An infectious molecular clone of a Japanese genotype 1b hepatitis C virus.

We describe an infectious molecular clone of a Japanese genotype 1b strain of hepatitis C virus (HCV-N). The molecularly cloned sequence of HCV-N was compared with alignments of other HCV sequences, leading to the identification of 15 unique, nonconservative amino acid substitutions within the HCV-N open reading frame (ORF). These were repaired to the consensus genotype 1b residue, and the infectivity of RNA transcribed from the repaired clone was assessed by intrahepatic inoculation of a chimpanzee. Viral RNA was first detected in the serum of this chimpanzee 3 weeks following inoculation, and was intermittently present over the next 14 weeks. A strong and persistent anti-HCV serological response developed 13 weeks following inoculation, with seroconversion in the recombinant immunoblot assay (RIBA). A weaker, transient serological response, characterized by seroconversion in a third-generation enzyme-linked immunosorbent assay (ELISA) but not RIBA, occurred between weeks 1 and 5. This may have represented an anamnestic response to HCV antigens translated directly from the intrahepatically inoculated RNA, because the animal previously had undergone 2 unsuccessful attempts at rescue of HCV by intrahepatic RNA inoculation. There was neither biochemical nor histological evidence of liver disease. Although this is within the range of expected outcomes in an HCV-naive chimpanzee, prior immunologic priming may have modified the infection in this animal. The HCV-N clone is the first infectious molecular clone of HCV that is comprised entirely of genotype 1b sequence, and it contains an ORF sequence that is significantly divergent from that of a previously described genotype 1a/1b chimera.     

Epidemiological profile of 806 Italian children with hepatitis C virus infection over a 15-year period

BACKGROUND/AIMS: To evaluate the epidemiological profile of Italian children with hepatitis C virus (HCV) infection over a 15-year period. METHODS: Fifteen tertiary care centers, belonging to a national Observatory established in 1998, retrospectively/prospectively recruited 806 consecutive HCV-infected, otherwise healthy, children seen from 1990 to 2004. RESULTS: Seven hundred and sixty four were Italian and 42 from foreign countries. Newly-diagnosed cases declined from 332 in 1995-1999 to 196 in 2000-2004, while the proportion of foreign children rose from 3% to 13%. Transfusion-transmitted infection disappeared after 1992. Maternal infection (with drug abuse in 63% of cases in the North) has become the most important mode of HCV diffusion throughout Italy and the exclusive source for all children infected in 2000-2004. The prevalence of HCV genotypes 3 and 4 increased and that of genotype 1b decreased significantly (p<0.02). Male/female ratio was significantly (p<0.001) lower among vertically infected (0.6) than in transfused children (1.3). CONCLUSIONS: The number of children with newly-diagnosed HCV infection is declining in Italy and most post-transfusion cases are now young adults. Thus foreign children could significantly contribute to the reservoir of pediatric infection in years to come. New infections result from maternal transmission and seem to privilege females and genotypes 3 and 4.     

Prospective study of a high-intensity interferon-alpha regimen for chronic hepatitis C virus genotype 1b infection

BACKGROUND/AIMS: To evaluate efficacy of high-intensity interferon administration for patients chronically infected with hepatitis C virus genotype 1b, we administered interferon-alpha with different regimens according to viral load. METHODOLOGY: Eighty-eight patients with hepatitis C virus genotype 1b were treated with recombinant interferon alpha-2b. The 70 patients with pretreatment hepatitis C virus RNA concentration > or = 10(6) copies/mL were given 10(7) units of interferon daily for the first 8 weeks and then three times weekly for 16 weeks (group A). The 18 patients with smaller pretreatment hepatitis C virus RNA concentration received the same dose daily for the first 2 weeks and then three times weekly for 14 weeks (group B). We analyzed tolerance of therapy, responses, and long-term outcome in the two groups. RESULTS: Fifteen of 70 patients (21.4%) in group A could not continue treatment and dropped out, while all patients in group B completed the entire course of therapy. The rate of sustained response in group A was 10.0%, being significantly less than in group B (72.2%; p < 0.0001). However, 12 patients in group A showed a biochemical sustained response despite presence of viremia. Long-term outcome did not differ between groups. CONCLUSIONS: Many patients could not tolerate high-intensity therapy, which showed the limitation of tolerance of patients receiving interferon monotherapy. High-intensity therapy could not improve eradication of hepatitis C virus in patients with high pretreatment hepatitis C virus RNA concentration. However, this therapy may increase the rate of sustained biochemical response, improving long-term outcome.     

