Distribution of hepatitis c virus (hcv) genotypes in patients with chronic infection from Rondonia, Brazil

Background: Hepatitis C virus (HCV) is an important human pathogen affecting around 3% of the human population. In Brazil, it is estimated that there are approximately 2 to 3 million HCV chronic carriers. There are few reports of HCV prevalence in Rondonia State (RO), but it was estimated in 9.7% from 1999 to 2005. The aim of this study was to characterize HCV genotypes in 58 chronic HCV infected patients from Porto Velho, Rondonia (RO), Brazil. Methods: A fragment of 380 bp of NS5B region was amplified by nested PCR for genotyping analysis. Viral sequences were characterized by phylogenetic analysis using reference sequences obtained from the GenBank (n = 173). Sequences were aligned using Muscle software and edited in the SE-AL software. Phylogenetic analyses were conducted using Bayesian Markov chain Monte Carlo simulation (MCMC) to obtain the MCC tree using BEAST v. 1.5.3. Results: From 58 anti-HCV positive samples, 22 were positive to the NS5B fragment and successfully sequenced. Genotype 1b was the most prevalent in this population (50%), followed by 1a (27.2%), 2b (13.6%) and 3a (9.0%). Conclusions: This study is the first report of HCV genotypes from Rondonia State and subtype 1b was found to be the most prevalent. This subtype is mostly found among people who have a previous history of blood transfusion but more detailed studies with a larger number of patients are necessary to understand the HCV dynamics in the population of Rondonia State, Brazil.

Development of Hepatitis C Virus Genotyping by Real-Time PCR Based on the NS5B Region

Background: Hepatitis C virus (HCV) genotyping is the most significant predictor of the response to antiviral therapy. The aim of this study was to develop and evaluate a novel real-time PCR method for HCV genotyping based on the NS5B region. Methodology/Principal Findings: Two triplex reaction sets were designed, one to detect genotypes 1a, 1b and 3a; and another to detect genotypes 2a, 2b, and 2c. This approach had an overall sensitivity of 97.0%, detecting 295 of the 304 tested samples. All samples genotyped by real-time PCR had the same type that was assigned using LiPA version 1 (Line in Probe Assay). Although LiPA v. 1 was not able to subtype 68 of the 295 samples (23.0%) and rendered different subtype results from those assigned by real-time PCR for 12/295 samples (4.0%), NS5B sequencing and real-time PCR results agreed in all 146 tested cases. Analytical sensitivity of the real-time PCR assay was determined by end-point dilution of the 5000 IU/ml member of the OptiQuant HCV RNA panel. The lower limit of detection was estimated to be 125 IU/ml for genotype 3a, 250 IU/ml for genotypes 1b and 2b, and 500 IU/ml for genotype 1a. Conclusions/Significance: The total time required for performing this assay was two hours, compared to four hours required for LiPA v. 1 after PCR-amplification. Furthermore, the estimated reaction cost was nine times lower than that of available commercial methods in Brazil. Thus, we have developed an efficient, feasible, and affordable method for HCV genotype identification.

