 systems use cloned, synthetic HCV RNA expressed in hepatocellular carcinoma cells to produce virions, but these cells cannot be infected with naturally occurring HCV obtained from infected patients.

Methodology/Principal Findings

Here, we describe a human hepatocyte culture permissible to the direct infection with naturally occurring HCV genotypes 1, 2, 3 and 4 in the blood of HCV-infected patients. The culture system mimics the biology and kinetics of HCV infection in humans, and produces infectious virions that can infect naïve human hepatocytes.

Conclusions/Significance

This culture system should complement the existing systems, and may facilitate the understanding of the HCV life cycle, its effects in the natural host cell, the hepatocyte, as well as the development of novel therapeutics and vaccines.     

Initial Fitness Recovery of HIV-1 Is Associated with Quasispecies Heterogeneity and Can Occur without Modifications in the Consensus Sequence

Background

Fitness recovery of HIV-1 “in vitro” was studied using viral clones that had their fitness decreased as a result of plaque-to-plaque passages.

Principal Findings

After ten large population passages, the viral populations showed an average increase of fitness, although with wide variations among clones. While 5 clones showed significant fitness increases, 3 clones showed increases that were only marginally significant (p<0.1), and 4 clones did not show any change. Fitness recovery was not accompanied by an increase in p24 production, but was associated with an increase in viral titer. Few mutations (an average of 2 mutations per genome) were detected in the consensus nucleotide sequence of the entire genome in all viral populations. Five of the populations did not fix any mutation, and three of them displayed marginally significant fitness increases, illustrating that fitness recovery can occur without detectable alterations of the consensus genomic sequence. The investigation of other possible viral factors associated with the initial steps of fitness recovery, showed that viral quasispecies heterogeneity increased between the initial clones and the passaged populations. A direct statistical correlation between viral heterogeneity and viral fitness was obtained.

Conclusions

Thus, the initial fitness recovery of debilitated HIV-1 clones was mediated by an increase in quasispecies heterogeneity. This observation, together with the invariance of the consensus sequence despite fitness increases demonstrates the relevance of quasispecies heterogeneity in the evolution of HIV-1 in cell culture.     

Low-volume jet injection for intradermal immunization in rabbits

Background

This study tested a low-volume (20–30 μl/20–30 μg DNA) jet injection method for intradermal delivery of a DNA vaccine. Jet injection offers the advantages of a needle-less system, low-cost, rapid preparation of the injected DNA solution, and a simple delivery system. More than one construct can be injected simultaneously and the method may be combined with adjuvants.

Results

Low-volume jet injection targeted delivery of a DNA solution exclusively to the dermis and epidermis of rabbits. A three injection series of plasmid DNA, encoding the Hepatitis B Surface Antigen stimulated a humoral immune response in 2/5 rabbits. One rabbit developed a significant rise in antibody titer after 1 injection and one following 2 injections. There were no significant differences between jet injection and particle bombardment in the maximal antibody titers or number of injections before response. A three injection series of the same plasmid DNA by particle bombardment elicited a significant rise in antibody titer in 3/5 rabbits. One rabbit developed antibody after 1 injection and two after 3 injections. In contrast, 0/5 rabbits receiving DNA by needle and syringe injection responded. In the jet injection and particle bombardment groups, gene expression levels in the skin did not predict response. While immune responses were similar, luciferase gene expression levels in the skin following particle bombardment were 10–100 times higher than jet injection.

Conclusion

Low-volume jet injection is a simple, effective methodology for intradermal DNA immunization.     

Genetic heterogeneity of hepatitis C virus in association with antiviral therapy determined by ultra-deep sequencing

Background and Aims

The hepatitis C virus (HCV) invariably shows wide heterogeneity in infected patients, referred to as a quasispecies population. Massive amounts of genetic information due to the abundance of HCV variants could be an obstacle to evaluate the viral genetic heterogeneity in detail.

Methods

Using a newly developed massive-parallel ultra-deep sequencing technique, we investigated the viral genetic heterogeneity in 27 chronic hepatitis C patients receiving peg-interferon (IFN) α2b plus ribavirin therapy.

Results

Ultra-deep sequencing determined a total of more than 10 million nucleotides of the HCV genome, corresponding to a mean of more than 1000 clones in each specimen, and unveiled extremely high genetic heterogeneity in the genotype 1b HCV population. There was no significant difference in the level of viral complexity between immediate virologic responders and non-responders at baseline (p = 0.39). Immediate virologic responders (n = 8) showed a significant reduction in the genetic complexity spanning all the viral genetic regions at the early phase of IFN administration (p = 0.037). In contrast, non-virologic responders (n = 8) showed no significant changes in the level of viral quasispecies (p = 0.12), indicating that very few viral clones are sensitive to IFN treatment. We also demonstrated that clones resistant to direct-acting antivirals for HCV, such as viral protease and polymerase inhibitors, preexist with various abundances in all 27 treatment-naïve patients, suggesting the risk of the development of drug resistance against these agents.

