In vivo analysis of the 3' untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone.
Large sections of the 3' untranslated region (UTR) of hepatitis C virus (HCV) were deleted from an infectious cDNA clone, and the RNA transcripts from seven deletion mutants were tested sequentially for infectivity in a chimpanzee. Mutants lacking all or part of the 3' terminal conserved region or the poly(U-UC) region were unable to infect the chimpanzee, indicating that both regions are critical for infectivity in vivo. However, the third region, the variable region, was able to tolerate a deletion that destroyed the two putative stem-loop structures within this region. Mutant VR-24 containing a deletion of the proximal 24 nt of the variable region of the 3' UTR was viable in the chimpanzee and seemed to replicate as well as the undeleted parent virus. The chimpanzee became viremic 1 week after inoculation with mutant VR-24, and the HCV genome titer increased over time during the early acute infection. Therefore, the poly(U-UC) region and the conserved region, but not the variable region, of the 3' UTR seem to be critical for in vivo infectivity of HCV. (+info)
Hepatitis virus infection in haemodialysis patients from Moldavia.
BACKGROUND: Although the epidemiology of hepatitis B (HBV) and C (HCV) now seems well established for Western European countries, in Central and Eastern Europe < 50% of all dialysis centres routinely test for hepatitis C antibodies since testing is not available or is not applied to all patients. This study describes the prevalence, risk factors and clinical significance of HBV and HCV infection for the haemodialysis population of the North Eastern region of Romania, Moldavia. METHODS: The presence of HBV antigens was determined with an ELISA kit (Wellcome, Abbot) and HCV antibodies with the ELISA-3 Ortho-HCV, third generation test. The following individual data were collected: gender, age, duration of dialysis, rural/urban domicile, actual and previous HBV status, actual HCV status, known acute, clinically evident hepatitis episodes in the last 3 years, monthly alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) levels, complete biochemical hepatic assessment at the time of the study, transfusions for the past 3 years and family history. RESULTS: HBV and HCV prevalences were 17% (stable over the last 3 years) and 75%, respectively; co-infection was seen in 10% of the subjects. Hospitalization (nosocomial infection) for HBV, blood transfusions and duration on dialysis for HCV, emerged as the main risk factors for hepatitis infection. Socio-economic conditions appear to be equally important for HCV infection, since the prevalence was significantly higher among patients from rural, underdeveloped areas than urban areas (80.8 vs 60.3%), and infection was already present in a large proportion of patients (47%) before starting dialysis, without being related to previous disease duration or blood transfusions. HBV and/or HCV was not associated with a worse clinical or biochemical profile at the time of the study. However, infected patients had significantly more previous cytolytic episodes, with higher, transient increases in ALAT and ASAT levels. CONCLUSIONS: HCV infection is endemic among dialysis centres in Moldavia. Apart from previously well-known risk factors for hepatitis infection, our study demonstrates the negative impact of socio-economic underdevelopment. Simple measures such as enforced general asepsia rules, careful disinfection and equipment sterilization, routine testing of patients from economically disadvantaged areas and monthly, serial determination of hepatic enzymes should be the common practice in dialysis centres in Romania. (+info)
Restricted isotypic antibody reactivity to hepatitis C virus synthetic peptides in immunocompromised patients.
An enzyme immunoassay based on three synthetic peptides from the core, NS4, and NS5 regions of hepatitis C virus allowed the detection of antibodies in 100% of immunocompetent infected patients and in 91% of immunocompromised patients (hemodialysis and hemophiliac patients). Immune impairment seemed to restrict the spectrum of antibody isotypes reacting to the core peptide. (+info)
Experimental transmission of hepatitis C virus-associated fulminant hepatitis to a chimpanzee.
Hepatitis C virus (HCV) was transmitted from a patient with fulminant hepatitis C to a chimpanzee. The patient had developed two episodes of fulminant hepatitis C, each occurring after a separate liver transplantation. Serial serum and liver samples from the patient and the chimpanzee were analyzed for HCV replication, genotype, quasispecies heterogeneity, and antibodies. In the patient, the levels of HCV replication in serum and liver correlated with the degree of hepatocellular necrosis and the clinical expression of fulminant hepatitis. The same HCV strain, genotype 1a, was recovered from both episodes of fulminant hepatitis. An unusually severe acute hepatitis was also observed in the chimpanzee. The viruses recovered from the patient and the chimpanzee were almost identical and displayed relatively little quasispecies heterogeneity. Thus, the same HCV strain induced two episodes of fulminant hepatitis in a single patient and severe hepatitis in a chimpanzee, suggesting that the pathogenicity or virulence of a specific HCV strain may be important in the pathogenesis of fulminant hepatitis C. (+info)
Cryoglobulinaemia and rheumatic manifestations in patients with hepatitis C virus infection.