Outcome in a hepatitis C (genotype 1b) single source outbreak in Germany--a 25-year multicenter study

BACKGROUND/AIMS: The natural course of the hepatitis C virus genotype 1b (HCV-1b) infection is still unclear but important for therapeutic decisions. There are few unbiased long-term follow-up studies with known dates of infection. METHODS: Between August 1978 and March 1979, 14 HCV-1b contaminated batches of anti-D immunoglobulin had been administered to 2867 women for prophylaxis of rhesus isoimmunization throughout East Germany. We reexamined 1980 women, representing 70% of the total cohort of 15 centers. RESULTS: After application of the contaminated anti-D, 93% of the recipients developed an acute hepatitis C. After 25 years, 86% of the 1833 affected women still tested positive for hepatitis C virus antibodies and 46% for HCV RNA. Only nine (0.5%) had overt liver cirrhosis, 30 women (1.5%) developed pre-cirrhotic stages and one HCC was diagnosed. Ten (0.5%) died of HCV related complications, half of these related to additional comorbidity. In the last 5 years, a continuous, but low increase of fibrotic scores was observed. CONCLUSIONS: Young women without comorbidity may clear HCV (1b) infection in more than half of the cases, or develop mild chronic hepatitis C. We confirmed the low risk of progression to cirrhosis in this cohort within 25 years.     

Characterization of naturally occurring protease inhibitor-resistance mutations in genotype 1b hepatitis C virus patients

Background and aims

Protease inhibitor (PI)-resistant hepatitis C virus (HCV) variants may be present in substantial numbers in PI-untreated patients according to recent reports. However, influence of these viruses in the clinical course of chronic hepatitis C has not been well characterized.

Methods

The dominant HCV nonstructural 3 (NS3) amino acid sequences were determined in 261 HCV genotype 1b-infected Japanese patients before pegylated interferon plus ribavirin (PEG-IFN/RBV) therapy, and investigated the patients?? clinical characteristics as well as treatment responses including sustained virological response (SVR) rate. HCV-NS3 sequences were also determined in 39 non-SVR patients after completion of the therapy.

Results

Four single mutations (T54S, Q80K, I153V, and D168E) known to confer PI resistance were found in 35 of 261 patients (13.4%), and double mutations (I153V plus T54S/D168E) were found in 6 patients (2.3%). Responses to PEG-IFN/RBV therapy did not differ between patients with and without PI-resistance mutations (mutation group, SVR 48%; wild-type group, SVR 40%; P?=?0.38). On the other hand, two mutations appeared in two non-SVR patients after PEG-IFN/RBV therapy (I153V and E168D, 5.1%).

Conclusions

PI-resistance-associated NS3 mutations exist in a substantial proportion of untreated HCV-1b-infected patients. The impact of these mutations in the treatment of PIs is unclear, but clinicians should pay attention to avoid further development of PI resistance.     

Impact of weight-based ribavirin with peginterferon alfa-2b in African Americans with hepatitis C virus genotype 1

WIN-R (Weight-based dosing of pegINterferon alfa-2b and Ribavirin) was a multicenter, randomized, open-label, investigator-initiated trial involving 236 community and academic sites in the United States, comparing response to pegylated interferon (PEG-IFN) alfa-2b plus a flat or weight-based dose of ribavirin (RBV) in treatment-naive patients with chronic hepatitis C and compensated liver disease. Patients were randomized to receive PEG-IFN alfa-2b at 1.5 microg/kg/week plus flat-dose (800 mg/day) or weight-based-dose RBV (800 mg/day for weight <65 kg, 1000 mg/day for 65-85 kg, 1200 mg/day for >85-105 kg, or 1400 mg/day for >105-<125 kg). Sustained virologic response (SVR; undetectable [<125 IU/mL] hepatitis C virus [HCV] RNA at end of follow-up) in patients > or =65 kg was the primary end point. Low SVR rates have been reported among African American individuals, in whom there is a preponderance of HCV genotype 1. This subanalysis of WIN-R was conducted to evaluate the efficacy of weight-based dosing among African American individuals with genotype 1 infection enrolled in the trial. Of 362 African American patients in the primary efficacy analysis, 188 received RBV flat dosing and 174 received weight-based dosing. SVR rates were higher (21% versus 10%; P = 0.0006) and relapse rates were lower (22% versus 30%) in the weight-based-dose group than in the flat-dose group. Safety and rates of drug discontinuation were similar between the 2 groups. CONCLUSION: Weight-based dosing of RBV is more effective than flat dosing in combination with PEG-IFN alfa-2b in African American individuals with HCV genotype 1. Even with weight-based dosing, response rates in African American individuals are lower than reported in other ethnic groups.     

