Hepatitis C viral quasispecies

Analysing significant numbers of cDNA clones of the hepatitis C virus (HCV) from single isolates provides unquestionable proof that the viral genome cannot be defined by a single sequence, but rather by a population of variant sequences closely related to one another. This way of organizing the genetic information is referred to as quasispecies. Throughout HCV infection, the number and composition of the variants in the viral population keeps changing owing to environmental influences, resulting in a virus that is constantly redefining itself both genetically and phenotypically. Therefore, the virus has often been investigated in population terms. Many clinical studies have tried to unravel, through the parameters that characterize the HCV quasispecies, prognostic markers of the disease and its response to treatment. Other investigations have focused on discovering how the virus and host interact during chronic infection. The consensus sequence, the rate of fixation of mutations and the complexity of the viral population are useful parameters for describing the viral population behaviour and its interaction with the host. In addition to sequencing, several other methods, based on electrophoretic mobility, have been used to study these parameters, such as temperature gradient-gel electrophoresis, single-strand conformation polymorphism and gel-shift analysis. The viral region examined, the source of clinical specimen, as well as the methodology employed, will be decisive in interpreting the information obtained.     

Viral, host and interferon-related factors modulating the effect of interferon therapy for hepatitis C virus infection

The estimated prevalence of hepatitis C virus infection in the US is approximately 1.8%. Although interferon monotherapy and combination therapy of interferon with ribavirin represent mainstay for treating HCV infection, the rate of sustained virologic response remains suboptimal. The growing evidence suggested that the clinical sequence and treatment response of chronic hepatitis C are determined by a dynamic, complex tripartite relationship among HCV infection, the host immune response, and the effect of different interferon regimens. The treatment response is associated with various viral factors including the pretreatment viral level, dynamic change of viral level during treatment, viral genotype quasispecies and nucleotide mutation in nonstructural protein 5A of hepatitis C virus. Host factors that may affect treatment response include age, gender, race, HLA alleles and the host immune responses. Interferon regimens, including type, dose, frequency and duration of treatment and combination of interferon with other anti-HCV agents also alter the therapeutic response. Understanding these complicated interaction may provide better insights into the mechanism(s) of interferon response, leading to more effective clinical application of interferon therapy.     

Therapy Implications of Hepatitis C Virus Genetic Diversity

Hepatitis C virus (HCV) is an important human pathogen with a high chronicity rate. An estimated 71 million people worldwide are living with chronic hepatitis C (CHC) infection, which carries the risk of progression to hepatic fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Similar to other RNA viruses, HCV has a high rate of genetic variability generated by its high mutation rate and the actions of evolutionary forces over time. There are two levels of HCV genetic variability: intra-host variability, characterized by the distribution of HCV mutant genomes present in an infected individual, and inter-host variability, represented by the globally circulating viruses that give rise to different HCV genotypes and subtypes. HCV genetic diversity has important implications for virus persistence, pathogenesis, immune responses, transmission, and the development of successful vaccines and antiviral strategies. Here we will discuss how HCV genetic heterogeneity impacts viral spread and therapeutic control.     

Hepatitis C virus infection: in vivo and in vitro models

Summary.  Major advances in the understanding of the molecular biology of hepatitis C virus (HCV) have been made recently. While the chimpanzee is the only established animal model of HCV infection, several in vivo and in vitro models have been established that allow us to study various aspects of the viral life cycle. In particular, the replicon system and the production of recombinant infectious virions revolutionized the investigation of HCV-RNA replication and rendered all steps of the viral life cycle, including entry and release of viral particles, amenable to systematic analysis. In the following we will review the different in vivo and in vitro models of HCV infection.     

