Hepatitis C Virus 1b Research Articles

Mutations of the interferon sensitivity-determining region (ISDR) correlate with the complexity of hypervariable region (HVR)-1 in the Japanese variant of hepatitis C virus (HCV) type 1b.

Hepatitis C virus (HCV) genotype 1b comprises mainly two subtypes in Japan, each named for its geographic prevalence (Japan-specific, J type; worldwide, W type). Because the newly identified subtypes have not been fully characterized, the present study directed this issue from virological viewpoints such as hypervariable region (HVR)-1 as well as interferon (IFN) sensitivity-determining region (ISDR). Fifty chronic hepatitis patients with HCV 1b (31 men and 19 women; mean age 50.5 years) were enrolled, and J/W type was determined according to envelope 1 (E1) sequence as described previously (23 J type and 27 W type). Correlations between age, number of HVR-1 clones, HVR-1 diversity, and ISDR mutations were analyzed in J and W type patients independently. In addition, the sequences of the three HCV regions obtained for the determination of the above genetic factors were studied phylogenetically. The number of HVR-1 clones was significantly higher for J type in comparison with W type (P = 0.044). In the J type-infected patients, the ISDR mutation number was correlated inversely with HVR-1 clone number (P = 0.0001, r = -0.734) and HVR-1 diversity (P = 0.0001, r = -0.722). However, this correlation was not observed in the W type patients. W type patients showed a significant correlation between age and HVR-1 clone number (P = 0.015, r = 0.462). Phylogenetic study revealed that the nonstructural (NS) 5A sequence, which is obtained for ISDR type determination, can distinguish between J and W types. The inverse correlation in J type patients between ISDR mutations and HVR-1 complexity may explain the usefulness of the ISDR for prediction of IFN response only in Japanese patients. This suggests that the ISDR is not directly related to IFN responsiveness, but the degree of HVR-1 complexity may be more important.

Human monoclonal antibody to hepatitis C virus E1 glycoprotein that blocks virus attachment and viral infectivity.

Human antibodies elicited in response to hepatitis C virus (HCV) infection are anticipated to react with the native conformation of the viral envelope structure. Isolation of these antibodies as human monoclonal antibodies that block virus binding and entry will be useful in providing potential therapeutic reagents and for vaccine development. H-111, an antibody to HCV envelope 1 protein (E1) that maps to the YEVRNVSGVYH sequence and is located near the N terminus of E1 and is able to immunoprecipitate E1E2 heterodimers, is described. Binding of H-111 to HCV E1 genotypes 1a, 1b, 2b, and 3a indicates that the H-111 epitope is highly conserved. Sequence analysis of antibody V regions showed evidence of somatic and affinity maturation of H-111. Finally, H-111 blocks HCV-like particle binding to and HCV virion infection of target cells, suggesting the involvement of this epitope in virus binding and entry.

Humoral immunity to immunodominant epitopes of Hepatitis C virus in individuals infected with genotypes 1a or 1b

Cellular immunity against multiple Hepatitis C virus (HCV) proteins is observed in patients acutely infected with HCV most of whom later resolve infection. We wished to assess humoral immunity in patients infected with HCV 1a or 1b genotypes in relation to viral load using plasma samples from HCV-infected individuals and a panel of peptides representing immunodominant epitopes of HCV structural and nonstructural proteins. Plasma from HCV 1a- and 1b-infected patients, respectively, were divided into two groups: patients with low viral load (≤100,000 RNA copies/ml) and patients with high viral load (≥10,000,000 RNA copies/ml). The antigens were peptides representing epitopes from immunodominant regions of HCV core, E2, NS3, and NS4 proteins, as well as the hypervariable (HVR) epitopes in E2 from genotypes 1a and 1b. Individuals infected with HCV 1a evoked a stronger immune response to many immunodominant epitopes of HCV relative to individuals infected with HCV 1b. Moreover, among individuals infected with HCV 1a, those with low viral loads mounted significantly greater responses against these epitopes than did individuals with high viral loads. Our observations demonstrate that quantitatively different antibody responses are elicited against HCV depending on the genotype of infecting virus, and suggest that humoral immunity directed against multiple immunodominant epitopes in HCV 1a-infected individuals may help lower viral load in vivo.

Control of heterologous hepatitis C virus infection in chimpanzees is associated with the quality of vaccine-induced peripheral T-helper immune response.

Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-gamma), protein-specific lymphoproliferative responses, IFN-gamma, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.

