Antiviral properties from plants of the Mediterranean flora.

Natural products are a successful source in drug discovery, playing a significant role in maintaining human health. We investigated the in vitro cytotoxicity and antiviral activity of extracts from 18 traditionally used Mediterranean plants. Noteworthy antiviral activity was found in the extract obtained from the branches of Daphne gnidium L. against human immunodeficiency virus type-1 (EC50 = 0.08 µg/mL) and coxsackievirus B5 (EC50 = 0.10 µg/mL). Other relevant activities were found against BVDV, YFV, Sb-1, RSV and HSV-1. Interestingly, extracts from Artemisia arborescens L. and Rubus ulmifolius Schott, as well as those from D. gnidium L., showed activities against two different viruses. This extensive antiviral screening allowed us to identify attractive activities, offering opportunities to develop lead compounds with a great pharmaceutical potential.

The use of molecular assays in the management of viral hepatitis.

Molecular assays are instrumental in the clinical management of viral hepatitis. During the past years, a wide variety of molecular assays have been developed and implemented. This considerably improved the understanding of the natural history and pathogenesis of Hepatitis B virus (HBV), Hepatitis C virus (HCV) or Hepatitis delta virus (HDV) hepatitis, but also caused uncertainties in the selection of the most appropriate assays for clinical requirements. Indeed, a rational choice and application of these assays requires adequate knowledge of the performance of the single test. Moreover, the choice of the most accurate assay for patients' needs and physicians' objectives, needs to be oriented to specific contexts, such as diagnosis, management or treatment. In the past, a hurdle in the routine use of assays for hepatitis viruses nucleic acid quantification was represented by the availability of only "home brew" methods which lacked standardization. Major improvement in addressing the use of molecular assays for viral hepatitis has been derived from recent standardization procedures that allowed a comparison between different tests after results were given as International Units. In addition, it should be reminded that, before getting into the market, molecular assays should be approved by European regulation authorities and validated using internationally recognized standards. A subsequent clinical validation should address the diagnostic accuracy of the assay. These proceedings have the aim of identifying which molecular tests, among those currently available, meet clinical requirements for each specific application.

Treatment of chronic hepatitis D.

Despite recent advances in the treatment of chronic viral hepatitis, therapy of chronic hepatitis D is not yet satisfactory. The only option currently available is interferon-alpha (IFN), whose efficacy is related to the dose and duration of treatment. However, the rate of sustained hepatitis D virus (HDV) clearance after a 1-year course with high doses of standard IFN is low. Better results have recently been reported with pegylated IFN both in IFN-naïve and in previous nonresponders to standard IFN, suggesting the use of pegylated IFN as a first-line therapy in chronic hepatitis D. Nucleoside analogues that inhibit hepatitis B virus (HBV) are ineffective against HDV and combination therapy with lamivudine or ribavirin has not shown significant advantages over monotherapy with either standard or pegylated IFN. Because the ultimate goal of treatment is eradication of both HDV and HBV, in responders IFN therapy should be continued as long as possible until the loss of hepatitis B surface antigen, adjusting the dose to patient tolerance. However, because side-effects are common, continuous monitoring is mandatory. Although the first results obtained with pegylated IFN have been encouraging, the rate of sustained virological response is still low and the rate of relapse high, emphasizing the need for developing novel classes of antivirals specifically interfering with the life cycle of this unique virus.

Predicting response to peginterferon alpha-2a, lamivudine and the two combined for HBeAg-negative chronic hepatitis B.

OBJECTIVE: In a trial of patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B, 24 week post-treatment biochemical and virological response rates with peginterferon alpha-2a with or without lamivudine were significantly higher than with lamivudine alone. The effect of pre-treatment factors on post-treatment responses was investigated. METHODS: Multivariate analyses were performed using available data from 518 patients treated with peginterferon alpha-2a with or without lamivudine, or with lamivudine alone. A post-treatment response was defined as alanine aminotransferase (ALT) normalisation and hepatitis B virus (HBV) DNA level of <20,000 copies/ml. RESULTS: In logistic regression analyses across all treatment arms, peginterferon alpha-2a (with or without lamivudine) therapy, younger age, female gender, high baseline ALT, low baseline HBV DNA and HBV genotype were identified as significant predictors of combined response at 24 weeks post-treatment. In the peginterferon alpha-2a and lamivudine monotherapy arms, patients with genotypes B or C had a higher chance of response than genotype D infected patients (p<0.001), the latter responding better to the combination than to peginterferon alpha-2a monotherapy (p = 0.015). At 1 year post-treatment, response rates by intention-to-treat analysis were 19.2% for the peginterferon alpha-2a, 19.0% for the combination, and 10.0% for the lamivudine groups, with genotypes B or C associated with a sustained combined response to peginterferon alpha-2a with or without lamivudine therapy. CONCLUSIONS: Baseline ALT and HBV DNA levels, patient age, gender, and infecting HBV genotype significantly influenced combined response at 24 weeks post-treatment, in patients treated with peginterferon alpha-2a and/or lamivudine. At 1 year post-treatment HBV genotype was significantly predictive of efficacy for patients treated with peginterferon alpha-2a with or without lamivudine.

