Cohort effect of HCV infection in liver cirrhosis assessed by a 25 year study.

BACKGROUND: The role of HCV infection in the development of chronic liver disease is still unclear. OBJECTIVES: Assess the presence of HCV infection in patients with liver cirrhosis. STUDY DESIGN: 123 cases of cirrhotic liver randomly selected over a 25 years period (1969-1994) from the autopsy archives of the Pathology Department of the University of Trieste, Italy, were analyzed for the presence of HCV viral genome. METHODS: Total RNA was extracted from formalin-fixed paraffin-embedded tissues of the cirrhotic liver. Genotype analysis for HCV was performed after RT-PCR by dot-blot hybridization with the three major genotype-specific probes (G1, G2 and G3). RESULTS: The overall HCV genome frequency was 50.4% (62/123). The positivity was quite constant in the 1969-1979 period (35-38%), rose to 65% in 1984, peaked to 77% in 1989 (P<0.005 vs. the previous decade), and decreased to 50% in 1994. HCV genotype G1 was found in 89% of the 62 positive samples. The mean age of death of HCV-positive and HCV-negative patients was comparable (69+/-12 vs. 67+/-16 years, NS). CONCLUSIONS: These data show an increasing frequency of HCV infection in cirrhotic liver tissues from 1969 to 1994, which peaked in 1989. The genotype G1 was the almost uniquely associated with cirrhosis. These findings indicate that the HCV infection occurred around the late 1950s-early 1960s, thus supporting the hypothesis of a cohort effect. HCV infection seems not to alter the natural history of liver cirrhosis as indicated by the comparable age at death of HCV positive and HCV negative patients.

Influence of correct secondary and tertiary RNA folding on the binding of cellular factors to the HCV IRES.

Structural integrity of the hepatitus C virus (HCV) 5' UTR region that includes the internal ribosome entry site (IRES) element is known to be essential for efficient protein synthesis. The functional explanation for this observation has been provided by the recent evidence that binding of several cellular factors to the HCV IRES is dependent on the conservation of its secondary structure. In order to better define the relationship between IRES activity, protein binding and RNA folding of the HCV IRES, we have focused our attention on its major stem-loop region (domain III) and the binding of several cellular factors: two subunits of eukaryotic initiation factor eIF3 and ribosomal protein S9. Our results show that binding of eIF3 p170 and p116/p110 subunits is dependent on the ability of the domain III apical stem-loop region to fold in the correct secondary structure whilst secondary structure of hairpin IIId is important for the binding of S9 ribosomal protein. In addition, we show that binding of S9 ribosomal protein also depends on the disposition of domain III on the HCV 5' UTR, indicating the presence of necessary inter-domain interactions required for the binding of this protein (thus providing the first direct evidence that tertiary folding of the HCV RNA does affect protein binding).

Immunoreactivity to putative B-cell epitopes of hepatitis G virus polyprotein in viremic and nonviremic subjects.

The hepatitis G virus (HGV) polyprotein was scanned by computer-aided prediction of antigenicity to search for B-cell epitopes. Four polypeptide sequences, V37D (amino acids [aa] 1685 to 1721), V36S (aa 2102 to 2137), P37R (aa 2156 to 2192), and C40P (aa 2280 to 2319), were identified and synthesized for use in immunoassays. Antibodies to these peptides were searched for in a panel of 239 serum samples, which were also tested for anti-E2 antibodies and HGV RNA. Furthermore, the course of HGV markers was studied prospectively in four patients who had been transfused with HGV RNA-positive blood. There was a negative association between immunoreactivity to V37D and P37R and presence of HGV RNA (2 of 53 and 1 of 53, respectively; P < 0.05); none of the subjects with dual antibody positivity was HGV RNA positive. Anti-V37D and anti-P37R antibodies compared favorably with anti-E2 antibodies as markers of recovery from HGV infection. These results might be useful for the development of new, more sensitive diagnostic assays.

IgM antibody response to the hepatitis C virus core protein in intravenous drug users.

