Differences in HCV antibody patterns in haemodialysis patients infected with the same virus isolate.

Eight cases of de novo hepatitis C virus (HCV) infection in a haemodialysis unit in Rio de Janeiro, Brazil, were retrospectively studied. HCV viraemia was demonstrated by RT nested PCR in seven of the seroconverters. Genotyping showed that six patients were infected with a genotype 1b strain and one with a genotype 1a strain. A phylogenetic analysis of nucleotide sequences of the HCV core region revealed that five of the six 1b isolates form a separate cluster when compared with other 38 HCV 1b core sequences randomly chosen from the GenBank. The revealed sequence similarities indicated the nosocomial spread of a single HCV strain within the unit. To investigate whether the patients infected with the same viral isolate display similar patterns of antibody response to individual proteins, serial serum samples were examined. A line immunoassay for qualitative and semi-quantitative determination of specific antibodies against recombinant and synthetic HCV antigens was employed. Despite infection with the same virus strain, the patients sera demonstrated different patterns of reactivity against individual structural and nonstructural HCV proteins immediately after seroconversion. For each patient, however, antibody responses remained mostly stable throughout the follow-up of 8 to 24 months.

Hepatitis C virus variability: sequence analysis of an isolate after 10 years of chronic infection.

Hepatitis C virus (HCV) variability was analyzed based upon an isolate which had caused the infection of more than 2500 women in 1978/79. Genome consensus sequences of two isolates obtained from the infectious source (HCV-AD78) and from a chronic hepatitis patient 10 years after the acute infection were determined. The entire open reading frame (ORF) exhibited 3.2 x 10(-3) nucleotide substitutions per site per year (deltant). Core (0.7 x 10(-3) deltant) and NS5B (1.9 x 10(-3) deltant) were found to be most conserved genes, while E2 (4.7 x 10(-3) deltant) with hypervariable region 1 (HVR1) (23 x 10(-3) deltant) was the most variable followed by p7 (4.2 x 10(-3) deltant). In the entire ORF transitions were 4.5 times more frequent than transversions while for the HVR1 this bias was turned. As an indicator of relative selective pressure on the proteins the rates of nonsynonymous to synonymous substitutions (dN/dS) were determined. The obtained values exceeded 1.0 only for E2 (dN/dS = 1.3). A subdivision of the entire ORF into 88 overlapping sections, each containing 300 nucleotides, led to a more precise analysis of HCV diversity. Besides for E2 an increased variability was mainly detected for three other regions: (a) the C terminal neighbouring region of E2 including p7, (b) the genome fragment extending from approximately the middle of NS3 to NS4B, and (c) the segment corresponding to the C-terminus of the NS5A protein. The variable region in NS5A was situated carboxyterminal to the predicted interferon sensitivity determining region (ISDR). These results suggest which regions other than HVR1 might contribute to persistence of the virus by the mechanism of immunescape.

The "interferon sensitivity determining region" of hepatitis C virus is a stable sequence element.

BACKGROUND/AIMS: A sequence of 40 amino acids within the nonstructural protein 5A of hepatitis C virus (HCV) has been suggested to be an interferon sensitivity determining region (ISDR). The variations in the ISDR after 12-14 years of chronic infection and the correlation between ISDR and interferon response were studied in patients who were infected by the same HCV isolate. METHODS: We determined the HCV-ISDRs of 13 chronically infected patients by direct sequencing of polymerase chain reaction products. All patients were infected by isolate HCV-AD78, but differed with respect to their sensitivity to interferon. Four patients were complete responders, two patients were non-responders, and seven showed a partial response. RESULTS: The ISDR of HCV-AD78 differed from a prototypical HCV-1b sequence in one amino acid and was therefore classified as an intermediate type. Direct sequencing of the HCV-ISDRs of the patients 12-14 years after infection, but before interferon therapy, revealed a rate of 2.2x10(-3) nucleotide substitutions per site per year, resulting in only single intermediate type amino acid exchanges. All sequences ranked with the intermediate type. Moreover, during interferon treatment no selection to a wild type ISDR was observed in five partial responders. CONCLUSIONS: Within the homogeneous patient group examined here, no correlation was found between the ISDR and the interferon response. Recent studies found only a small number of mutant type ISDRs in Europe. Additionally, our results indicate that the ISDR is a stable sequence element. This provides an explanation for the divergent data relating to the importance of the ISDR in different geographical regions.

Antibodies directed to envelope proteins of hepatitis C virus outside of hypervariable region 1.

The relatively high variability of the hepatitis C virus (HCV) envelope proteins E1 and E2 suggests that parts of these proteins other than the hypervariable region 1 (HVR1) might be involved in the induction of virus neutralizing antibodies. To test this hypothesis, two HCV proteins, pE1 and pE2 delta, were generated by in vitro translation. They represent amino acids 174-337 of E1 and 411-688 of E2, respectively, of isolate HCV-AD78; the protein pE2 delta contained no HVR1. As a control, protein pG.HVR1, which represents amino acids 384-410 of HVR1 of isolate HCV-AD78, was expressed separately. These three proteins were used in an immunoprecipitation assay to detect the presence of antiviral antibodies in sera of patients infected with the same isolate of HCV (HCV-AD78). Sera were obtained 4-8 months postinfection from patients who later resolved an acute infection or developed chronic liver disease. A high prevalence of antibodies (up to 85.7%) against pE1 and pE2 delta could be detected in both groups of patients, suggesting that these forms of the HCV envelope proteins contain B-cell epitopes. The antibody responses against proteins pE1 and pE2 delta did not differ significantly between patients with resolving or chronic infection, whereas antibodies against protein pG.HVR1 were associated with resolution of infection. Rabbit antisera raised against pE1 and pE2 delta were tested for their ability to neutralize the binding of HCV to susceptible cells in tissue cultures. The results suggested that although a few B-cell epitopes outside of HVR1 can induce virus neutralizing antibodies, these antibodies are probably not associated with the resolution of infection.

Cloning and characterization of a complete open reading frame of the hepatitis C virus genome in only two cDNA fragments.

The synthesis of long cDNA molecules encoding the complete genome of RNA viruses has recently been demonstrated; this major improvement has numerous practical applications such as construction of infectious cDNA clones or study of sequence variability at the level of a single RNA molecule. Using hepatitis C virus (HCV) as a model, we established an RT-PCR technique for amplification of cDNA fragments with a length of about 5 kb. The RT reaction was carried out with a Moloney murine leukaemia virus reverse transcriptase lacking detectable RNase H activity. For PCR reactions an enzyme mix containing Taq and Pwo DNA polymerases was used. Hot start and addition of 5% DMSO were also important to efficiently achieve long PCR products. About 10(6) HCV genome equivalents/ml in serum were needed in order to amplify the HCV genome in only two cDNA fragments covering about 98% of the complete genome. Analysis of the HCV quasi-species is also possible by this method as shown by sequencing of the hypervariable region 1 (HVR1) after cloning of cDNAs. The integrity of the long cDNA clones was proven by (1) restriction analyses, (2) partial sequencing and (3) expression of respective gene products. In vitro transcribed cDNAs were translated in rabbit reticulocyte lysate. Structural and nonstructural HCV proteins were identified by immunoprecipitation using patient serum. These results suggest that the two cDNA clones encode a complete and functional open reading frame of HCV.