Unusual Multiple Recoding Events Leading to Alternative Forms of Hepatitis C Virus Core Protein from Genotype 1b

In addition to its involvement in the formation of the capsid shell of the virus particles, the core protein of hepatitis C virus (HCV) is believed to play an important role in the pathogenesis and/or establishment of persistent infection. We describe here alternative forms of genotype 1b HCV core protein identified after purification of various products of core protein segment 1-169 expressed in Escherichia coli and their analysis by proteolysis, mass spectrometry, and amino acid sequencing. These proteins all result from a +1 frameshift at codon 42 (a different position than that previously reported in genotype 1a) and, for some of them, from a rephasing in the normal open reading frame at the termination codon 144 in the +1 open reading frame. To test the relevance of these recoding events in a eukaryotic translational context, the nucleotide sequences surrounding the two shift sites were cloned in the three reading frames into expression vectors, allowing the production of a C-terminally fused green fluorescent protein, and expressed both in a reticulocyte lysate transcription/translation assay and in culture cells. Both recoding events were confirmed in these expression systems, strengthening the hypothesis that they might occur in HCV-infected cells. Moreover, sera from HCV-positive patients of genotype 1a or 1b were shown to react differently against synthetic peptides encoded in the +1 open reading frame. Together, these results indicate the occurrence of distinct recoding events in genotypes 1a and 1b, pointing out genotype-dependent specific features for F protein.

Nonstructural protein precursor NS4A/B from hepatitis C virus alters function and ultrastructure of host secretory apparatus.

The nonstructural proteins of hepatitis C virus (HCV) have been shown previously to localize to the endoplasmic reticulum (ER) when expressed singly or in the context of other HCV proteins. To determine whether the expression of HCV nonstructural proteins alters ER function, we tested the effect of expression of NS2/3/4A, NS4A, NS4B, NS4A/B, NS4B/5A, NS5A, and NS5B from genotype 1b HCV on anterograde traffic from the ER to the Golgi apparatus. Only the nominal precursor protein NS4A/B affected the rate of ER-to-Golgi traffic, slowing the rate of Golgi-specific modification of the vesicular stomatitis virus G protein expressed by transfection by approximately threefold. This inhibition of ER-to-Golgi traffic was not observed upon expression of the processed proteins NS4A and NS4B, singly or in combination. To determine whether secretion of other cargo proteins was inhibited by NS4A/B expression, we monitored the appearance of newly synthesized proteins on the cell surface in the presence and absence of NS4A/B expression; levels of all were reduced in the presence of NS4A/B. This reduction is also seen in cells that contain genome length HCV replicons: the rate of appearance of major histocompatibility complex class I (MHC-I) on the cell surface was reduced by three- to fivefold compared to that for a cured cell line. The inhibition of protein secretion caused by NS4A/B does not correlate with the ultrastructural changes leading to the formation a "membranous web" (D. Egger et al., J. Virol. 76:5974-5984, 2002), which can be caused by expression of NS4B alone. Inhibition of global ER-to-Golgi traffic could, by reducing cytokine secretion, MHC-I presentation, and transport of labile membrane proteins to the cell surface, have significant effects on the host immune response to HCV infection.

High‐density lipoprotein binding rate differs greatly between genotypes 1b and 2a/2b of hepatitis C virus

The hepatitis C virus (HCV) virion is associated with lipoproteins and immunoglobulins in the sera of patients with chronic hepatitis C; however, an accurate binding rate of HCV to lipoproteins or immunoglobulins has not yet been elucidated. Therefore, the accurate binding rate of HCV to low-density lipoproteins (LDL), high-density lipoproteins (HDL), and immunoglobulins was measured quantitatively by a real-time PCR assay. The immunoglobulin binding rate of HCV was found to be greater than 97.5% in most patients, as compared with an LDL binding rate of greater than 80% in most patients. In contrast, the HDL binding rate was greater than 98% in the genotype 2a/2b patients, while it varied in the genotype 1b patients. The genotype 2a/2b HCV not only had a higher LDL binding rate but also had a strikingly higher HDL binding rate than that of the genotype 1b HCV. These lipoprotein binding rates correlated neither to any patient's variables, including the serum apolipoprotein levels, nor to the viral load or the hypervariable region 1 (HVR 1) amino acid sequences. Most of the HCV virions in the sera of such patients have been shown to be associated simultaneously with immunoglobulins and LDL and/or HDL, but not exclusively.