Peginterferon alpha-2b plus ribavirin for chronic hepatitis C virus mixed genotype infection

Background and aim. The treatment efficacy of peginterferon plus ribavirin for patients with HCV genotype 1 is inferior to that in patients with HCV genotype 2, but the efficacy among patients with mixed HCV genotype 1 + 2 is less clear. We compared the treatment outcome of peginterferon alpha-2b plus ribavirin among naïve chronic hepatitis C patients in Taiwan with HCV genotype 1 and 2, and mixed genotype 1 + 2.Material and methods. In this retrospective cohort study, 150 patients were treated with peginterferon alpha-2b once weekly, plus ribavirin, for 24 weeks. The endpoint was sustained virological response after receiving at least one dose of the study medication.Results. There were no differences in clinical characteristics among the 3 groups. There were significant differences in rapid virological response rate between patients with genotype 1 and genotype 2 (64.7 vs. 85.5%, respectively; p < 0.05) and a sustained virological response rate (55.9 vs. 83.6%, respectively; p = 0.001). The rapid virological response rate differed between the genotype 1 and mixed genotype 1 + 2 groups (64.7 vs. 85.2%, respectively; p < 0.05), but the sustained virological response rate was similar (55.9 vs. 74.1%; p = 0.101). Conclusions. Using peginterferon alpha-2b plus ribavirin for 24 weeks to treat patients with HCV genotype 1 + 2 achieved a 74.1% sustained virological response rate; the treatment efficacy was not inferior to patients with HCV genotype 1, but the percentage of liver cirrhosis in mixed genotype 1 + 2 group was higher to 22%, it is worth to be appropriately valued and studied.     

HIV-1 epidemic trends in rural south-west Uganda over a 10-year period

The objective of this study was to examine the epidemic trends of HIV-1 infection in a rural population cohort in Uganda followed for 10 years. The methods used were to assess incidence and prevalence trends in adults in this longitudinal cohort study. The results showed that incidence of infection has fallen significantly in all adults, and separately in males, females, young adults and older adults over the course of the study period. There was also a reduction in prevalence, especially in young men and women. There was some evidence of a cohort effect in women. The conclusions are that this study provides the first evidence of a falling incidence in a rural general population in Africa. This was an observational cohort exposed to national health education messages, giving hope that similar campaigns elsewhere in Africa could be used effectively in efforts to control the HIV epidemic.     

Clearance of genotype 1b hepatitis C virus in chimpanzees in the presence of vaccine-induced E1-neutralizing antibodies

Accumulating evidence indicates that neutralizing antibodies play an important role in protection from chronic hepatitis C virus (HCV) infection. Efforts to elicit such responses by immunization with intact heterodimeric E1E2 envelope proteins have met with limited success. To determine whether antigenic sites, which are not exposed by the combined E1E2 heterodimer structure, are capable of eliciting neutralizing antibody responses, we expressed and purified each as separate recombinant proteins E1 and E2, from which the immunodominant hypervariable region (HVR-1) was deleted. Immunization of chimpanzees with either E1 or E2 alone induced antigen-specific T-helper cytokines of similar magnitude. Unexpectedly, the capacity to neutralize HCV was observed in E1 but not in animals immunized with E2 devoid of HVR-1. Furthermore, in vivo only E1-vaccinated animals exposed to the heterologous HCV-1b inoculum cleared HCV infection.     

SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders

BACKGROUND & AIMS: SCH 503034 is a novel and potent oral hepatitis C virus (HCV) protease inhibitor. In this phase Ib study, we assessed safety parameters and virologic response of combination of SCH 503034 plus pegylated (PEG) interferon (IFN) alpha-2b in patients with HCV genotype 1 infections who were previously nonresponders to PEG-IFN-alpha-2b +/- ribavirin therapy. METHODS: This was a multicenter, open-label, 2-dose level, 3-way crossover, randomized (to crossover sequence) study carried out in 3 medical centers in Europe. Adult patients received SCH 503034 200 mg (n = 14) or 400 mg (n = 12) 3 times daily orally and PEG-IFN-alpha-2b 1.5 microg/kg subcutaneously once each week. Patients received SCH 503034 as monotherapy for 1 week, PEG-IFN-alpha-2b as monotherapy for 2 weeks, and combination therapy for 2 weeks with washout periods between each treatment period. RESULTS: Combination therapy with SCH 503034 and PEG-IFN-alpha-2b was well tolerated, with no clinically significant changes in safety parameters. Mean maximum log(10) changes in HCV RNA were -2.45 +/- 0.22 and -2.88 +/- 0.22 for PEG-IFN-alpha-2b plus 200 mg and 400 mg SCH 503034, respectively, compared with -1.08 +/- 0.22 and -1.61 +/- 0.21 for SCH 503034 200 mg and 400 mg, respectively, and -1.08 +/- 0.22 and -1.26 +/- 0.20 for PEG-IFN-alpha-2b alone in the 200 mg and 400 mg SCH 503034 groups, respectively. CONCLUSIONS: SCH 503034 plus PEG-IFN-alpha-2b was well tolerated in patients with HCV genotype 1 nonresponders to PEG-IFN-alpha-2b +/- ribavirin. These preliminary results of antiviral activity of the combination suggest a potential new therapeutic option for this hard-to-treat, nonresponder patient population.     