To verify whether a solid-phase enzyme immunoassay for serum IgM antibodies to the hepatitis C virus (HCV) core protein (IgM anti-HCVcore) might be proposed as a surrogate test for serum HCV RNA, we studied 86 anti-HCV antibody-positive intravenous drug users. Serum HCV RNA was demonstrated by RT-PCR with primers derived from the 5' non-coding and the core region. IgM anti-HCVcore antibodies were found in 62/86 (72%) subjects; circulating HCV RNA was detected by the 5' noncoding assay in 53/86 samples (62%) and by the core region assay in 35/86 samples (41%). IgM anti-HCVcore reactivity was associated with core HCV RNA seropositivity (p < 0.05) but not with 5' noncoding HCV RNA seropositivity (p = NS). Patients infected by HCV type 1a were more-often positive for IgM anti-HCVcore (p < 0.05) and for core HCV RNA (p = 0.005) than patients infected by other HCV genotypes. IgM anti-HCVcore reactivity was significantly more common in subjects positive for core HCV RNA (p < 0.005) and in subjects aged > 30 years (p < 0.05). In conclusion, the IgM anti-HCVcore assay frequently tests positive in intravenous drug users, particularly when infected by HCV 1a, but is not a surrogate of testing for serum HCV RNA.

Factors associated with serum HCV RNA positivity in anti-HCV antibody positive intravenous drug users.

Serum hepatitis C virus (HCV) RNA, HCV genotypes and liver function tests were evaluated in a series of 189 unselected, consecutive anti-HCV positive intravenous drug users (IVDUs). Serum HCV RNA was detected in 106/189 patients. Abnormal liver function tests were associated with alcohol abuse, but not with the presence of serum HCV RNA. Among 109 patients retested after a mean follow-up of 21 months, 41 were intermittently serum HCV RNA positive. Patients persistently negative had more commonly a past history of acute hepatitis. A history of prostitution and/or a pattern of abuse involving >30 injections per week were related to infection by genotype 3a. In conclusion, serum HCV RNA is either transiently or persistently detectable in most anti-HCV positive IVDUs, but bears no association with abnormal liver biochemistry. Infection by HCV-3a is more common in IVDUs with more deviant life styles. In those cases where serum HCV RNA is found repeatedly negative, HCV infection may have been cleared, possibly through an episode of acute hepatitis.

Localization of the different hepatitis C virus core gene products expressed in COS-1 cells.

In this study we have investigated the subcellular localization in transfected COS-1 cells of the two major forms of the hepatitis C virus core protein: the immature protein of 191 residues (p21) and its proteolytically cleaved product of 173 residues (p19). In this study, and unlike previous investigations, we have been able to distinguish separately the localization of p21 from p19. This was achieved by the addition of a C-terminal HSV Tag to the p21 full coding sequence, and exploiting the fact that it is subsequently lost in the p19 product. In order to obtain an accurate localization of both p21 and p19 we used a mouse anti-HSV Tag MAb together with a human anti-core MAb (B12.F8) to perform double immunofluorescence studies. The results have shown that p21 is always localized around the nuclear envelope. On the other hand, p19 can be found diffused in the cytoplasm to different degrees. These in vivo results reinforce the proposed links between the regulated processing of the hepatitis C virus core protein and the possibility that this may contribute towards the regulation of its diverse biological functions.

High prevalence of infection with hepatitis G virus in patients with hepatic and extrahepatic malignancies.

BACKGROUND/AIMS: The pathogenic role of hepatitis G virus, the recently discovered blood-borne agent, is controversial. Our aim was to ascertain the prevalence of hepatitis G virus infection in hepatic and in extrahepatic malignancies. METHODS: We studied 166 Italian patients (112 male, 54 female, mean age 61.8+/-9.3, mean+/-SD, range 34-85). One hundred and eighteen had cirrhosis, which was complicated by hepatocellular carcinoma in 66 cases. Forty-eight patients had extra-hepatic malignancies. Circulating HGV RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) of both the nonstructural-3 and 5'noncoding regions of the hepatitis G virus genome. Antibodies to the E2 protein of hepatitis G virus were detected by means of an enzyme-linked immunosorbent assay. RESULTS: Ongoing HGV infection was detected in 30/66 (46%) patients with hepatocellular carcinoma, 12/52 (23%) patients with cirrhosis, and 14/48 (29%) patients with extrahepatic malignancies (p<0.05). Evidence of exposure to hepatitis G virus (detection of either HGV RNA or anti-E2 antibodies) was found in 46% of patients with cirrhosis, 66% of patients with hepatocellular carcinoma, and 39% of patients with extrahepatic malignancies. Serum HGV RNA positivity was associated with a hematocrit value < or = 0.35 and with history of exposure to blood products (p<0.005). CONCLUSIONS: Ongoing hepatitis G virus infection is detected at a very high rate in patients with hepatocellular carcinoma, but is also fairly common in extrahepatic malignancies. Hepatitis G virus infection in these patients is likely to originate from exposure to blood products, and to persist because of deficient immune surveillance.