Serum lipid profile of patients with genotype 1b hepatitis C viral infection in Japan

Hepatitis C virus (HCV) infection is associated with the development of steatosis in the liver. Recently, infection with genotype 3a HCV has been reported to have a closer association with hepatic steatosis than that with genotype 1 or 2 HCV. Moreover, infection with genotype 3a HCV but not with genotype 1 has been shown to be associated with serum hypocholesterolemia or hypobetalipoproteinemia in European countries. We conducted a case control study to characterize the serum lipid profile in patients infected with genotype 1b HCV, which is the most prevalent HCV genotype in Japan. These patients had significantly lower serum cholesterol levels than those infected with HBV or genotype 2a HCV who had similar liver disease progression and body mass index. Further analysis of serum apolipoproteins revealed that not only apolipoprotein B but also apolipoprotein CII and apolipoprotein CIII levels were significantly reduced, while apolipoprotein AI, AII and E levels were similar in patients infected with genotype 1b HCV and those with HBV or genotype 2a HCV. These results indicate that, in Japan, infection with genotype 1b HCV is a cause of lipid metabolism disturbances, which may be associated with the pathogenesis of hepatitis C liver disease.

Hepatitis C virus molecular epidemiology in Uzbekistan.

The aim of this study was to identify hepatitis C virus (HCV) genotypes and to estimate their prevalence in various risk groups and the regional distribution in Uzbekistan. Preliminary serological screening of 1,269 subjects revealed 6.5% anti-HCV-positive in a general population, 27.1% in patient groups, and 51.7% among intravenous drug users. HCV genotypes of 104 anti-HCV-positive subjects were determined using a PCR-genotyping system in core region, and the results were supported by nucleotide sequencing of the NS5B region. Genotype 1b identified in total 64.2%, was the most prevalent. The genotype 3a identified in 25.0% was the second one distributed. HCV genotypes 2a, 1a, 2b, and 3b were identified in 3.8%, 2.9%, 2.9%, and 1.0% of cases, respectively. The intravenous drug users were distinguished from other groups by having the highest prevalence of genotype 3a, i.e., 50.0%, higher than the 33.3% for genotype 1b in this group. Geographically, genotype 1b was common; genotype 3a was also found frequently in all three regions. Uncommon HCV genotypes (1a, 2a, 2b, and 3b) were found in comparatively greater variability in the western region. Molecular evolutionary analysis based on the NS5B region did not reveal specific clustering or indigenous strains among Uzbekistan HCV isolates. In summary, two main mechanisms of HCV infection distribution were observed in Uzbekistan: HCV 1b genotype infection is widespread through blood products, and HCV 3a genotype infection is spreading through the growing number of intravenous drug users.

Hepatitis C virus core protein expression in human B-cell lines does not significantly modify main proliferative and apoptosis pathways

Hepatitis C virus (HCV) chronic infection has been associated with many lymphoproliferative disorders. Several studies performed on hepatoma and fibroblast cell lines suggest a role of the HCV core protein in activation of cellular transduction pathways that lead to cell proliferation and inhibition of apoptosis. However, no data are available concerning the effects of HCV core expression on B-lymphocyte proliferation and apoptosis. B-lymphocyte cell lines permanently expressing full-length HCV 1b core sequences isolated from chronically infected patients were established using B-cell lines at different degrees of differentiation. Clones and pools of clones permanently expressing the HCV core were selected and characterized for protein expression by Western blot and FACS. Expression of HCV core proteins did not significantly enhance cell proliferation rates under normal culture conditions or under mitogenic stimulation. Analysis of NF-kappa B, CRE, TRE and SRE pathways by luciferase reporter genes did not show a significant influence of HCV core expression on these signal transduction cascades in B-lymphocytes. The effects of HCV core on anti-IgM and anti-FAS-induced apoptosis in B-cell lines was also analysed. In this experimental model, HCV core expression did not significantly modify the apoptotic profile of the B-lymphocyte cell lines tested. These data underline a cell type-specific effect of HCV core expression. In fact, it was not possible to show a significant contribution of the HCV core protein in activation of the major B-cell signal transduction pathways involved in the regulation of proliferation and programmed cell death, which is in contrast with the results reported in hepatoma cell lines.