A Novel Hepatitis C Virus Genotyping Method Based on Liquid Microarray

The strategy used to treat HCV infection depends on the genotype involved. An accurate and reliable genotyping method is therefore of paramount importance. We describe here, for the first time, the use of a liquid microarray for HCV genotyping. This liquid microarray is based on the 5'UTR - the most highly conserved region of HCV - and the variable region NS5B sequence. The simultaneous genotyping of two regions can be used to confirm findings and should detect inter-genotypic recombination. Plasma samples from 78 patients infected with viruses with genotypes and subtypes determined in the Versant (TM) HCV Genotype Assay LiPA (version I; Siemens Medical Solutions, Diagnostics Division, Fernwald, Germany) were tested with our new liquid microarray method. This method successfully determined the genotypes of 74 of the 78 samples previously genotyped in the Versant (TM) HCV Genotype Assay LiPA (74/78, 95%). The concordance between the two methods was 100% for genotype determination (74/74). At the subtype level, all 3a and 2b samples gave identical results with both methods (17/17 and 7/7, respectively). Two 2c samples were correctly identified by microarray, but could only be determined to the genotype level with the Versant (TM) HCV assay. Genotype ""1'' subtypes (1a and 1b) were correctly identified by the Versant (TM) HCV assay and the microarray in 68% and 40% of cases, respectively. No genotype discordance was found for any sample. HCV was successfully genotyped with both methods, and this is of prime importance for treatment planning. Liquid microarray assays may therefore be added to the list of methods suitable for HCV genotyping. It provides comparable results and may readily be adapted for the detection of other viruses frequently co-infecting HCV patients. Liquid array technology is thus a reliable and promising platform for HCV genotyping.

Social Networks Shape the Transmission Dynamics of Hepatitis C Virus

Hepatitis C virus (HCV) infects 170 million people worldwide, and is a major public health problem in Brazil, where over 1% of the population may be infected and where multiple viral genotypes co-circulate. Chronically infected individuals are both the source of transmission to others and are at risk for HCV-related diseases, such as liver cancer and cirrhosis. Before the adoption of anti-HCV control measures in blood banks, this virus was mainly transmitted via blood transfusion. Today, needle sharing among injecting drug users is the most common form of HCV transmission. Of particular importance is that HCV prevalence is growing in non-risk groups. Since there is no vaccine against HCV, it is important to determine the factors that control viral transmission in order to develop more efficient control measures. However, despite the health costs associated with HCV, the factors that determine the spread of virus at the epidemiological scale are often poorly understood. Here, we sequenced partial NS5b gene sequences sampled from blood samples collected from 591 patients in Sao Paulo state, Brazil. We show that different viral genotypes entered Sao Paulo at different times, grew at different rates, and are associated with different age groups and risk behaviors. In particular, subtype 1b is older and grew more slowly than subtypes 1a and 3a, and is associated with multiple age classes. In contrast, subtypes 1a and 3b are associated with younger people infected more recently, possibly with higher rates of sexual transmission. The transmission dynamics of HCV in Sao Paulo therefore vary by subtype and are determined by a combination of age, risk exposure and underlying social network. We conclude that social factors may play a key role in determining the rate and pattern of HCV spread, and should influence future intervention policies.

Molecular Characterization, Distribution, and Dynamics of Hepatitis C Virus Genotypes in Blood Donors in Colombia

Hepatitis C virus (HCV) is a frequent cause of acute and chronic hepatitis and a leading cause for cirrhosis of the liver and hepatocellular carcinoma. HCV is classified in six major genotypes and more than 70 subtypes. In Colombian blood banks, serum samples were tested for anti-HCV antibodies using a third-generation ELISA. The aim of this study was to characterize the viral sequences in plasma of 184 volunteer blood donors who attended the ""Banco Nacional de Sangre de la Cruz Roja Colombiana,`` Bogota, Colombia. Three different HCV genomic regions were amplified by nested PCR. The first of these was a segment of 180 bp of the 5`UTR region to confirm the previous diagnosis by ELISA. From those that were positive to the 5`UTR region, two further segments were amplified for genotyping and subtyping by phylogenetic analysis: a segment of 380 bp from the NS5B region; and a segment of 391 bp from the E1 region. The distribution of HCV subtypes was: 1b (82.8%), 1a (5.7%), 2a (5.7%), 2b (2.8%), and 3a (2.8%). By applying Bayesian Markov chain Monte Carlo simulation, it was estimated that HCV-1b was introduced into Bogota around 1950. Also, this subtype spread at an exponential rate between about 1970 to about 1990, after which transmission of HCV was reduced by anti-HCV testing of this population. Among Colombian blood donors, HCV genotype 1b is the most frequent genotype, especially in large urban conglomerates such as Bogota, as is the case in other South American countries. J. Med. Virol. 82: 1889-1898, 2010. (C) 2010 Wiley-Liss, Inc.