Hepatitis C virus Core Antigen as a predictor of non-response in genotype 1 chronic hepatitis C patients treated with peginterferon alpha-2b plus ribavirin

BACKGROUND/AIMS: Chronic hepatitis C patients infected by genotype 1 are the least responsive to combination therapy and therefore monitoring response is important in identifying non-responders quickly, permitting therapy discontinuation and avoiding side effects and costs. We examined the usefulness of measuring total HCV Core Ag in early treatment with peginterferon alpha-2b and ribavirin in genotype 1 patients in the prediction of response and compared the results with those from HCV RNA quantification. METHODS: Two hundred and sixty-eight serum samples from 46 genotype 1 patients receiving combination therapy were examined for HCV Core Ag and quantitative HCV RNA. RESULTS: At baseline, mean HCV RNA and HCV Core Ag concentrations were significantly lower in sustained virologic responders than in non-responders. The negative predictive value of HCV Core Ag testing in predicting non-response at week 12 is 100%, and for a 2 log drop in HCV RNA, using two quantitative tests, it is 88%. CONCLUSIONS: HCV Core Ag determination allows the identification of non-responders with only one test at week 12 and permits stopping therapy in these patients. HCV Core Antigen testing is cheaper and easier to perform than HCV RNA quantification.     

Successful treatment of three patients with human immunodeficiency virus and hepatitis C virus genotype 1b co-infection by daclatasvir plus asunaprevir

Co-infection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) often accelerates the course of HCV-associated liver disease. Daclatasvir (DCV) plus asunaprevir (ASV) have been shown to be highly effective for HCV-infected patients with genotype 1b. Three patients co-infected with HIV/HCV genotype 1b were enrolled in this study. Prior to initiation of HCV treatment, the variants associated with L31 and Y93 in the non-structural protein 5A (NS5A) region of the HCV genome were confirmed to be absent using a direct sequencing method. Taking into consideration the lower risk of drug–drug interaction and the need for immediate treatment, the patients received 60 mg DCV once daily plus 100 mg ASV twice daily for 24 weeks. In one patient, the alanine aminotransferase level was elevated to 228 IU/L at 24 weeks after the start of treatment, but he completed the 24-week treatment course. All three patients achieved sustained viral response, without severe complications (including HIV virological rebound). Thus, in cases where NS5A variants are confirmed to be absent and patients are antiretroviral therapy-naïve, with CD4+ over 500/μL or HIV well controlled by RAL-based cART, DCV plus ASV may represent a good treatment option for HIV and HCV genotype 1b co-infected patients.     

Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in germany: a 20-year multicenter study

From August 1978 until March 1979, 14 batches of anti-D immune globulin contaminated with hepatitis C virus (HCV) genotype 1b (20, 000-480,000 copies/dose) from a single erythrocyte donor had been administered for prophylaxis of rhesus isoimmunization throughout East Germany. All 2,867 women involved had been recalled after January 12, 1979 for repeated screening of alanine transaminase (ALT). They were prospectively followed in regional centers. We have reexamined a cohort of 1,018 women (median age 24, range 16-38 years at infection) on follow-up for 20 years in 9 representative centers. Within 6 months after anti-D administration, 10% of these women had no evidence of disease and 90% had acute hepatitis C (n = 917) including 49% with symptomatic and 22% with icteric course. After 20 years, 85% of the 917 affected women still tested positive for HCV antibodies (among them 3% responded to interferon treatment) and 55% were positive for HCV RNA (among them 7% were nonresponders to interferon and 3% were apparent HCV carriers). Only 4 (0.4%) had overt cirrhosis. Two (0.2%) died of superinfected fulminant hepatitis B or alcoholism and cirrhosis, respectively. Histology obtained in 44% of the viremic women showed hepatitis of minimal to moderate grade in 96%, portal fibrosis in 47%, and septal fibrosis in 3% of the cases. In conclusion, formerly healthy young women, without hepatic comorbidity, may clear HCV (1b) infection in half of the cases or develop mild chronic hepatitis C with low risk of progression to cirrhosis within 20 years.     