Conclusion

Use of the ultra-deep sequencing technology revealed massive genetic heterogeneity of HCV, which has important implications regarding the treatment response and outcome of antiviral therapy.     

Hepatitis B and C in dialysis units in Kosova

Background

The complement system is one of the most potent weapons of innate immunity. It is not only a mechanism for direct protection against invading pathogens but it also interacts with the adaptive immunity to optimize the pathogen-specific humoral and cellular defense cascades in the body. Complement-mediated lysis of HIV is inefficient but the presence of HIV particles results in complement activation by the generation of many C3-fragments, such as C3dg and C3d. It has been demonstrated that activation of complement can enhance HIV infection through the binding of special complement receptor type 2 expression on the surface of mature B cells and follicular dendritic cells.

Presentation of the hypothesis

Previous studies have proven that the complement-mediated antibody-dependent enhancement of HIV infection is mediated by the association of complement receptor type 2 bound to the C3 fragment and deposited on the surface of HIV virions. Thus, we hypothesize that a new activator of complement, consisting of a target domain (C3-binding region of complement receptor type 2) linked to a complement-activating human IgG1 Fc domain (CR2-Fc), can target and amplify complement deposition on HIV virions and enhance the efficiency of HIV lysis.

Testing the hypothesis

Our hypothesis was tested using cell-free HIV-1 virions cultivatedin vitro and assessment of virus opsonization was performed by incubating appropriate dilutions of virus with medium containing normal human serum and purified CR2-Fc proteins. As a control group, viruses were incubated with normal human serum under the same conditions. Virus neutralization assays were used to estimate the degree of CR2-Fc-enhanced lysis of HIV compared to untreated virus.

Implications of the hypothesis

The targeted complement activator, CR2-Fc, can be used as a novel approach to HIV therapy by abrogating the complement-enhanced HIV infection of cells.     

Purification and characterization of a serine protease (CESP) from mature coconut endosperm

Background

Identifying the endogenous RNA induced silencing complex(RISC)-associated RNAs is essential for understanding the cellular regulatory networks by miRNAs. Recently, isolation of RISC-associated mRNAs using antibody was reported, but their method needs a large amount of initial materials. We tried to improve the protocol and constructed an efficient and convenient system for analyzing miRNA and mRNA contents in RISC.

Findings

With our protocol, it is possible to clone both miRNAs and mRNAs from the endogenous RISC-associated RNAs immunoprecipitated from less than 10⁷ cells, and we show the ability of our system to isolate the particular target mRNAs for a specific miRNA from the RISC-associated mRNAs using well-characterized miR-122 as an example. After introduction of miR-122 into HepG2 cells, we found several cDNA clones that have miR-122 target sequences. Four of these clones that were concentrated in RISC but decreased in total RNA fraction are expected to be miR-122 target candidates. Interestingly, we found substantial amounts of Alu-related sequences, including both free Alu RNA and Alu-embedded mRNA, which might be one of the general targets for miRNA, in the cDNA clones from the RISC-associated mRNAs.

Conclusion

Our method thus enables us to examine not only dynamic changes in miRNA and mRNA contents in RISC but also the relationship of miRNA and target mRNA. We believe that our method can contribute to understanding cellular regulatory networks by miRNAs.     

Enhancement of the expression of HCV core gene does not enhance core-specific immune response in DNA immunization: advantages of the heterologous DNA prime, protein boost immunization regimen

Background

Hepatitis C core protein is an attractive target for HCV vaccine aimed to exterminate HCV infected cells. However, although highly immunogenic in natural infection, core appears to have low immunogenicity in experimental settings. We aimed to design an HCV vaccine prototype based on core, and devise immunization regimens that would lead to potent anti-core immune responses which circumvent the immunogenicity limitations earlier observed.

