A novel stop codon mutation within the hepatitis B surface gene is detected in the liver but not in the peripheral blood mononuclear cells of HIV-infected individuals with occult HBV infection.

To characterize occult HBV infection (OHB) in different compartments of HIV+ individuals. This retrospective study involved 38 consecutive HIV+ patients; 24 HBsAg negative (HBV-) and 14 HBsAg positive (HBV+). OHB was assessed in serum samples, liver tissue (LT) and peripheral blood mononuclear cells (PBMC) by genomic amplification of the partial S, X and precore/core regions. HBV genomic analysis was inferred by direct sequencing of PCR products. The intracellular HBV-DNA was measured by a quantitative real-time PCR. HBV+ patients were used as a control for HBV replication and genomic profile. In HBV- patients, HBV-DNA was undetectable in all serum samples, while it was found positive in 7/24 (29%) LT in which genotype D prevailed (57%). HBV-DNA was found in 6/7 (86%) PBMC of occult-positive and none of occult-negative LT. Significantly lower HBV-DNA load was present in both compartments in OHB+ with respect to the HBV+ group (LT: P = 0.002; PBMC: P = 0.026). In the occult-positive cases, HBV replication was significantly higher in LT than in PBMC (P = 0.028). A hyper-mutated S gene in PBMC and a nucleotide mutation at position C695 in LT that produces a translational stop codon at amino acid 181 of the HBs gene characterized OHB. In this group of HIV+ persons, OHB is frequent and exhibits lower replication levels than chronic HBV in the different compartments examined. HBV-DNA detection in PBMC may offer a useful tool to identify OHB in serum-negative cases. The novel HBs gene stop codon found in LT could be responsible for reduced production leading to undetectability of HBsAg.

Low replication and variability of HBV pre-core in concomitant infection with hepatitis B and hepatitis C viruses.

In an attempt to define the virological profile of HBV in HCV co-infection, we analysed the viral load, the infecting genotype, and the mutational pattern of the HBV pre-core region (pre-C), which is involved in viral encapsidation and DNA replication. Eighty-six patients were studied: 32 with serological HBV/HCV-1b co-infection (group BC), 32 infected by HBV alone (group B), and 22 by HCV-1b alone (group C). Sequence analysis of the HBV pre-S and pre-C regions identified genotypes and mutational patterns. The HBV viral load was significantly lower in group BC than in group B (p < 0.001), and the distribution of HBV pre-C mutations showed a higher prevalence of wild type in concomitant infection than in the control group (p < 0.006). The predominant HBV infecting strain was genotype D in both the BC (96%) and B (87%) groups. No difference was observed in HCV viremia levels between the two groups, whereas in HBV/HCV infection, the low levels of circulating HBV were closely associated with the low degree of variability of pre-C domain (p = 0.005). In conclusion, in HBV/HCV infection, the virological pattern was characterised by the dominance of HCV associated with lower HBV replication capacity and decreased emergence of HBV pre-C variants.

Virological analysis, genotypes and mutational patterns of the HBV precore/core gene in HBV/HCV-related hepatocellular carcinoma.

We investigated the replicative profile of hepatitis B (HBV) and hepatitis C (HCV) viruses and the mutational pattern of the HBV precore/core (pre-C/C) domain in hepatocellular carcinoma (HCC). Thirty-eight consecutive patients with HCC were included in the study - 18 of them with HBV/HCV co-infection and 20 with HBV single infection. Twenty-three additional patients with co-infection, without HCC were recruited as the control group. Replication activity was evaluated by detecting and quantitating both HBV and HCV genomes. The HBV pre-C/C region, encompassing the pregenome encapsidation signal involved in viral replication, was analysed by direct sequencing. HBV viraemia levels were significantly lower (P = 0.04) in patients with co-infection in comparison with single-infected HCC, whereas two different HBV viraemia profiles were detected in co-infection with or without circulating HCV. HBV genotype D was prevalent in the three groups and HCV genotype 1b was found to be the infecting strain in all patients. Lower variability in the pre-C/C region was found in co-infection in comparison with HBV single infection (P = 0.0004). A synonymous T1936C mutation was found in all co-infected HCC cases not related to the presence or absence of circulating HCV, and a hypermutated pre-C strain, characterized by the same mutational pattern, was identified in three HCC cases. The mutational pattern of the pre-C/C region was closely related to HBV replication efficiency, and specific HBV mutations selectively associated with HCV co-infection could be linked with accelerated HBV/HCV-related disease progression.

Mutations in the E2-PePHD region of hepatitis C virus type 1b in patients with hepatocellular carcinoma.

An interaction between the protein kinase (PKR)-eIF2-alpha phosphorylation homology domain (PePHD) within the E2 protein of hepatitis C virus (HCV) and cell protein kinase (PKR) may affect the control of protein synthesis and cell growth. In an attempt to investigate the genetic variability of the E2-PePHD domain in hepatocellular carcinoma (HCC), we studied sera and liver tissues from HCC patients. The partial E2-PePHD region was analysed by direct sequencing of the sera of 47 HCCs in cirrhotic livers and 31 cases of chronic active hepatitis (CAH), and tumoral and non-tumoral liver tissues from 13 HCC patients. A similar number of mutations was detected within the E2 domain in the HCC and CAH cases, but nine of the 47 HCCs (19%) showed an amino acid (aa) mutation at position 660, eight of which involved a change in the same aa (alanine instead of serine; A/S). No such mutation was detected in any of the PePHD sequences from the CAH patients: this difference was statistically significant (P = 0.008). The aa change at position 660 was also found in two sequences from tumoral but not non-tumoral tissue from the same liver. The analysis of 461 sequences obtained from GenBank supports the conclusion that the observed aa change is an infrequent event in HCV-infected patients, thus suggesting that it could be associated with HCC.