Hepatitis C virus infection diagnosis using metabonomics

Summary. Metabonomics based on nuclear magnetic resonance (NMR) can reveal the profile of endogenous metabolites of low molecular weight in biofluids related to disease. The profile is identified a ‘metabolic fingerprint’ like from the pathological process, why this metabonomics has been used as a diagnostic method. The aim of the present study was to apply metabonomics to identify patients infected with the hepatitis C virus (HCV) through an analysis of ¹H NMR spectra of urine samples associated with multivariate statistical methods. A pilot study was carried out for the diagnostic test evaluation, involving two groups: (i) 34 patients positive for anti‐HCV and HCV‐RNA and negative for anti‐HBc (disease group); and (ii) 32 individuals positive for anti‐HBc and negative for HBsAg and anti‐HCV. The urine samples were analyzed through ¹H NMR, applying principal component analysis and discriminant analysis for classification. The metabonomics model was capable of identifying 32 of the 34 patients in the disease group as positive and 31 of the 32 individuals in the control group as negative, demonstrating 94% sensitivity and specificity of 97% as well as positive and negative predictive values of 97% and 94%, respectively, and 95% accuracy (P < 0.001). In conclusion, the metabonomics model based on ¹H NMR spectra of urine samples in this preliminary study discriminated patients with HCV infection with high sensitivity and specificity, thereby demonstrating this model to be a potential tool for use in medical practice in the near future.     

Hepatitis C virus infection among HIV-1 infected individuals from northern Mexico

Aims: The prevalence of hepatitis C virus (HCV) infection, risk factors and HCV genotypes in 140 HIV-1 infected individuals from northern Mexico was determined. Methods: Hepatitis C infection was confirmed by the detection of anti-HCV antibodies and HCV-RNA in sera, and genotyping was performed by the InnoLiPA-HCV genotype assay. Results: Seventeen (12.1%) out of 140 HIV-infected individuals were found to be HCV-positive. Coinfected individuals were more likely to be male (87%). The most frequent genotype was 1a (41%), followed by 1b (29.4%), 2a/c (17.6%), 2b (5.9%) and 3 (5.9%). Serum transaminase concentrations (AST and ALT) were higher in coinfected patients. Among the risk factors for coinfection: sexual transmission was the most frequently observed (men who have sex with men (MSM); 64.7% and bisexual behavior; 64.7%) followed by intravenous drug users (IVDU) (53%). There was no association of the HCV genotypes with the age and risk factors for HIV-1 and HCV infection observed in the studied patients. Conclusion: The results suggest that the prevalence of HIV-1/HCV coinfection in Mexico is lower than in other American countries.     

Hepatitis C virus infection in patients with clinically diagnosed alcoholic liver diseases

Summary To determine the incidence of hepatitis C virus (HCV) infection in patients with alcoholic liver disease (AID), serum samples from 252 patients with AID were tested for anti-HCV and HCV RNA. Serial sera of these patients were collected and stored under optimal conditions to allow exact quantification of HCV RNA. Fifteen patients who visited our hospital during the same period of time with chronic HCV infections served as controls. In those with AID, anti-HCV and HCV RNA were positive in 55.5% and 41.2%, respectively. Patients with histologically diagnosed chronic hepatitis and hepatocellular carcinoma had much higher prevalence rates of HCV RNA (84% and 100%, respectively) compared to those with fatty liver (4.3%), hepatic fibrosis (10.1%) and alcoholic hepatitis (22.2%) ( P < 0.01). Although no difference in serum HCV RNA levels was observed between the patients with both AID and chronic HCV infection and those with chronic HCV infection alone, HCV RNA levels significantly (10-fold) dropped after abstinence in nearly half of the patients ( P < 0.01). These data indicate that HCV infection in patients with AID promotes progression of liver disease, and abstinence from alcohol is associated with a reduction in serum HCV RNA levels.     