The economics of treating chronic hepatitis C patients with peginterferon alpha-2a (40 kDa) plus ribavirin presenting with persistently normal aminotransferase.

Peginterferon alpha-2a (40 kDa) plus ribavirin is effective at achieving sustained viral response compared with no treatment in patients with chronic hepatitis C (CHC) and persistently normal aminotransferase levels (PNALT). The cost-effectiveness of treating CHC in the setting of PNALT has not been assessed. Disease progression in patients with PNALT was simulated in a Markov model. The rate of fibrosis progression, quality of life and costs for each health state were based on literature estimates. The perspective of the Italian National Health Service was adopted and costs (euro 2003) and benefits were discounted at 3%. Sensitivity analyses were performed on important parameters. The primary analysis compared combination therapy with peginterferon alpha-2a (40 kDa) plus ribavirin to no treatment in a cohort of patients with mean age 45 years, and was based on findings from a multinational, randomized trial in patients with PNALT. In genotype 1 patients, the risk of cirrhosis at 30 years is forecast to fall from 32% with no treatment to 19% with combination therapy, increasing quality-adjusted life years (QALYs) by 0.74 years at an incremental cost per QALY gained of euro 16,831. The 30-year risk of cirrhosis in genotype 2 or 3 is projected to fall to 9% with combination therapy, an increase in QALYs of 1.34 years, at an incremental cost per QALY gained of euro 3,000. Thus treatment of PNALT with peginterferon alpha-2a (40 kDa) plus ribavirin is projected to reduce the incidence of cirrhosis, increase life expectancy and have an acceptable cost-effectiveness ratio from a societal perspective.

Hepatitis C virus. The importance of viral heterogeneity.

Although recent evidence indicates that the quasispecies nature of HCV constitutes a critical strategy for the virus to survive in the host, the mechanisms of viral persistence remain unknown. Similarly, the correlates of immune protection in a limited proportion of individuals who succeed in clearing HCV are still largely undefined. Understanding the mechanisms of sterilizing immunity is essential for devising preventive measures against HCV and unraveling how the virus eludes such immunity. As in other viral infections, the complex interactions between the virus and the host early in the course of HCV infection probably determine the outcome of the disease (i.e., resolution or persistence). The evidence now accumulated on HCV and other models of viral infection is compatible with the hypothesis that both cellular and humoral components are needed for definitive viral clearance. Nevertheless, detailed studies of the specific cellular and humoral immune responses during the incubation period and the acute phase of hepatitis C, in relation to the viral quasispecies evolution and the clinical outcome, are still lacking both in humans and in the chimpanzee model. Until such studies are performed, most ideas of viral clearance mechanisms remain hypothetical, and the immunologic basis of HCV clearance will continue to be inferred from associations rather than from causal relationships.

Clinical significance of hepatitis C virus genotypes and quasispecies.

Hepatitis C virus (HCV) is a major cause of morbidity and mortality worldwide. The infection becomes chronic in about 85% of infected individuals, in the face of a strong humoral and cellular immune response. One of the most important features of HCV is its high degree of genetic variability, which is due to the inherent low fidelity of the viral replication machinery. As a consequence, HCV circulates in vivo as a population of divergent, albeit closely related, genomes exhibiting a distribution that follows the model referred to as a quasispecies. The genetic variability of HCV is complex and has been classified into four hierarchical strata: genotypes, subgenotypes, isolates, and quasispecies. Over the past few years, an extraordinary interest has been focused on the biologic and clinical implications of the genetic variability of HCV. Although there is consensus that the genotypes may influence the out come of antiviral therapy, their clinical significance in the natural history of the disease, as well as in transmission, infectivity, and pathogenesis of HCV infection, remains elusive. Conversely, evidence has accumulated that the quasispecies nature of HCV provides a large reservoir of biologically different viral variants that may have important clinical implications for viral persistence by immune escape mechanisms, for the generation of antiviral drug resistance, and for the development of an effective vaccine. This article reviews the state of the art on the biologic and clinical implications of the genetic variability of HCV.

