Mapping of serotype-specific, immunodominant epitopes in the NS-4 region of hepatitis C virus (HCV):use of type-specific peptides to serologically differentiate infections with HCV types 1, 2, and 3

The effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenicity of the NS-4 protein was investigated by epitope mapping and by enzyme-linked immunosorbent assay with branched oligopeptides. Epitope mapping of the region between amino acid residues 1679 and 1768 in the HCV polyprotein revealed two major antigenic regions (1961 to 1708 and 1710 to 1728) that were recognized by antibody elicited upon natural infection of HCV. The antigenic regions were highly variable between variants of HCV, with only 50 to 60% amino acid sequence similarity between types 1, 2, and 3. Although limited serological cross-reactivity between HCV types was detected between peptides, particularly in the first antigenic region of NS-4, type-specific reactivity formed the principal component of the natural humoral immune response to NS-4. Type-specific antibody to particular HCV types was detected in 89% of the samples from anti-HCV-positive blood donors and correlated almost exactly with genotypic analysis of HCV sequences amplified from the samples by polymerase chain reaction. Whereas almost all blood donors appeared to be infected with a single virus type (97%), a higher proportion of samples (40%) from hemophiliacs infected from transfusion of non-heat-inactivated clotting factor contained antibody to two or even all three HCV types, providing evidence that long-term exposure may lead to multiple infection with different variants of HCV

Hepatitis C virus genotype frequency in Isfahan province of Iran: a descriptive cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Hepatitis C is an infectious disease affecting the liver, caused by the hepatitis C virus (HCV). The hepatitis C virus is a small, enveloped, single-stranded, positive sense RNA virus with a large genetic heterogeneity. Isolates have been classified into at least eleven major genotypes, based on a nucleotide sequence divergence of 30-35%. Genotypes 1, 2 and 3 circulate around the world, while other genotypes are mainly restricted to determined geographical areas. Genotype determination of HCV is clinically valuable as it provides important information which can be used to determine the type and duration of therapy and to predict the outcome of the disease.</p> <p>Results</p> <p>Plasma samples were collected from ninety seven HCV RNA positive patients admitted to two large medical laboratory centers in Isfahan province (Iran) from the years 2007 to 2009. Samples from patients were subjected to HCV genotype determination using a PCR based genotyping kit. The frequency of HCV genotypes was determined as follows: genotype 3a (61.2%), genotype 1a (29.5%), genotype 1b (5.1%), genotype 2 (2%) and mixed genotypes of 1a+3a (2%).</p> <p>Conclusion</p> <p>Genotype 3a is the most frequent followed by the genotype 1a, genotype 1b and genotype 2 in Isfahan province, Iran.</p

Geographical distribution of hepatitis C virus genotypes in blood donors:an international collaborative survey

The frequency of infection with the six classified major genotypes of hepatitis C virus (HCV) was investigated in 447 infected volunteer blood donors from the following nine countries: Scotland, Finland, The Netherlands, Hungary, Australia, Egypt, Japan, Hong Kong, and Taiwan. Viral sequences in plasma from blood donors infected with HCV were amplified in the 5'-noncoding region and were typed by restriction fragment length polymorphism analysis. Electrophoresis of DNA fragments produced by cleavage with HaeIII-RsaI and ScrFI-HinfI allowed HCV types 1 (or 5), 2, 3, 4, and 6 to be identified. Further analysis with MvaI-HinfI allowed sequences of the type 5 genotype to be distinguished from sequences of type 1 genotype. Types 1, 2, and 3 accounted for almost all infections in donors from Scotland, Finland, The Netherlands, and Australia. Types 2 and 3 were not found in the eastern European country (Hungary), where all but one of the donors were infected with type 1. Donors from Japan and Taiwan were infected only with type 1 or 2, while types 1, 2, and 6 were found in those from Hong Kong. HCV infection among Egyptians was almost always by type 4. Donors infected with HCV type 1 showed broad serological reactivity with all four antigens of the second generation Chiron RIBA-2 assay (Chiron Corporation, Emeryville, Calif.), while infection with divergent HCV genotypes elicited antibodies mainly reactive to c22-3 and c33c. Reactivities with antibodies 5-1-1 and c100-3 were infrequent and were generally weak, irrespective of the geographical origin of the donor. Because the envelope region of HCV is even more variable than the NS-4 region, it is likely that vaccines based on these proteins need to be multivalent and perhaps specifically adapted for different geographical regions.link_to_subscribed_fulltex

Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68 to 79 % overall sequence similarity to one another. Phylogenetic analysis ofnucleofide sequences derived from part of the gene encoding a non-structural protein (NS-5) has provided evidence for six major genotypes of HCV amongst a worldwide collection of 76 samples from HCV-infected blood donors and patients with chronic hepatitis. Many of these HCV types comprised a number of more closely related subtypes, leading to a current total of 11 genetically distinct viral populations. Phylo-genetic analysis of other regions of the viral genome produced relationships between published sequences equivalent to those found in NS-5, apart from the more highly conserved 5 &apos; non-coding region in which only the six major HCV types, but not subtypes, could be differentiated. A new nomenclature for HCV variants is proposed in this communication that reflects the twotiered nature of sequence differences between different viral isolates. The scheme classifies all known HCV variants to date, and describes criteria that would enable new variants to be assigned within the classification as they are discovered