Methods

Plasmids encoding core with no translation initiation signal (pCMVcore); with Kozak sequence (pCMVcoreKozak); and with HCV IRES (pCMVcoreIRES) were designed and expressed in a variety of eukaryotic cells. Polyproteins corresponding to HCV 1b amino acids (aa) 1–98 and 1–173 were expressed in E. coli. C57BL/6 mice were immunized with four 25-μg doses of pCMVcoreKozak, or pCMV (I). BALB/c mice were immunized with 100 μg of either pCMVcore, or pCMVcoreKozak, or pCMVcoreIRES, or empty pCMV (II). Lastly, BALB/c mice were immunized with 20 μg of core aa 1–98 in prime and boost, or with 100 μg of pCMVcoreKozak in prime and 20 μg of core aa 1–98 in boost (III). Antibody response, [³H]-T-incorporation, and cytokine secretion by core/core peptide-stimulated splenocytes were assessed after each immunization.

Results

Plasmids differed in core-expression capacity: mouse fibroblasts transfected with pCMVcore, pCMVcoreIRES and pCMVcoreKozak expressed 0.22 ± 0.18, 0.83 ± 0.5, and 13 ± 5 ng core per cell, respectively. Single immunization with highly expressing pCMVcoreKozak induced specific IFN-γ and IL-2, and weak antibody response. Single immunization with plasmids directing low levels of core expression induced similar levels of cytokines, strong T-cell proliferation (pCMVcoreIRES), and antibodies in titer 10³(pCMVcore). Boosting with pCMVcoreKozak induced low antibody response, core-specific T-cell proliferation and IFN-γ secretion that subsided after the 3rd plasmid injection. The latter also led to a decrease in specific IL-2 secretion. The best was the heterologous pCMVcoreKozak prime/protein boost regimen that generated mixed Th1/Th2-cellular response with core-specific antibodies in titer ≥ 3 × 10³.

Conclusion

Thus, administration of highly expressed HCV core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. Instead, the latter is induced by a heterologous DNA prime/protein boost regimen that circumvents the negative effects of intracellular core expression.     

丙型肝炎病毒准种在NS2区的变异状况初探

探讨HCV准种在NS2区的基因结构特征及变异状况。利用逆转录－巢式PCR从1份HCV慢性携带者的阳性血清及1份丙肝患者的血清中获得HCV NS2全长cDNA,将其克隆于T载体,各随机挑取5个阳性克隆进行序列测定,结果显示克隆到HCV NS2全长基因,所测克隆在核苷酸水平和氨基酸水平互不相同。该慢性携带者HCV NS2区序列以完整读码框架（ORF）为主,一个于HCV多聚蛋白第835位氨基酸的位置出现终止信号,而该丙型肝炎患者以NS2N端发现终止信号的序列为主,其中三个于第835位氨基酸的位置出现终止信号,一个于第887位氨基酸的位置出现终止信号,仅一个克隆的序列为完整ORF。对ORF完整的序列进行比较,发现丙型肝炎患者氨基酸变异主要集中于N端,蛋白二级结构模拟显示丙肝患者NS2与慢性携带者的优势二级结构类似,研究表明从我们选择的两种感染者的HCV NS2序列看,不同临床类型的HCV病人体内的HCV准种在NS2区存在差异,这种差异可能与病毒存在于机体的状态一定的一致性。     

HCV Genotypes,Characterization of Mutations Conferring Drug Resistance to Protease Inhibitors,and Risk Factors among Blood Donors in S?o Paulo,Brazil

Background

Hepatitis C virus (HCV) infection is a global health problem estimated to affect almost 200 million people worldwide. The aim of this study is to analyze the subtypes and existence of variants resistant to protease inhibitors and their association with potential HCV risk factors among blood donors in Brazil.

Methods

Repeat anti-HCV reactive blood donors are systematically asked to return for retest, notification, and counseling in which they are interviewed for risk factors for transfusion-transmitted diseases. We analyzed 202 donors who returned for counseling from 2007 to 2010 and presented enzyme immunoassay- and immunoblot-reactive results. The HCV genotypes and resistance mutation analyses were determined by the direct sequencing of the NS5b and NS3 regions, respectively. The HCV viral load was determined using an in-house real-time PCR assay targeting the 5′-NCR.

Results

HCV subtypes 1b, 1a, and 3a were found in 45.5%, 32.0%, and 18.0% of the donors, respectively. The mean viral load of genotype 1 was significantly higher than that of the genotype 3 isolates. Subtype 1a was more frequent among young donors and 3a was more frequent among older donors. Protease inhibitor-resistant variants were detected in 12.8% of the sequenced samples belonging to genotype 1, and a higher frequency was observed among subtype 1a (20%) in comparison to 1b (8%). There was no difference in the prevalence of HCV risk factors among the genotypes or drug-resistant variants.

Conclusions

We found a predominance of subtype 1b, with an increase in the frequency of subtype 1a, in young subjects. Mutations conferring resistance to NS3 inhibitors were frequent in treatment-naïve blood donors, particularly those infected with subtype 1a. These variants were detected in the major viral population of HCV quasispecies, have replicative capacities comparable to nonresistant strains, and could be important for predicting the response to antiviral triple therapy.     