The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies.

The mechanisms by which hepatitis C virus (HCV) induces chronic infection in the vast majority of infected individuals are unknown. Sequences within the HCV E1 and E2 envelope genes were analyzed during the acute phase of hepatitis C in 12 patients with different clinical outcomes. Acute resolving hepatitis was associated with relative evolutionary stasis of the heterogeneous viral population (quasispecies), whereas progressing hepatitis correlated with genetic evolution of HCV. Consistent with the hypothesis of selective pressure by the host immune system, the sequence changes occurred almost exclusively within the hypervariable region 1 of the E2 gene and were temporally correlated with antibody seroconversion. These data indicate that the evolutionary dynamics of the HCV quasispecies during the acute phase of hepatitis C predict whether the infection will resolve or become chronic.

Independent expression of serological markers of thyroid autoimmunity and hepatitis virus C infection in the general population: results of a community-based study in north-western Sardinia.

To assess the relationship between serological markers of thyroid autoimmunity and chronic hepatitis C, we surveyed the general population of two villages in the region of Sardinia, Italy, where infection with hepatitis viruses is endemic and the prevalence of autoimmune diseases is elevated. A total of 1310 subjects aged 6-88 years (65% of the total resident population) participated in the survey, and 1233 (94%; 444 males and 789 females) agreed to provide a blood sample. Autoantibodies to thyroid peroxidase (anti-TPO) were measured by radioimmunoassay; antibodies to HCV (anti-HCV) by a third generation enzyme immunoassay and borderline positive results confirmed by recombinant immunoblot assay. For both anti-HCV and anti-TPO the age- and gender-standardized prevalence rates (SPR) were calculated and the significance of the association between the two antibodies tested by Yates corrected chi2 test. The overall SPR for anti-HCV was 50.7x10(-3) (86/1,233), similar between men [49.1x10(-3) (22/444)] and women [52.3x10(-3) (64/789)]. The overall SPR for anti-TPO was 136.9x10(-3) (204/1,233), and that among women [201x10(-3) (174/789)] was almost 3-fold that among men [71.6x10(-3) (30/444)]. A concurrent anti-HCV and anti-TPO positivity was found in a small minority of subjects [8/1,233 (0.65%)], all women aged 57-81 years. The SPR for the two concurrent events was 3.3x10(-3), which was not significantly different (Yates corrected chi2 test = 0.65) from that expected under the assumption of unrelated events. To explore whether HCV infection is a risk factor for anti-TPO positivity, we designed a case-control study with anti-TPO positive subjects as the cases, and anti-TPO negative subjects as the controls. The age- and gender-adjusted odd ratio (OR) was 0.4 (95% CI 0.2,0.7), indicating a negative association. In conclusion, no evidence for epidemiological association of circulating thyroid autoantibodies and antibodies to HCV was found. Our findings do not therefore support a pathogenetic link between HCV infection and thyroid autoimmunity.

Longitudinal analysis of serum chemokine levels in the course of HIV-1 infection.