OBJECTIVES: To investigate the association of cryoglobulinaemia and rheumatic manifestations in Korean patients with hepatitis C virus (HCV) infection. METHODS: Forty nine Korean patients with HCV infection were recruited. The prevalence, concentration, and type of cryoglobulin (by immunofixation), rheumatoid factor (RF), antinuclear antibody (ANA), and various rheumatological symptoms were investigated and HCV genotype was determined by polymerase chain reaction with genotype specific primer. RESULTS: The prevalence of cryoglobulin was 59% in Korean HCV patients and the concentration of cryoglobulin was 9.8 (7.9) g/l (mean (SD)). The type of cryoglobulinaemia was identified in 23 (80%) of 29 HCV patients with cryoglobulinaemia and they were all type III. There were no differences in age, sex, history of operation and transfusion, proportion of liver cirrhosis between the patients with cryoglobulinaemia and those without cryoglobulinaemia. The frequencies of RF and ANA were 14% and 3.4% respectively in HCV patients with cryoglobulinaemia. There was no difference in HCV genotype between the patients with cryoglobulinaemia and those without cryoglobulinaemia. Clinical features of HCV patients were as follows: arthralgia/arthritis (35%), cutaneous manifestation (37%), Raynaud's phenomenon (8%), paresthesia (44%), dry eyes (22%), dry mouth (10%), oral ulcer (33%), and abdominal pain (14%). However, these rheumatological symptoms did not differ between the two groups. CONCLUSION: Although the rheumatological symptoms were not different between HCV patients with and without cryoglobulinaemia, HCV patients showed various rheumatological manifestations. These result suggests that HCV infection could be included as one of the causes in patients with unexplained rheumatological symptoms. (+info)
Acute hepatitis C virus structural gene sequences as predictors of persistent viremia: hypervariable region 1 as a decoy.
We hypothesized that hepatitis C virus (HCV) persistence is related to the sequence variability of putative envelope genes. This hypothesis was tested by characterizing quasispecies in specimens collected every six months from a cohort of acutely HCV-infected subjects (mean duration of specimen collection, 72 months after seroconversion). We evaluated 5 individuals who spontaneously cleared viremia and 10 individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2, including hypervariable region 1 (HVR1). To assess the quasispecies complexity and to detect variants for sequencing, the first PCR-positive sample was examined by using a previously described method that combines heteroduplex analysis and analysis of single-stranded conformational polymorphisms. The ratio of nonsynonymous to synonymous substitutions (dN/dS) within each sample was evaluated as an indicator of relative selective pressure. Amino acid sequences were analyzed for signature patterns, glycosylation signals, and charge. Quasispecies complexity was higher and E1 dN/dS ratios (selective pressure) were lower in those with persistent viremia; the association with persistence was strengthened by the presence of a combination of both characteristics. In contrast, a trend toward higher HVR1 dN/dS ratios was detected among those with persistent viremia. We did not detect any such association for factors that may affect complexity such as serum HCV RNA concentration. HVR1 had a lower positive charge in subjects with persistent viremia, although no consistent motifs were detected. Our data suggest that HCV persistence is associated with a complex quasispecies and immune response to HVR1. (+info)
Long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis C virus.
Two chimpanzees (Ch1535 and Ch1536) became infected with hepatitis C virus (HCV) following intrahepatic inoculation with RNA transcribed from a full-length cDNA clone of the virus. Both animals were persistently infected and have been followed for 60 weeks. They showed similar responses to infection, with transient liver enzyme elevations and liver inflammatory responses, which peaked at weeks 17 (Ch1535) and 12 (Ch1536) postinoculation (p.i.). Antibody responses to structural and nonstructural proteins were first detected at weeks 13 (Ch1535) and 10 (Ch1536) p.i. Serum RNA titers increased steadily during the first 10 to 13 weeks but decreased sharply in both animals following antibody and inflammatory responses. Despite direct evidence of humoral immune responses to multiple viral antigens, including hypervariable region 1 (HVR1), both animals remained chronically infected. Detailed sequence analysis of serum HCV RNA revealed no change in the majority HVR1 sequence in Ch1535 and a single-amino-acid mutation in Ch1536, with very little clonal variation in either animal. Full-length genome analysis at week 60 revealed several amino acid substitutions localized to antigens E1, E2, p7, NS3, and NS5. Of these, 55.6 and 40% were present as the majority sequence in serum RNA isolated at week 26 p.i. (Ch1535) and week 22 p.i. (Ch1536), respectively, and could represent immune escape mutations. Mutations accumulated at a rate of 1.57 x 10(-3) and 1.48 x 10(-3) nucleotide substitutions/site/year for Ch1535 and Ch1536, respectively. Taken together, these data indicate that establishment of a persistent HCV infection in these chimpanzees is not due to changes in HVR1; however, the possibility remains that mutations arising in other parts of the genome contributed to this persistence. (+info)
The natural course of hepatitis C virus infection 18 years after an epidemic outbreak of non-A, non-B hepatitis in a plasmapheresis centre.
BACKGROUND: The natural history of hepatitis C virus (HCV) infection is variable and factors determining the course of the illness are unclear. AIMS: To determine the natural course of HCV infection in a well characterised group of patients 18 years after an epidemic outbreak of non-A, non-B hepatitis at a plasmapheresis centre. METHODS: Between 1994 and 1996, 20 of 30 affected individuals were studied. HCV infection was confirmed using second and third generation ELISA test kits. HCV RNA was detected by a polymerase chain reaction (PCR) method and HCV genotyping was performed by analysing amplicons from the conserved 5'-non-translated region generated by nested PCR. Thirty two liver biopsies were carried out in 14 patients. RESULTS: HCV antibodies were detected in all subjects. Eighteen patients had abnormal liver enzymes and 17 were HCV RNA positive, all of whom were infected with genotype 1a. Ninety per cent of this cohort showed evidence of chronic HCV infection with 50% having progressive liver disease and 20% cirrhosis 18 years after acute onset of non-A, non-B hepatitis. Considerable variation in disease outcome occurred between individuals and no correlation with clinical features of the acute illness was found. CONCLUSIONS: Variability in the consequences of HCV infection in cases infected with the same virus suggests that host factors are important in determining disease outcome. The factors which determine differences in the natural history of the disease still remain to be elucidated. (+info)