Opt-Out Panel Testing for HIV,Hepatitis B and Hepatitis C in an Urban Emergency Department: A Pilot Study

Objectives

Studies suggest 2 per 1000 people in Dublin are living with HIV, the level above which universal screening is advised. We aimed to assess the feasibility and acceptability of a universal opt-out HIV, Hepatitis B and Hepatitis C testing programme for Emergency Department patients and to describe the incidence and prevalence of blood-borne viruses in this population.

Methods

An opt-out ED blood borne virus screening programme was piloted from March 2014 to January 2015. Patients undergoing blood sampling during routine clinical care were offered HIV 1&2 antibody/antigen assay, HBV surface antigen and HCV antibody tests. Linkage to care where necessary was co-ordinated by the study team. New diagnosis and prevalence rates were defined as the new cases per 1000 tested and number of positive tests per 1000 tested respectively.

Results

Over 45 weeks of testing, of 10,000 patient visits, 8,839 individual patient samples were available for analysis following removal of duplicates. A sustained target uptake of >50% was obtained after week 3. 97(1.09%), 44(0.49%) and 447(5.05%) HIV, Hepatitis B and Hepatitis C tests were positive respectively. Of these, 7(0.08%), 20(0.22%) and 58(0.66%) were new diagnoses of HIV, Hepatitis B and Hepatitis C respectively. The new diagnosis rate for HIV, Hepatitis B and Hepatitis C was 0.8, 2.26 and 6.5 per 1000 and study prevalence for HIV, Hepatitis B and Hepatitis C was 11.0, 5.0 and 50.5 per 1000 respectively.

Conclusions

Opt-out blood borne viral screening was feasible and acceptable in an inner-city ED. Blood borne viral infections were prevalent in this population and newly diagnosed cases were diagnosed and linked to care. These results suggest widespread blood borne viral testing in differing clinical locations with differing population demographic risks may be warranted.     

Cost-Effectiveness of HBV and HCV Screening Strategies – A Systematic Review of Existing Modelling Techniques

Introduction

Studies evaluating the cost-effectiveness of screening for Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) are generally heterogeneous in terms of risk groups, settings, screening intervention, outcomes and the economic modelling framework. It is therefore difficult to compare cost-effectiveness results between studies. This systematic review aims to summarise and critically assess existing economic models for HBV and HCV in order to identify the main methodological differences in modelling approaches.

Methods

A structured search strategy was developed and a systematic review carried out. A critical assessment of the decision-analytic models was carried out according to the guidelines and framework developed for assessment of decision-analytic models in Health Technology Assessment of health care interventions.

Results

The overall approach to analysing the cost-effectiveness of screening strategies was found to be broadly consistent for HBV and HCV. However, modelling parameters and related structure differed between models, producing different results. More recent publications performed better against a performance matrix, evaluating model components and methodology.

Conclusion

When assessing screening strategies for HBV and HCV infection, the focus should be on more recent studies, which applied the latest treatment regimes, test methods and had better and more complete data on which to base their models. In addition to parameter selection and associated assumptions, careful consideration of dynamic versus static modelling is recommended. Future research may want to focus on these methodological issues. In addition, the ability to evaluate screening strategies for multiple infectious diseases, (HCV and HIV at the same time) might prove important for decision makers.     

The spectrum of thyroid dysfunction in an Australian hepatitis C population treated with combination Interferon-α2β and Ribavirin

Background

The study aims to assess the pattern of thyroid response to combination Interferon-α2β (IFN-α) and Ribavirin (RBV) anti-viral therapy in an Australian hepatitis C cohort. These include the prevalence of thyroid dysfunction (TD) including hyperthyroidism and hypothyroidism and their possible predictors, the common overall pattern of thyroid function tests whilst receiving therapy and TD outcomes, and the correlation with HCV status outcome.

Methods

A retrospective analysis of all medical records was performed to assess thyroid function in Hepatitis C Virus (HCV) patients who were treated at the Hunter Area hepatitis C treatment center between 1995 and March 2004. The centre is part of the John Hunter hospital, a major tertiary referral centre in New South Wales, Australia.

Results

There were 272 cases available for review. The prevalence of TD is 6.7 percent and is made up predominantly of females (80 percent). There were 3 (1.1 percent) cases of hyperthyroidism with 2 (67 percent) females. Thirteen out of fifteen (80 percent) cases of hypothyroidism were females with the overall prevalence of 5.5 percent. The majority of hypothyroid patients still required Thyroxine supplement at the end of follow up.