OBJECTIVES: To investigate the correlation between the serum levels of the CC-chemokines RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, and the progression of HIV-1 disease. DESIGN: Retrospective analysis of serial serum samples from HIV-1 seroconverters selected according to clinical outcome. METHODS: Twenty-one patients, derived from a cohort recruited between 1985 and 1996 for a prospective study of the natural history of HIV infection, were analysed. All patients had at least one HIV-1-seronegative sample within 1 year prior to the first seropositive test and were followed longitudinally throughout the course of HIV-1 infection (mean follow-up, 73.5 months). Nine were rapid progressors (RP; patients who developed AIDS within 60 months of antibody seroconversion), seven were slow progressors (SP; patients who developed AIDS after 60 months), and five were long-term asymptomatic (LTA; patients with circulating CD4+ cells higher than 400 x 10(6)/l, no signs of HIV disease, no antiretroviral therapy for more than 96 months). A total of 339 serum samples was studied (mean, 16.1 per patient). The levels of RANTES, MIP-1alpha and MIP-1beta were measured by enzyme-linked immunosorbent assay and correlated with different immunological and clinical parameters. RESULTS: Over the entire follow-up period, the geometric mean of serum RANTES was significantly higher in RP [68.6 ng/ml; 95% confidence interval (CI), 56.9-82.7] than in SP (23.7 ng/ml; 95% CI, 20.0-28.2; P < 0.001) and LTA (19.5 ng/ml; 95% CI, 15.5-24.5; P < 0.001). This difference was already significant during the early clinical stages, when patients had peripheral blood CD4+ cell counts still greater than 400 x 10(6)/l (P < 0.001). By contrast, the mean serum levels of MIP-1alpha and MIP-1beta did not differ significantly between the three study groups. Multivariate analysis using the Cox proportional hazard model demonstrated that the mean serum concentration of RANTES before the development of AIDS was independently associated with the time to AIDS (relative risk, 4.5; 95% CI, 1.1-18.2; P = 0.035). In patients with low versus high mean serum RANTES before the fall of CD4+ cells below 400 x 10(6)/l, the median AIDS-free time was 117.5 and 42.7 months, respectively (P = 0.037). CONCLUSION: These data suggest that an elevation of serum RANTES predicts a rapid progression of the disease since the early stages of HIV-1 infection.

Experimental transmission of hepatitis C virus-associated fulminant hepatitis to a chimpanzee.

Hepatitis C virus (HCV) was transmitted from a patient with fulminant hepatitis C to a chimpanzee. The patient had developed two episodes of fulminant hepatitis C, each occurring after a separate liver transplantation. Serial serum and liver samples from the patient and the chimpanzee were analyzed for HCV replication, genotype, quasispecies heterogeneity, and antibodies. In the patient, the levels of HCV replication in serum and liver correlated with the degree of hepatocellular necrosis and the clinical expression of fulminant hepatitis. The same HCV strain, genotype 1a, was recovered from both episodes of fulminant hepatitis. An unusually severe acute hepatitis was also observed in the chimpanzee. The viruses recovered from the patient and the chimpanzee were almost identical and displayed relatively little quasispecies heterogeneity. Thus, the same HCV strain induced two episodes of fulminant hepatitis in a single patient and severe hepatitis in a chimpanzee, suggesting that the pathogenicity or virulence of a specific HCV strain may be important in the pathogenesis of fulminant hepatitis C.

Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein.

The identification of the neutralization domains of hepatitis C virus (HCV) is essential for the development of an effective vaccine. Here, we show that the hypervariable region 1 (HVR1) of the envelope 2 (E2) protein is a critical neutralization domain of HCV. Neutralization of HCV in vitro was attempted with a rabbit hyperimmune serum raised against a homologous synthetic peptide derived from the HVR1 of the E2 protein, and the residual infectivity was evaluated by inoculation of HCV-seronegative chimpanzees. The source of HCV was plasma obtained from a patient (H) during the acute phase of posttransfusion non-A, non-B hepatitis, which had been titered for infectivity in chimpanzees. The anti-HVR1 antiserum induced protection against homologous HCV infection in chimpanzees, but not against the emergence of neutralization escape mutants that were found to be already present in the complex viral quasispecies of the inoculum. The finding that HVR1 can elicit protective immunity opens new perspectives for the development of effective preventive strategies. However, the identification of the most variable region of HCV as a critical neutralization domain poses a major challenge for the development of a broadly reactive vaccine against HCV.

A hyperimmune serum against a synthetic peptide corresponding to the hypervariable region 1 of hepatitis C virus can prevent viral infection in cell cultures.

To investigate whether a principal neutralization epitope exists in hypervariable region 1 (HVR1) within the putative envelope of hepatitis C virus (HCV), we generated a hyperimmune rabbit serum against a synthetic peptide corresponding to HVR1 of HCV isolate H77. The reactivity of the serum in the enzyme-linked immunosorbent assay was correlated with the 13 amino acids (position 398-410) in HVR1. The serum prevented infection with H77 virus in cell cultures but did not prevent infection with H90 virus, a genetically divergent isolate from the same patient. The study demonstrated that neutralization of HCV was mediated, in part, by isolate-specific antibody recognizing HVR1.

Immunity elicited by hepatitis C virus.