Molecular evidence of horizontal transmission of hepatitis C virus within couples

Hepatitis C virus (HCV) transmission has decreased with the adoption of universal blood donor screening and social policies to reduce the risk of infection in intravenous drug users, but remains a worldwide health problem. The objective of this study was to evaluate the phylogenetic relationships among sequences from different HCV genomic regions from sexual partners of infected patients. Nine couples with a stable relationship and without other risk factors for HCV infection and 42 control patients were selected, and the NS3 and NS5B regions were analysed. Phylogenetic analysis showed that viruses from five of the couples had a common origin, clustering in the same monophyletic group, with bootstrap values greater than 70. For the other couples, monophyletic groups were observed, but without bootstrap support. Thus, using two different viral genome regions, a common source of infection was observed in both members of five couples. These data strongly support HCV transmission within couples.

Post-transplant recurrent hepatitis C: immunohistochemical detection of hepatitis C virus core antigen and possible pathogenic implications

Introduction: The mechanisms by which severe cholestatic hepatitis develops after liver transplantation are not fully understood. Reports on immunohistochemical distribution of hepatitis C virus (HCV) antigens are still scarce, but recently, HCV immunostaining was suggested for early diagnosis of cholestatic forms of recurrent hepatitis C in liver grafts. After purification, Rb246 pab anticore (aa1-68) yielded specific, granular cytoplasmic staining in hepatocytes. Signal amplification through the Envision-Alkaline Phosphatase System avoided endogenous biotin and peroxidase. Aims/Methods: Rb246 was applied to liver samples of explants of 12 transplant recipients, six with the most severe form of post-transplantation recurrence, severe cholestatic hepatitis (group 1) and six with mild recurrence (group 2). We also assessed immuno-reactivity at two time-points post-transplantation (median 4 and 22 months) in both groups. HCV-core Ag was semiquantified from 0 to 3+ in each time point. Serum HCV-RNA was also measured on the different time points by branched DNA. Results: In the early post-transplant time point, one patient had a mild staining (1+), two patients had a moderate staining (2+) and the other three had no staining in group 1, compared with five patients with no staining (0) and one patient with mild staining (1+) in group 2. Late post-transplant liver samples were available in nine patients, and two out of four samples in group 1 showed a mild staining, compared with no staining patients in five patients in group 2. Strikingly, on the explant samples, HCV immunostaining was strongly positive in group 1, and mildly positive in group 2. Two out of five samples showed 3+ staining, and three samples showed 2+ staining in group 1; two out of five samples showed no staining, two samples showed 1+ staining and one sample showed 2+ staining in group 2. Serum HCV-RNA was significantly higher in group 1, on both time-points post-transplantation. HCV-core Ag was not directly associated with serum HCV-RNA on the different time points. Conclusion: These preliminary results suggest that strong HCV immunostaining in the explant is predictive of more severe disease recurrence.

Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection in children and adolescents