A Possible Geographic Origin of Endemic Hepatitis C Virus 6a in Hong Kong: Evidences for the Association with Vietnamese Immigration

BACKGROUND: Hepatitis C virus (HCV) 6a accounts for 23.6% of all HCV infections of the general population and 58.5% of intravenous drug users in Hong Kong. However, the geographical origin of this highly predominant HCV subgenotype is largely unknown. This study explores a hypothesis for one possible transmission route of HCV 6a to Hong Kong. METHODS: NS5A sequences derived from 26 HCV 6a samples were chosen from a five year period (1999-2004) from epidemiologically unrelated patients from Hong Kong. Partial-NS5A sequences (513-bp from nt 6728 to 7240) were adopted for Bayesian coalescent analysis to reconstruct the evolutionary history of HCV infections in Hong Kong using the BEAST v1.3 program. A rooted phylogenetic tree was drawn for these sequences by alignment with reference Vietnamese sequences. Demographic data were accessed from "The Statistic Yearbooks of Hong Kong". RESULTS: Bayesian coalescent analysis showed that the rapid increase in 6a infections, which had increased more than 90-fold in Hong Kong from 1986 to 1994 correlated to two peaks of Vietnamese immigration to Hong Kong from 1978 to 1997. The second peak, which occurred from 1987 through 1997, overlapped with the rapid increase of HCV 6a occurrence in Hong Kong. Phylogenetic analyses have further revealed that HCV 6a strains from Vietnam may be ancestral to Hong Kong counterparts. CONCLUSIONS: The high predominance of HCV 6a infections in Hong Kong was possibly associated with Vietnamese immigration during 1987-1997

Serum Islet Cell Autoantibodies During Interferon α Treatment in Patients With HCV-Genotype 4 Chronic Hepatitis

Chronic hepatitis C virus (HCV) infection is a leading cause of end-stage liver disease worldwide and HCV genotype 4 (HCV4) is predominant in African and Middle Eastern countries. It is well established that interferon-α (IFNa) treatment for HCV may trigger serum autoantibodies against pancreatic islet cells (ICA) in a subgroup of patients. Available data on the incidence of ICA during IFNa therapy for chronic HCV4 infection are not conclusive. We investigated the appearance of ICA in 40 naïve Egyptian patients (38 males, 32 ± 6 years) with histologically defined chronic HCV4 infection undergoing IFNa treatment at a dose of 9-million U/week for 24 weeks. Serum samples were collected at baseline and following IFNa therapy and ICA were detected using indirect immunofluorescence. Baseline evaluation indicated that 2/40 (5%) patients had detectable serum ICA. After the completion of the treatment scheme, 12/38 (32%) previously ICA negative patients became ICA positive; however, no patient developed impaired glucose tolerance (IGT) or diabetes during follow-up. In conclusion, we submit that IFNa treatment for chronic hepatitis C (CHC) may induce serum ICA in one-third of Egyptian patients with HCV4. These autoantibodies, however, do not lead to alterations in glucose metabolism

Lysosomotropic agents as HCV entry inhibitors

HCV has two envelop proteins named as E1 and E2 which play an important role in cell entry through two main pathways: direct fusion at the plasma membrane and receptor-mediated endocytosis. Fusion of the HCV envelope proteins is triggered by low pH within the endosome. Lysosomotropic agents (LA) such as Chloroquine and Ammonium chloride (NH4Cl) are the weak bases and penetrate in lysosome as protonated form and increase the intracellular pH. To investigate the antiviral effect of LA (Chloroquine and NH4Cl) on pH dependent endocytosis, HCV pseudoparticles (HCVpp) of 1a and 3a genotype were produced and used to infect liver cells. The toxicological effects of Chloroquine and NH4Cl were tested in liver cells through MTT cell proliferation assay. For antiviral screening of Chloroquine and NH4Cl, liver cells were infected with HCVpp of 3a and 1a genotype in the presence or absence of different concentrations of Chloroquine and NH4Cl and there luciferase activity was determined by using a luminometer. The results demonstrated that Chloroquine and NH4Cl showed more than 50% reduction of virus infectivity at 50 μM and 10 mM concentrations respectively. These results suggest that inhibition of HCV at fusion step by increasing the lysosomal pH will be better option to treat chronic HCV

In-vitro model systems to study Hepatitis C Virus

Hepatitis C virus (HCV) is a major cause of chronic liver diseases including steatosis, cirrhosis and hepatocellular carcinoma. Currently, there is no vaccine available for prevention of HCV infection due to high degree of strain variation. The current treatment of care, Pegylated interferon α in combination with ribavirin is costly, has significant side effects and fails to cure about half of all infections. The development of in-vitro models such as HCV infection system, HCV sub-genomic replicon, HCV producing pseudoparticles (HCVpp) and infectious HCV virion provide an important tool to develop new antiviral drugs of different targets against HCV. These models also play an important role to study virus lifecycle such as virus entry, endocytosis, replication, release and HCV induced pathogenesis. This review summarizes the most important in-vitro models currently used to study future HCV research as well as drug design