Phylogenetic analysis indicates transmission of hepatitis C virus from an infected orthopedic surgeon to a patient

During recent years, a controversial discussion has emerged in the medical community on the real number and possible public health implications of hepatitis C virus (HCV) transmissions from infected medical staff to susceptible patients. We report here on molecular virological and epidemiological analyses involving 229 patients who underwent exposure-prone operations by an HCV-infected orthopedic surgeon. Of the 229 individuals affected, 207 could be tested. Three were positive for HCV antibodies. Molecular and epidemiological investigation revealed that two of them were not infected by the surgeon. The third patient, a 50-year-old man, underwent complicated total hip arthroplasty with trochanteric osteotomy. He harbored an HCV 2b isolate that in phylogenetic analysis of the hypervariable region 1 (HVR 1) was closely related to the HCV strain recovered from the infected surgeon, indicating that HCV-provider-to-patient transmission occurred intraoperatively. To our knowledge, this is the first documented case of HCV transmission by an orthopedic surgeon. The recorded transmission rate of 0.48% (95% confidence interval: 0.09–2.68%) was within the same range reported previously for the spread of hepatitis B virus during orthopedic procedures. Since the result of our investigation sustains the notion that patients may contract HCV from infected health-care workers during exposure-prone procedures, a series of further retrospective exercises is needed to assess more precisely the risk of HCV provider-to-patient transmission and to delineate from these studies recommendations for the guidance and management of HCV-infected medical personnel. J. Med. Virol. 66:461–467, 2002. © 2002 Wiley-Liss, Inc.

Phylogenetic analysis indicates transmission of hepatitis C virus from an infected orthopedic surgeon to a patient

During recent years, a controversial discussion has emerged in the medical community on the real number and possible public health implications of hepatitis C virus (HCV) transmissions from infected medical staff to susceptible patients. We report here on molecular virological and epidemiological analyses involving 229 patients who underwent exposure-prone operations by an HCV-infected orthopedic surgeon. Of the 229 individuals affected, 207 could be tested. Three were positive for HCV antibodies. Molecular and epidemiological investigation revealed that two of them were not infected by the surgeon. The third patient, a 50-year-old man, underwent complicated total hip arthroplasty with trochanteric osteotomy. He harbored an HCV 2b isolate that in phylogenetic analysis of the hypervariable region 1 (HVR 1) was closely related to the HCV strain recovered from the infected surgeon, indicating that HCV-provider-to-patient transmission occurred intraoperatively. To our knowledge, this is the first documented case of HCV transmission by an orthopedic surgeon. The recorded transmission rate of 0.48% (95% confidence interval: 0.09-2.68%) was within the same range reported previously for the spread of hepatitis B virus during orthopedic procedures. Since the result of our investigation sustains the notion that patients may contract HCV from infected health-care workers during exposure-prone procedures, a series of further retrospective exercises is needed to assess more precisely the risk of HCV provider-to-patient transmission and to delineate from these studies recommendations for the guidance and management of HCV-infected medical personnel.

ISDR Pattern and Evolution in Patients with Chronic Hepatitis C Treated with Standard Or Peg-Ifn plus Ribavirin

The aim of the study was to characterize the interferon sensitivity determining region (ISDR) mutation pattern and its changes at 4 weeks of treatment in a population of patients infected with hepatitis C virus (HCV) genotype 1b receiving standard or PEG-IFN plus ribavirin (RBV), to find possible early correlates of therapy outcome. Forty-five patients with chronic hepatitis due to HCV 1b were treated by PEG-IFN-alpha2b (n=23) or IFN-alpha2b (n=22) plus RBV 1000-1200 mg/day. They were classified 24 weeks after stopping therapy as sustained responders (SR), relapsers (REL) or non-responders (NR). Sixteen patients were SR, 12 REL and 17 NR. ISDR mutations were evaluated by direct sequencing at baseline in all and after 4 weeks in patients with detectable viraemia (n=30). The frequency of the three ISDR types was 26.7% wild-type, 64.4% intermediate-type and 8.9% mutant-type, without significant difference in their frequency in SR, REL and NR, independent of IFN formulation. Average numbers of mutations in SR, REL and NR were 1.88 +/- 0.54, 1.33 +/- 0.33 and 0.94 +/- 0.25, respectively, P>0.05. The baseline number of ISDR mutations was not related to the extent of viral load decline in the first month of therapy. Sequence analysis of ISDR region performed 4 weeks after starting therapy revealed qualitative or quantitative changes of ISDR sequence in only seven patients, without correlation with response. Thus, in our patients the baseline pattern of ISDR was unrelated to treatment outcome. Selection towards a dominant IFN-resistant strain did not occur under standard or PEG-IFN plus RBV.