Conclusion

Ninety three percent of HCV treated patients have intact thyroid function at the end of treatment. The predominant TD is hypothyroidism. The predominant pattern of thyrotoxicosis (TTX) is that of thyroiditis although the number is small. Graves' like disease was not observed. People with pre-existing thyroid auto-antibodies should be closely monitored for thyroid dysfunction, particularly hypothyroidism.     

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.     

Multigene Tracking of Hepatitis C Virus Quasispecies after Liver Transplantation: Correlation of Genetic Diversification in the Envelope Region with Asymptomatic or Mild Disease Patterns

To investigate the role of hepatitis C virus (HCV) quasispecies mutation in the pathogenesis of HCV infection, we analyzed changes in the genetic diversity of HCV genomes in 22 patients before and after liver transplantation by using heteroduplex mobility assay (HMA) technology. All patients were infected with HCV genotype 1 and developed high-titer posttransplant viremia. Each patient was classified according to the severity of posttransplant hepatitis, as assessed by standard biochemical and histological criteria. HCV quasispecies were characterized by HMA analysis of eight separate subgenomic regions of HCV, which collectively comprise 44% of the entire genome. The glycoprotein genes E1 and E2, as well as the nonstructural protein genes NS2 and NS3, had the greatest genetic divergence after liver transplantation (the change in the heteroduplex mobility ratio [HMR] ranged from 2.5 to 7.0%). In contrast, genes encoding the core, NS4, and NS5b proteins had the least amount of genetic divergence after liver transplantation (range, 0.3 to 1.2%). The E1/E2 region showed the greatest change in genetic diversity after liver transplantation, and the change in HMRs was 2.5- to 3.3-fold greater in patients with asymptomatic or moderate disease than in those with severe disease. The E1-5′ region of HCV quasispecies isolated from patients in the asymptomatic group had a significantly greater degree of diversification after liver transplantation than the same regions of HCV quasispecies isolated from patients in the severe disease group (P = 0.05). While changes in the genetic diversity of some nonstructural genes were also greater in asymptomatic patients or in patients with mild disease than in patients with severe disease, the results were not significant. Data from this cohort demonstrate that greater rates of HCV quasispecies diversification are associated with mild or moderate liver disease activity in this immunosuppressed population.

Hepatitis C virus (HCV), a member of the Flaviviridae family, is known to be a major causative agent of chronic liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (27). Chronic hepatitis C is now recognized as the leading indication for orthotopic liver transplantation in the United States, with nearly 100% of HCV-infected liver transplant recipients developing recurrent viremia after transplantation (4, 17, 22, 32, 44).The HCV genome is a single-stranded, positive-sense RNA of about 9.5 kb, which exists as a viral quasispecies in infected humans (6, 25, 31, 41). HCV quasispecies are characterized by extensive genetic mutation in the hypervariable region 1 (HVR1) of the second envelope glycoprotein gene (E2) (25, 41). Mutation of this region of the genome is believed to be associated with viral persistence via immune escape mechanisms (15, 16, 29, 42). The role of evolution of HCV quasispecies in the development of chronic hepatitis C is currently unknown.HCV-infected liver transplant recipients offer an opportunity to study the evolution of HCV quasispecies in a new host tissue and to assess the role of quasispecies diversification in the development of posttransplant hepatitis. In a previous study (23) of HCV-infected liver transplant recipients, we found that in the three patients who developed severe, recurrent hepatitis, quasispecies major variants present in pretransplant serum samples were efficiently propagated after liver transplantation and during acute and chronic posttransplant hepatitis. In contrast, in the two asymptomatic cases, we observed rapid depletion of pretransplant quasispecies major variants from posttransplant serum samples, followed by the emergence of quasispecies minor variants. These data suggested that the evolution of HCV quasispecies after liver transplantation may be related to posttransplant disease severity.In order to extend our previous findings and to address the hypothesis that mutation of other HCV genes also correlates with severity of posttransplant hepatitis C, we analyzed the pre- and posttransplant HCV quasispecies in 22 HCV-infected liver transplant recipients. The 22 patients were selected based on two virological criteria: all patients were infected with HCV genotype 1 before and after liver transplantation, and all patients developed recurrent, high-titer HCV viremia within 30 days posttransplant. Additionally, at least five posttransplant liver biopsies were available in each case to evaluate the histopathologic course of posttransplant hepatitis C. Pre- and posttransplant HCV quasispecies were characterized by using the heteroduplex mobility assay (HMA) to analyze eight different regions of the viral genome.