Hepatitis C virus infection and genotypes in Japanese hemophiliacs

Abstract: Liver function and antibodies to hepatitis C virus and to human immunodeficiency virus-1 were examined in 195 Japanese patients with hemophilia. One hundred and seventy-three were positive for antibody to HCV and 61 for antibody to human immunodeficiency virus-1. In 63 patients, we examined HCV genotypes according to the double polymerase chain reaction method. Forty cases (63%) were infected with hepatitis C virus with a single genotype, including type 1a in five, type 1b in 21, type 2a in seven and type 2b in seven; 16 (25%) were infected with double genotypes, including types 1a+1b in 14, types 1b+2a in one and types 1b+2b in one; and four (6%) were infected with triple genotypes, including types 1a+1b+2a in two and types 1a+1b+2b in two. Genotype could not be determined in three patients by this method. In the 191 non-hemophiliac patients with chronic hepatitis C, HCV genotyping was as follows: type 1a in 0, type 1b in 121, type 2a in 40 and type 2b in 10 of 171 cases (89.5%) with single infection and types 1b+2a in five and types 2a+2b in one of six (5.5%) with double infection. In the remaining 14 patients, genotype could not be determined. Frequent transfusion of domestic and/or imported coagulation factor concentrates probably caused the high incidence of HCV infection with rare or mixed genotypes in Japanese hemophiliacs.     

Antibodies to a novel antigen in acute hepatitis C virus infections

BACKGROUND: Conformational viral proteins potentially play an important role in the immunobiology of acute hepatitis C virus (HCV) infection and may enable earlier antibody detection. MATERIALS AND METHODS: HCV RNA was detected using nucleic acid testing. Early antibody production was evaluated using three enzyme immunoassays (EIAs) containing antigenic proteins not present in licensed EIAs. Respectively, these contained: (1) multiple-epitope fusion antigen (MEFA) 7.1-NS3/4a, (2) F and Core, and (3) E1/E2 proteins. NS3/4a is a conformational antigen retaining protease and helicase enzymatic activities. MEFA 7.1 contains the linear epitopes used in licenced EIAs, including the latest EIA-3.0, in combination with genotype 1-3 specific epitopes. Forty-two RNA positive, EIA-3.0 negative samples, including two persistently serosilent cases, were used to evaluate these research EIAs. As controls, 54 EIA-3.0 negative/RNA negative and three HCV RNA+/antibody positive specimens were included. RESULTS: Only the MEFA 7.1-NS3/4a EIA was positive in seven (17%) of the 42 HCV RNA + specimens, in all three EIA-3.0 positive controls but in none of 54 EIA-3.0 negative/HCV RNA negative controls. Notably, six of the seven (86%) specimens had evidence of active hepatitis (ALT > 210 IU/l). The two serosilent cases were research EIA negative. CONCLUSION: A novel EIA with conformational and linear epitopes detected HCV antibodies in 17% of viraemic specimens missed by the standard reference EIA-3.0. Our research EIA appears to detect HCV antibodies closer to the initiation of acute hepatitis. Given that the average RNA-positive, antibody-negative window period is 56.4 days, this 17% yield would translate into a 10-day earlier detection of antibodies.     

Serological response to infection with different isolates of hepatitis C virus

Different isolates of hepatitis C virus (HCV) show nucleotide sequence variability throughout the genome. Detection of antibodies to recombinant proteins derived from hepatitis C virus genotype 1, the prototype HCV clone HCV-PT, constitutes the main method for screening HCV infection. The influence of the genomic variability on the serological diagnosis of HCV by enzyme immunoassay remains poorly defined. The aim of this study was to assess the serological reactivity of a panel of well characterized French HCV isolates typed by sequence analysis from patients with chronic hepatitis. The 73 sera samples were tested in three third generation EIA tests and three confirmatory assays. HCV isolates were determined by RT-PCR and sequencing in NS5B region of the genome. The 73 sera were positive in the three EIA tests. The three confirmatory tests showed a weaker reactivity with NS5 protein whatever the genotype, and a lower reactivity in NS4 antigens of non-type 1 sequences, particularly for genotype 3. Even though the reactivity of the antigens differed among the HCV isolates, the 73 isolates from genotype 1-6 were reactive with the three commercial screening assays. These results demonstrate that using a single test is adequate in the routine diagnosis of HCV infection in clinical laboratory, as recommended by the last French and European consensus conference.     