In vivo translational efficiency of different hepatitis C virus 5'-UTRs.

Initiation of translation in hepatitis C virus (HCV) is dependent on the presence of an internal ribosome entry site (IRES) contained in its 341-nt-long 5'-untranslated region (UTR). This region is very conserved among different isolates and has been used to classify HCV isolates in six different genotypes. These genotypes differ in nucleotide sequence that generally preserve the IRES structure. However, the small differences seen may be biologically and clinically significant as the HCV strains seem to differ from each other in several important ways, such as the behaviour of the viral infection and the response to interferon therapy. Therefore, differences in translational initiation efficiency amongst the various genotypes could provide an explanation for these phenomena. Using a bicistronic expression system we have compared the in vivo translational ability of the three most common European genotypes of HCV (1, 2, and 3). The results show that genotype 3 is less able than 1 and 2 to promote translation initiation. In addition, by site-directed mutagenesis of the sequence of the IRES domain III apical stem loop structure, we have shown that the conservation of the primary nucleotide sequence and not only the structure, is important for the conservation of IRES function.

Endogenous interferon-alpha concentration and outcome of interferon treatment in patients with chronic hepatitis C.

To verify its value with regard to the outcome of therapy in chronic hepatitis C, serum interferon-alpha (IFN) was measured by ELISA in 70 patients (43 male, 27 female) with chronic hepatitis C, treated with IFN 9 MU/week subcutaneously for up to one year. Serum IFN was detectable in 49/70 patients, 16 of whom had IFN values >125 pg/ml. Only 1/22 patients who maintained a sustained response six months after the end of treatment had pretreatment serum IFN > 125 pg/ml, in comparison to 15/48 patients who did not respond or who relapsed (chi2 6.1, P < 0.02). At multivariate analysis the predictive value of serum IFN was independent of age, sex, presence of cirrhosis, infection by genotype 1b (improvement chi2 7.1, P < 0.01). In patients with chronic hepatitis C, measurement of serum IFN at baseline might be useful for the selection of patients with higher probability of long-term response.

Improved reactivity of hepatitis C virus core protein epitopes in a conformational antigen-presenting system.

Recent studies have identified several epitopes in the N-terminal portion of the nucleocapsid protein which are predominantly recognized by sera of patients infected with hepatitis C virus (HCV). The characterization of the sequences recognized by theses antibodies and the evaluation of their reactivities have been performed mainly with synthetic peptides. However, synthetic peptides are notoriously unreliable as antigens when the immune response is directed against conformational epitopes. In order to improve the detection of antibody responses in HCV-infected patients, we have evaluated the reactivities of three immunodominant regions of the HCV core protein (residues 1 to 20, 21 to 40, and 32 to 46) displayed in a conformation-specific manner on the surface of the Flock House virus (FHV) capsid protein. The results obtained with these proteins in the analysis of 94 serum samples positive by anti-HCV enzyme-linked immunosorbent assay where then compared with those obtained with the corresponding synthetic peptides. The sequence most reactive both with the peptide and with the FHV protein was the region from residues 1 to 20, confirming the low conformational requirements for the display of these residues. On the other hand, the already reported conformational nature of residues 32 to 46 is in keeping with its observed high reactivity when displayed by the FHV recombinant protein and with the low reactivity displayed by its corresponding synthetic peptide. Finally, the high reactivity observed for the chimeric protein displaying the region from residues 21 to 40, as opposed to the results obtained with the synthetic peptide, also suggests that this sequence contains one or more conformational epitopes whose structures cannot be mimicked correctly with synthetic peptides.