Enhancement of genotype 1 hepatitis C virus replication by bile acids through FXR

BACKGROUND/AIMS: Hepatitis C virus (HCV) infected patients with high serum levels of bile acids (BAs) usually fail to respond to antiviral therapy. Besides, BAs are essential factors for replication of the porcine enteric calicivirus by inhibiting interferon signaling. The role of BAs on HCV RNA replication was thus assessed. METHODS: BAs and other compounds were tested using an HCV-replication model containing a luciferase reporter gene. RESULTS: BAs, especially chenodeoxycholate and deoxycholate, up-regulated genotype 1 HCV RNA replication by more than tenfold. Only free but not conjugated BAs were active, suggesting that their effect was mediated by a nuclear receptor. Only farnesoid X receptor (FXR) ligands stimulated HCV replication while FXR silencing and FXR antagonism by guggulsterone blocked the up-regulation induced by BAs. Furthermore, guggulsterone alone inhibited basal level of HCV replication by tenfold. Modulation of HCV replication by FXR ligands occurred in the same proportion in presence or absence of type I interferon, suggesting a mechanism of action independent of this control of viral replication. However, BAs or guggulsterone did not affect replication of genotype 2a-JFH1. CONCLUSIONS: Exposure to routinely measured concentrations of BAs increases HCV replication by a novel mechanism involving activation of the nuclear receptor FXR.     

The status of blood pressure over a 10-year period

The aim of this study is to relate the evolution of blood pressure (BP) measured every 5 years during 10 years in 12,824 healthy adults and children from both sexes. At the first evaluation, 2,856 were 4-15 years of age, 1,471 were 15-25, 6,590 were 25-45 and 1,907 were 45-65. The result of BP were adjusted according to possible effects of age, height or year of examination. Correlation coefficients at 5 and 10 years for the male patients are 0.38 and 0.34 (systolic BP and 0.23 and 0.18 (diastolic BP); for the female patients they are 0.36 and 0.31 (systolic BP) and 0.23 and 0.17 (diastolic BP). The reliability of the correlations increases according to age at first examination: for an initial age between 4 and 10 years, the correlation coefficient between first and last evaluation is 0.14 while for an initial age of 45-65 years it is 0.38 (systolic BP). The predictive value of a relatively high BP defined as percentage of patients whose systolic BP is staying in the upper fifth of the BP distribution ten years later is for increasing age group: 28.7%, 31.1%, 39.7%, 42.5%; that means a relative risk moving from 1.34 to 3.17. Sensitivity of detection of male patients whose systolic BP will be in the upper fifth of the distribution varies from 22 to 42% according to age, with a specificity of 86.3 to 82.2%. The results are sensibly similar in the female patients and for diastolic BP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains

Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases worldwide, but treatment options are limited. Basic HCV research required for vaccine and drug development has been hampered by inability to culture patient isolates, and to date only the JFH1 (genotype 2a) recombinant replicates spontaneously in hepatoma cells and releases infectious virus. A JFH1 chimera with the 5' end through NS2 from another genotype 2a strain, J6, had enhanced infectivity. However, the full-length J6 clone (J6CF), which we previously found to be fully functional in vivo, was replication incompetent in vitro. Through a systematic approach of culturing J6 with minimal JFH1 sequences, we identified three mutations in NS3, NS4A, and NS5B that permitted full-length J6 propagation and adaptation with infectivity titers comparable to JFH1-based systems. The most efficient recombinant, J6cc, had six adaptive mutations and did not accumulate additional changes following viral passage. We demonstrated that HCV NS3/NS4A protease-, NS5A- and NS5B polymerase-directed drugs respectively inhibited full-length J6 infection dose dependently. Importantly, the three J6-derived mutations enabled culture adaptation of the genetically divergent isolate J8 (genotype 2b), which differed from the J6 nucleotide sequence by 24%. The most efficient recombinant, J8cc, had nine adaptive mutations and was genetically stable after viral passage. The availability of these robust JFH1-independent genotype 2a and 2b culture systems represents an important advance, and the approach used might permit culture development of other isolates, with implications for improved individualized treatments of HCV patients and for development of broadly efficient vaccines.