Hepatitis C virus (HCV), the major causative agent of post-transfusion and community-acquired non-A, non-B (NANB), is a single-stranded RNA virus characterized by a high degree of genetic heterogeneity. HCV is endemic worldwide and is a major cause of chronic liver disease and hepatocellular carcinoma. The development of a broadly reactive vaccine is a high priority for the control of HCV infection. In recent years, however, serious concerns have been raised regarding the degree of protective immunity elicited by HCV in the host. Several observations, both in patients and in the chimpanzee model, have suggested a lack of protective immunity against HCV. Chronic HCV infection develops in more than 80% of patients, suggesting that in most cases the immune response of the host fails to mediate resolution of the infection. Cross-challenge studies demonstrated that convalescent chimpanzees are not protected against re-infection with homologous or heterologous HCV strains. Similar evidence has been obtained in polytransfused beta-thalassemic children, in whom re-infection with HCV was associated with multiple episodes of acute hepatitis. Although most of the evidence thus far accumulated suggests that HCV does not elicit a protective immune response, recent studies have provided experimental evidence, both in vitro and in vivo, that HCV infection induces a neutralizing antibody response in humans. However, such antibodies are isolate-restricted and ineffective against variant HCV strains emerging in vivo. Recently, using recombinant envelope proteins of HCV, a successful vaccination of chimpanzees against challenge with a homologous viral strain was reported. Whether this vaccine can provide protection against challenge with a higher infectious dose of the homologous virus or against challenge with heterologous strains of HCV remains to be established. Overall, the data hitherto accumulated indicate that the genetic heterogeneity of HCV will be a major impediment for the development of a broadly reactive vaccine for the control of HCV infection.

Acute viral hepatitis in Saudi Arabia: seroepidemiological analysis, risk factors, clinical manifestations, and evidence for a sixth hepatitis agent.

We conducted a prospective, descriptive cohort study of all 217 cases of acute viral hepatitis (AVH) seen in adults during 1992 at the sole hospitals with infectious disease departments in the second and third largest cities in the Kingdom of Saudi Arabia. In addition, we undertook a nested case-control study. Our goals were (1) to determine the causes, demographics, risk factors, and clinical characteristics of AVH in the Kingdom; (2) to evaluate the reliability of diagnostic tests for acute hepatitis C and E; and (3) to assess the relative importance, characteristics, and risk factors of a sixth hepatitis agent, non-A-E. All cases and controls completed a questionnaire. Cases provided blood samples for studies of serum bilirubin, alanine and aspartate aminotransferases, and antibody to hepatitis viruses as well as genome detection studies. The results of serological and molecular tests were used to categorize each case as hepatitis A, B, C, D, E, or non-A-E. Historical, clinical, and laboratory determinants were statistically analyzed by comparisons between groups with different types of AVH and controls. Analysis of risk factors suggested that hepatitis C and D were parenterally transmitted, while hepatitis A, E, and non-A-E were not; the route of transmission of hepatitis B was unclear. Hepatitis E was strongly associated with living or traveling on the Indian subcontinent. The clinical disease caused by all six agents was indistinguishable. The putative sixth agent caused 13% of cases. The second-generation tests for antibody to HCV and HEV were relatively reliable for the diagnosis of AVH.

Growth of macrophage-tropic and primary human immunodeficiency virus type 1 (HIV-1) isolates in a unique CD4+ T-cell clone (PM1): failure to downregulate CD4 and to interfere with cell-line-tropic HIV-1.