Expression, purification, immunological characterization and application of Escherichia coli-derived hepatitis C virus E2 proteins.

The envelope glycoprotein E2 of hepatitis C virus (HCV) has been shown to bind human target cells. Anti-E2 antibodies have been associated with both recovery from natural infection in humans and protection from challenge with homologous HCV in chimpanzees. Therefore E2 has become a major target for the development of anti-HCV vaccines. Two E2 fragments [amino acids (aa) 450-565 and aa 385-565] derived from a subtype 1b HCV genome were expressed as N-terminally hexahistidine-tagged proteins in Escherichia coli and purified to over 85% purity. Both proteins were specifically recognized by homologous hepatitis-C-patient's serum on Western blotting, suggesting that these E. coli-derived E2 proteins displayed E2-specific antigenicity. E2-116 (aa 450-565) elicited strong antibody responses in BALB/c mice and rabbits. Rabbit antiserum raised against renatured E2-116 (R(E2-116R)) was able to recognize subtype 1b and 1a E2 glycoproteins expressed in mammalian cells on Western blotting. E2-181 (aa 385-565) reacted with 40% of anti-HCV(+) patients' sera in ELISA. R(E2-116R) and E2-181 were successfully used in preliminary modified vaccinia virus Ankara- and DNA-based E2 vaccine studies for detecting antigen expression in vitro and assessing induced humoral immune responses in mice. The E2 proteins and rabbit antiserum reported here could find wider applications in the development of effective diagnostic, prophylactic and therapeutic measures against HCV.

Japan‐specific subtype of hepatitis C virus genotype 1b, J subtype, has relatively low pathogenicity

The prognostic implication of viral genotype 1b for hepatitis C virus (HCV) infection has been controversial, possibly due to the pathogenetic heterogeneity of genotype 1b. We analyzed two newly delineated subtypes of HCV genotype 1b subtypes with respect to progression of liver disease. Patients with chronic HCV 1b infection (113 total, including 18 with chronic persistent hepatitis, 60 with chronic active hepatitis, 19 with cirrhosis, and 16 with hepatocellular carcinoma with cirrhosis) were studied to elucidate the factors associated with progression of liver disease. Factors evaluated included sex, age at diagnosis, blood transfusion history, and HCV genotype 1b subtype (W subtype or J subtype). W subtype was identified more often in association with chronic active hepatitis than with chronic persistent hepatitis (P = 0.0089), and more often in patients with cirrhosis than in those without (P = 0.0044). The age-, sex-, and transfusion history-adjusted odds ratios with respect to histological activity and presence of cirrhosis for W subtype compared to J subtype were 6.966 (1.856 to 26.145) and 6.397 (1.506 to 27.179), respectively. Age at diagnosis was the most important risk factor for predicting development of cirrhosis and carcinoma. In conclusion, the W subtype of HCV 1b is associated closely with histologically active disease and development of cirrhosis, whereas the Japan-specific J subtype has relatively low pathogenicity. HCV genotype 1b, therefore, is heterogeneous in its pathogenicity. J. Med. Virol. 65:45–51, 2001. © 2001 Wiley-Liss, Inc.

Japan‐specific subtype of hepatitis C virus genotype 1b, J subtype, has relatively low pathogenicity

The prognostic implication of viral genotype 1b for hepatitis C virus (HCV) infection has been controversial, possibly due to the pathogenetic heterogeneity of genotype 1b. We analyzed two newly delineated subtypes of HCV genotype 1b subtypes with respect to progression of liver disease. Patients with chronic HCV 1b infection (113 total, including 18 with chronic persistent hepatitis, 60 with chronic active hepatitis, 19 with cirrhosis, and 16 with hepatocellular carcinoma with cirrhosis) were studied to elucidate the factors associated with progression of liver disease. Factors evaluated included sex, age at diagnosis, blood transfusion history, and HCV genotype 1b subtype (W subtype or J subtype). W subtype was identified more often in association with chronic active hepatitis than with chronic persistent hepatitis (P = 0.0089), and more often in patients with cirrhosis than in those without (P = 0.0044). The age-, sex-, and transfusion history-adjusted odds ratios with respect to histological activity and presence of cirrhosis for W subtype compared to J subtype were 6.966 (1.856 to 26.145) and 6.397 (1.506 to 27.179), respectively. Age at diagnosis was the most important risk factor for predicting development of cirrhosis and carcinoma. In conclusion, the W subtype of HCV 1b is associated closely with histologically active disease and development of cirrhosis, whereas the Japan-specific J subtype has relatively low pathogenicity. HCV genotype 1b, therefore, is heterogeneous in its pathogenicity.