Background & Aims: This multi-center study aimed to prospectively evaluate the safety and efficacy of a genotype-based Pegylated Interferon alfa-2a/Ribavirin therapy in treatment-naive hepatitis C virus (HCV), positive HCV serology, and quantifiable HCV RNA, infected children. Methods: Eighteen children with genotypes 2 and 3 patients (group A) were assigned to medication for 24 weeks, and 47 children with genotypes 1, 4, 5 and 6 patients (group B) for 48 weeks. Results: Early response at week 12 was observed in 83% of group A patients and in 57% of group B patients (p <0.05). End of treatment response was achieved in 94% of patients in group A and in 57% in group B (p <0.001). Sustained virologic response was maintained in 89% of patients in group A and in 57% of patients in group B (p <0.01). Ten patients stopped prematurely the treatment, 2 for serious adverse event (acute hepatitis and thyrotoxicosis), and 8 because of no virologic response at week 24. Peginterferon alfa-2a and Ribavirin dose was adjusted in 15 patients (23%), 11 for neutropenia (17%), and 3 patients (5%), for anemia, respectively. Treatment-related adverse events included fever and flu-like symptoms (54%), irritability depression change of mood (34%), vomiting (23%), abdominal pain (38%), loss of appetite (21.5%) and dermatitis (29%). No influence on height growth was observed. Conclusions: Pegylated inteferon alfa-2a and Ribavirin treatment allowed to achieve SVR in 57% of pediatric patients with genotypes 1, 4, 5 and 6, and in 94% of genotypes 2 and 3. These results show an improved SVR as compared to reference series in adults with similar regimen. (c) 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Conservation of hepatitis C virus nonstructural protein 3 amino acid sequence in viral isolates during liver transplantation

As a consequence of selective pressure exerted by the immune response during hepatitis C virus (HCV) infection, a high rate of nucleotide mutations in the viral genome is observed which leads to the emergence of viral escape mutants. The aim of this study was to evaluate the evolution of the amino acid (aa) sequence of the HCV nonstructural protein 3 (NS3) in viral isolates after liver transplantation. Six patients with HCV-induced liver disease undergoing liver transplantation (LT) were followed up for sequence analysis. Hepatitis C recurrence was observed in all patients after LT. The rate of synonymous (dS) nucleotide substitutions was much higher than that of nonsynonymous (dN) ones in the NS3 encoding region. The high values of the dS/dN ratios suggest no sustained adaptive evolution selection pressure and, therefore, absence of specific NS3 viral populations. Clinical genotype assignments were supported by phylogenetic analysis. Serial samples from each patient showed lower mean nucleotide genetic distance when compared with samples of the same HCV genotype and subtype. The NS3 samples studied had an N-terminal aa sequence with several differences as compared with reference ones, mainly in genotype 1b-infected patients. After LT, as compared with the sequences before, a few reverted aa substitutions and several established aa substitutions were observed at the N-terminal of NS3. Sites described to be involved in important functions of NS3, notably those of the catalytic triad and zinc binding, remained unaltered in terms of aa sequence. Rare or frequent aa substitutions occurred indiscriminately in different positions. Several cytotoxic T lymphocyte epitopes described for HCV were present in our 1b samples. Nevertheless, the deduced secondary structure of the NS3 protease showed a few alterations in samples from genotype 3a patients, but none were seen in 1b cases. Our data, obtained from patients under important selective pressure during LT, show that the NS3 protease remains well conserved, mainly in HCV 3a patients. It reinforces its potential use as an antigenic candidate for further studies aiming at the development of a protective immune response.

Cutaneuos cryoglobulinemic vasculitis induced by chronic hepatitis C virus infection

Cutaneous vasculitis may represent a great clinical challenge, even after careful dermatological examination and laboratory assessment. The authors present a case of cutaneous cryoglobulinemic vasculitis associated to chronic hepatitis C virus infection, pointing out the importance of the dermatological examination for diagnostic investigation. They discuss about the importance of defining the etiology and making correct classification for appropriate prognosis and treatment of cutaneous vasculitis.

The increasing problem of hepatitis C virus infection

The widespread incidence of hepatitis C (HCV) infection throughout the community is of concern. Although many of those infected will not suffer significantly from their infection, up to one-third will have liver disease, fatigue and oral health problems. General dental practitioners need to be aware of the precautions necessary in treating people with severe liver disease. This paper discusses the issues associated with treating patients who have HCV infection including the importance of preventive programs to reduce dental pathology and maximise oral health.

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV Proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles. At times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

Inducible system in human hepatoma cell lines for hepatitis C virus production

We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetra cycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 In to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect naive Huh7 and stable Huh7-human CD81 cells. (C) 2002 Elsevier Science (USA).