Hepatitis C virus infection in renal failure patients in the absence of anti-hepatitis C virus antibodies

The magnitude and clinical significance of Hepatitis C virus (HCV) infection in dialysis patients is controversial and underestimated. This study was conducted in order to evaluate the correlation between HCV replication and antibody response to HCV in dialysis patients. HCV infection in dialysis patients was evaluated over a period of 3 years and compared to HCV infection in Liver Clinic patients. Sera were collected from 310 dialysis patients and tested for anti-HCV and HCV-RNA. In addition, HCV genotype and HCV viral load were determined in HCV-RNA-positive sera. Anti-HCV was detected in 43 (14%) of the dialysis patients. Of these, 37 (86%) were HCV-RNA-positive. Among the 267 HCV-seronegative dialysis patients, 25 (9%) were found to be HCV-RNA-positive in more than one sample during the study. These patients were characterized by low viral load; at least two orders of magnitude lower than in the group of HCV-seropositives. In contrast, in the Liver Clinic patients, HCV-RNA was found exclusively in HCV-seropositive patients. Comparison of the genotype pattern in the two groups did not reveal a difference.   Our results suggest that HCV infection in dialysis units may be underestimated due to cases of low viral load, depending on the method of RNA extraction and sensitivity of the test used. Low viral load might contribute to the lack of humoral immune response seen in some dialysis patients.     

Hepatitis C virus infection in a resident elderly population: A 10-year follow-up study

BACKGROUND: The natural history of hepatitis C virus infection in the elderly is poorly known. OBJECTIVE: To assess the mortality rate, the progression of liver disease, the hepatitis C virus carrier state and the co-morbidity in a cohort of 35 out of 1,063 anti-hepatitis C virus positive elderly people prospectively followed-up from 1992 to 2002. METHODS: Liver function tests, hepatitis C virus-RNA analysis, hepatitis C virus genotyping and abdominal ultrasonography were assessed at the beginning of the study, and then, liver function tests and ultrasonography were performed annually during the first 5 years of the follow-up. At the end of the 10-year period, causes of death were recorded, while surviving patients underwent again medical examination, liver function tests and abdominal ultrasonography. RESULTS: Out of 35 patients with a 10-year follow-up, 12 patients died: only 2 (5.7%) from liver-related disease (hepatocellular carcinoma and liver failure), whilst 10 (28.5%) from extrahepatic causes. Out of the two patients dying from liver-related causes, one was hepatitis C virus-RNA positive and one hepatitis C virus-RNA negative. Among the 23 living patients, 13 were hepatitis C virus-RNA positive (56.5%), the majority being infected with genotype 2 (69%); of them, 6 (46.1%) had persistently normal alanine aminotransferase levels. None of the hepatitis C virus-RNA positive individuals had excessive alcohol intake. CONCLUSION: Despite the presumably long duration of infection in our cohort, the liver-related mortality was five-fold lower than that from extrahepatic causes (five-fold higher). Lack of hepatic co-morbidity factors, such as alcohol consumption, seems to be relevant for the limited severity of liver disease.     

Viral hepatitis: virus/host interaction

Abstract   Hepatitis A virus is considered directly cytopathic to the liver cell. Severity of the liver damage is dictated by viral load. Acute infection is followed by sustained immunity to the virus. Hepatitis B (HBV) and C (HCV) viruses are noncytopathic, hepatotropic viruses that cause acute and chronic hepatitis and hepatoma. Cellular and humoral immune responses are responsible not only for viral clearance but also for hepatocyte damage. T-cell response to HBV is vigorous, polyclonal, and multispecific in acutely infected patients who clear the virus while it is weak and narrowly focused in chronically infected patients. It is mainly executed by cytotoxic T lymphocytes (CTL), which destroy infected hepatocytes and secrete antiviral cytokines that interrupt the HBV life cycle. T-cell response to HCV is strong and multispecific in both acutely and chronically infected patients. Whether HCV is susceptible to a cytokine-mediated type of control is unknown. The ability of HCV to persist despite a strong CTL response suggests that HCV is either less visible to the CTL or less responsive to cytokine-mediated antiviral signals than HBV. Both viruses, but especially HCV, have a high mutation rate, leading to the occurrence of variant viral genomes with growth advantage and the ability of escaping immune recognition.     