Human immunodeficiency virus type 1 (HIV-1) isolates derived directly from clinical samples are usually unable to grow in cytokine-independent continuous cell lines, thus hindering the study of their biological features and their sensitivity to humoral and cellular protective immunity. To overcome these limitations, we have derived from the Hut78 T-cell line a CD4+ clone (PM1) characterized by a unique susceptibility to a wide range of HIV-1 isolates, including primary and biologically pure macrophage (M phi)-tropic isolates (e.g., HIV-1BaL), which are unable to infect other human T- or promonocytic cell lines. Both primary and M phi-tropic HIV-1 establish persistent infection in PM1, with sustained levels of virus replication for prolonged periods. Experiments with chimeric viruses containing envelope fragments of HIV-1BAL inserted into the genetic framework of HXB2, a molecular clone derived from the cell-line-tropic isolate HIV-1IIIB, showed the third hypervariable domain (V3) of gp120 to be a critical determinant of the cell line tropism of HIV-1. Nevertheless, the V3 loop of HIV-1BaL was not sufficient to confer on the chimeras a bona fide M phi tropism. The biological characteristics of HIV-1BaL and of a primary isolate (HIV-1(573)) were investigated by using the PM1 clone. Infection of PM1 by HIV-1BaL was critically dependent on the CD4 receptor, as shown by competition experiments with an anti-CD4 monoclonal antibody (OKT4a) or with soluble CD4. However, the amount of soluble CD4 required for inhibition of HIV-1BaL was approximately 100-fold higher than for HIV-1IIIB, suggesting that the affinity of HIV-1BaL for CD4 is significantly lower. Infection of PM1 with either HIV-1BaL or HIV-1(573) failed to induce downregulation of surface CD4 expression and syncytium formation. Analogous results were obtained with a chimeric virus (HXB2[BaL PvuII-BamHI]) encompassing a large portion of gp120 and gp41 of HIV-1BaL, indicating that the env genes contain critical determinants for CD4 downregulation and syncytium formation. Consistent with the lack of CD4 downregulation, persistent infection of PM1 by HIV-1BaL or HIV-1(573) failed to interfere with HIV-1IIIB superinfection, as revealed by the expression of a type-specific V3 loop epitope (M77) and by the induction of extensive syncytium formation. This lack of interference suggests that a direct viral interaction may occur in vivo between biologically diverse HIV-1 strains.(ABSTRACT TRUNCATED AT 400 WORDS)

Hepatitis type C after orthotopic liver transplantation: reinfection and disease recurrence.

We determined the prevalence of hepatitis C virus markers and the clinical course in patients transplanted for terminal type C or non-A, non-B cirrhosis. Hepatitis C virus infection recurred in 16 of 17 patients (94%) with type C cirrhosis (seropositive for hepatitis C virus prior to surgery) and in 10 of 11 patients (91%) with non-A, non-B cirrhosis whose hepatitis C virus status prior to surgery had not been determined. Markers of hepatitis C virus were detected in 4 of 16 liver transplants whose donors tested negative for hepatitis C virus prior to surgery; this figure represents the risk of hepatitis C virus acquisition from external sources at or after transplantation. In 18 of 26 reinfected patients aminotransferases increased after grafting and remained elevated throughout the 14 to 79 (mean 46.5) months of follow up. The histological findings varied from mild or moderate hepatitis in 15 patients to severe active hepatitis in two patients. Two patients developed cirrhosis; one of them died of intercurrent infection while she was receiving immunosuppressive therapy for chronic rejection. Patients transplanted for hepatitis C virus or non-A, non-B liver disease are at high risk of hepatitis C virus reinfection. However the course of recurrent hepatitis C is most often mild and compatible with a normal life and an excellent survival rate.

Prevention of hepatitis C virus infection in chimpanzees after antibody-mediated in vitro neutralization.

Hepatitis C virus (HCV) is the most important etiologic agent of non-A, non-B hepatitis and is a major cause of chronic liver disease and hepatocellular carcinoma. Development of an effective vaccine would be the most practical method for prevention of the infection, but whether infection with HCV elicits protective immunity in the host is unclear. Neutralization of HCV in vitro was attempted with plasma of a chronically infected patient, and the residual infectivity was evaluated by inoculation of eight seronegative chimpanzees. The source of HCV was plasma obtained from a patient during the acute phase of posttransfusion non-A, non-B hepatitis, which had previously been titered for infectivity in chimpanzees. Neutralization was achieved with plasma obtained from the same patient 2 yr after the onset of primary infection but not with plasma obtained 11 yr later, although both plasmas contained antibodies against nonstructural and structural (including envelope) HCV proteins. Analysis of sequential viral isolates from the same patient revealed significant genetic divergence as early as 2 yr after infection. However, the HCV recovered from the patient 2 yr after the infection had a striking sequence similarity with the HCV recovered from one of the chimpanzees inoculated with the acute-phase virus, suggesting that the progenitor of the new strain was already present 2 yr earlier. This evidence, together with the different sequences of HCV recovered from the chimpanzees that received the same inoculum, confirms that HCV is present in vivo as a quasispecies. These results provide experimental evidence in vivo that HCV infection elicits a neutralizing antibody response in humans but suggest that such antibodies are isolate-specific. This result raises concerns for the development of a broadly reactive vaccine against HCV.