Hepatitis C virus genotypes and risk of cirrhosis in southern Italy.

Because hepatitis C virus (HCV) genotypes have raised considerable interest as variables that influence chronic hepatitis C progression, a case-control study was conducted to estimate their effects on patients with cirrhosis. Case patients (n = 46) had tested positive for anti-HCV antibody and HCV RNA and were residents of the study area who had cirrhosis recently diagnosed. Controls (n = 138) were drawn randomly from a residents' cohort from the same area. Demographic and other information were recorded. Presence of HCV infection, presence of HCV RNA, and HCV genotypes were assessed. Crude, stratified, and logistic regression analyses were performed. HCV genotype 2a/c occurred in 84 controls (60.9%) and 9 case patients (19.6%); HCV genotype 1b was found in 45 controls (32.6%) and 34 case patients (73.9%). HCV 1b genotype showed an independent effect on the risk of cirrhosis (odds ratio, 7.49; 95% confidence interval, 3.15--17.81). No significant effects related to other variables were observed. These results indicate that the genetic diversity of HCV phylogenetic variants may explain differences in biological behaviors.

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.

Ser2194 Is a Highly Conserved Major Phosphorylation Site of the Hepatitis C Virus Nonstructural Protein NS5A

Phosphorylation of the nonstructural NS5A protein is highly conserved among hepatitis C virus (HCV) genotypes. However, the precise site or sites of phosphorylation of NS5A have not been determined, and the functional significance of phosphorylation remains unknown. Here, we showed by two-dimensional phosphopeptide mapping that a protein kinase or kinases present in yeast, insect, and mammalian cells phosphorylated a highly purified HCV genotype 1b NS5A from insect cells on identical serine residues. We identified a major phosphopeptide (corresponding to amino acids 2193–2212 of the HCV 1b polyprotein) by using negative-ion electrospray ionization–microcapillary high performance liquid chromatography–mass spectrometry. The elution time of the phosphopeptide determined by negative-ion electrospray ionization–mass spectrometry corresponded with the elution time of the majority of 32P-label that was incorporated into the phosphopeptide by an in vitro kinase reaction. Subsequent analysis of the peak fraction by automated positive-ion electrospray ionization–tandem mass spectrometry revealed that Ser2194 was the major phosphorylated residue on the phosphopeptide GpSPPSLASSSASQLSAPSLK. Substitution for Ser2194 with Ala resulted in the concomitant disappearance of major in vivo phosphorylated peptides. Ser2194 and surrounding amino acids are highly conserved in all HCV genotypes, suggesting NS5A phosphorylation at Ser2194 may be an important mechanism for modulating NS5A biological functions.

Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in germany: a 20-year multicenter study.

From August 1978 until March 1979, 14 batches of anti-D immune globulin contaminated with hepatitis C virus (HCV) genotype 1b (20, 000-480,000 copies/dose) from a single erythrocyte donor had been administered for prophylaxis of rhesus isoimmunization throughout East Germany. All 2,867 women involved had been recalled after January 12, 1979 for repeated screening of alanine transaminase (ALT). They were prospectively followed in regional centers. We have reexamined a cohort of 1,018 women (median age 24, range 16-38 years at infection) on follow-up for 20 years in 9 representative centers. Within 6 months after anti-D administration, 10% of these women had no evidence of disease and 90% had acute hepatitis C (n = 917) including 49% with symptomatic and 22% with icteric course. After 20 years, 85% of the 917 affected women still tested positive for HCV antibodies (among them 3% responded to interferon treatment) and 55% were positive for HCV RNA (among them 7% were nonresponders to interferon and 3% were apparent HCV carriers). Only 4 (0.4%) had overt cirrhosis. Two (0.2%) died of superinfected fulminant hepatitis B or alcoholism and cirrhosis, respectively. Histology obtained in 44% of the viremic women showed hepatitis of minimal to moderate grade in 96%, portal fibrosis in 47%, and septal fibrosis in 3% of the cases. In conclusion, formerly healthy young women, without hepatic comorbidity, may clear HCV (1b) infection in half of the cases or develop mild chronic hepatitis C with low risk of progression to cirrhosis within 20 years.