Hepatitis C virus infection of salivary gland epithelial cells: Lack of evidence

Background: Hepatitis C virus genome (HCV-RNA) has been detected in whole salivary gland tissue of chronically infected patients. However, contamination of the tissue by plasma or blood cells was not excluded by the previous reports.Aims: To assess whether HCV infects the salivary gland epithelial cells in patients with chronic HCV liver disease.Methods: Twenty unselected patients with chronic active hepatitis (11 cases) or active cirrhosis (nine cases) were examined. Serum and saliva samples were obtained from all patients, 12 of whom (seven, chronic active hepatitis; five, active cirrhosis) underwent salivary gland biopsy. PCR for HCV-RNA was performed on RNA extracted from serum, saliva and salivary gland epithelial cells collected by isokinetic gradient separation after trypsin digestion of whole salivary gland tissue. Saliva samples were also examined for the presence of secretory IgA anti-HCV by gel chromatography and ELISA testing.Results: HCV-RNA was detected in all sera with titers ranging from 5.42×10⁵ genome equivalents/ml to 123.2×10⁵ genome equivalents/ml. Thirteen patients were infected with genotype 1b, four patients had genotype 1a, two patients had genotype 2a and one patient was unclassifiable. Low titer HCV-RNA (<2×10⁵ genome equivalents/ml) was detected in saliva samples (15%) from highly viremic patients infected with 1b genotype. RNA extracted from salivary gland epithelial cells consistently tested negative for HCV-RNA. In addition, all saliva specimens tested negative for secretory-IgA (S-IgA) anti-HCV, even after a 10-fold concentration of the samples.Conclusions: There was no evidence that HCV infects the salivary gland epithelial cells in our viremic patients with HCV chronic liver disease. Low level HCV-RNA in saliva is most probably due to virus spillover from blood.     

Hepatitis C virus infection among healthy Egyptian children: prevalence and risk factors

Summary. The aim of this study was to estimate the prevalence and identify the risk factors of hepatitis C virus (HCV) infection among healthy Egyptian children. A representative random sample of 500 children, age between 6 and 15 years, was selected from 10 schools in Alexandria, Egypt. A questionnaire was used to collect demographic data and potential risk factors, while blood samples were collected and analyzed for antibodies to HCV (anti‐HCV). Positive sera were further confirmed by HCV‐RNA. HCV seroprevalence of 5.8% was found, with HCV viraemia in 75% of the studied children. The prevalence of anti‐HCV increased with age from 0% in children aged 6–7 years to 16% in those of 15 years old. It was also shown that history of previous blood transfusion (odds ratio[OR] = 34.8, 95% CI = 4.39–272.95), intravenous injections (OR = 4.68, 1.89–11.59), surgical intervention (OR = 5.64, 2.55–12.52), dental treatment (OR = 6.81, 2.64–17.39), injection (OR = 2.29, 1.08–4.89) and circumcision for boys by informal health care providers (OR = 2.6, 1.0–6.73), age above 10 years (OR = 6.83, 2.44–19.07), very low socioeconomic class (OR = 5.92, 1.3–25.2) and rural area residence (OR = 2.49,1.61–5.29) are the most significant risk factors for HCV infection. Adjusting for all other risk factors by multivariate logistic regression analysis, it has been shown that blood transfusion, surgical procedures, dental treatment, and age above 10 years are still significant risk factors associated with anti‐HCV (P < 0.05). The current study reveals the extremely high HCV seroprevalence among Egyptian children. This mandates immediate preventive strategies to limit further HCV spread.