Genetic variability of hepatitis C virus in HBV/HCV co-infection and HCV single-infection.

To describe the virological profile of HCV in HBV/HCV co-infection, we investigated the variability of HVR-1 and NS5A domains, which may be involved in viral persistence and replication efficiency. We studied 95 patients: 37 with serological markers of HBV/HCV co-infection, 33 with single HBV and 25 with single HCV infection. HVR-1 complexity and NS5A gene variability were respectively explored by means of PCR-SSCP and direct sequencing. Serum HBV genomes were detected in all coinfected patients: 19 also had circulating HCV particles (group BC-I), whereas HCV were undetectable in the other 18 (group BC-II). Group BC-I was characterised by a significantly lower HBV replication capacity, that reflects the replicative dominance of HCV, although the dominant virus had the same degree of variability as the HCV in single infection. HBV viral load was higher in group BC-II, but not significantly different from that observed in the single infection. Our data indicate an alternation in replicative dominance in co-infection: HBV can suppress HCV replication to undetectable levels, whereas HCV may reduce but does not abrogate the replication capacity of HBV. Furthermore, in the cases of HCV dominance, circulating HBV genomes did not have a significant effect on the viral heterogeneity of HCV.

Genetic heterogeneity of hepatitis C virus (HCV) in clinical strains of HIV positive and HIV negative patients chronically infected with HCV genotype 3a.

The clinical correlation between the degree of HCV variability and the response to anti-HCV treatment in HIV positive patients infected with HCV genotype 3a is unknown. In this study, 27 HIV positive and 5 HIV negative patients with HCV genotype 3a infection were treated with interferon-alpha-2b with or without ribavirin. Nine patients (5 HIV positive) achieved a sustained virological response (SR) and 23 (only one HIV negative) were non-responders (NR). Sequence analyses of the partial E2 domain and the non-structural 5A protein were performed at baseline in all patients, and before and during treatment in the HIV positive NRs. There was no difference in the mean number of amino acid mutations from HCV 3a prototype, within E2 region, between the HIV positive and HIV negative patients: 17 (range 11-25) vs 16 (range 14-17). The mean baseline number of mutations in E2 region, was similar in HIV positive SRs and NRs: 18 (range 14-25) vs 16 (range 11-19). Phylogenetic analysis of HCV paired serum samples at baseline and during treatment revealed identical E2 sequence in 5/21 HIV positive NR patients, whereas 6 other sequences were strictly related to baseline E2 domain and the remaining 10 were divergent. The mean number of amino acid mutations in the NS5A protein at baseline, was 1 (range 0-3) in HIV negative patients and 2 (range 0-4) in HIV positive ones. This region was highly conserved in all isolates of HIV positive NRs analysed during treatment. These results suggest that genetic variability at baseline within the E2 region and NS5A protein of HCV 3a strain obtained from HIV positive and HIV negative patients is not associated with treatment response. Furthermore, the anti-HCV treatment did not influence HCV heterogeneity within the E2 and NS5A domains in HIV positive patients infected with HCV genotype 3a.

Sequence analysis of NS3 protease gene in clinical strains of hepatitis C virus.

The amino terminal region of the non structural gene 3 (NS3) of hepatitis C virus (HCV) is a chymotripsinlike serine-protease responsible for cleavage of the non structural proteins of Hepatitis C virus (HCV). In order to investigate the genetic variation of this region, we developed a nested PCR to obtain NS3 protease sequences from 54 patients chronically infected with HCV genotypes 1a, 1b and 3, respectively. Comparison of nucleotide and amino acids sequences of NS3 protease domain with consensus sequence obtained within the same genotype, showed 3.73% nucleotide divergence and 1.64% amino acid divergence in isolates of genotype 3a, whereas isolates 1a exhibited 4.45% nucleotide and 4% amino acid change, respectively. Finally, NS3 sequence from 1b isolates revealed 6.47% nucleotide and 3.5 % aa changes. Comparison of consensus amino acid sequences derived from isolates 1a, 1b and 3, with the HCV prototypes showed a low amino acid sequence diversity. However, the consensus sequence of HCV genotype 3 isolates showed an amino acid changed from the prototype, that was located within a region important for enzyme structure and activity. These results indicated that the NS3 protease gene is highly conserved within the same HCV genotype. The domains involved in enzyme function were highly conserved in 1a and 1b strains, whereas consensus sequence of isolates 3a showed that the majority of these strains were not perfectly conserved in one of such regions. These findings altogether suggested that the NS3 protease enzyme of HCV may constitute an important target for antiviral therapy, but the NS3 protease variability of isolates 3 within a region that is a potential target for antiviral therapy could pose a problem for structure based drug development.

Evolution of the E2 region of hepatitis C virus in an infant infected by mother-to-infant transmission.

To demonstrate vertical transmission of hepatitis C virus from an HCV infected woman and to assess the evolution of HCV quasispecies in the infant, the variable E2 region was analyzed in one mother-infant pair at birth and in serial samples from the infected baby. Sequence analysis of the E2 region obtained by means of direct sequencing of PCR products of mother-infant pair at birth, showed that the sequence of the dominant strain in the infant was related closely but not identical to that of her mother. The HCV population in mother-infant pair at birth and in serial samples of the infant was analyzed by polymerase-chain reaction-mediated Single Strand Conformational Polymorphism analysis (SSCP), which can distinguish DNA fragments of the same size as different electrophoretic migration of single stranded DNA. Single Strand Polymorphism analysis revealed that the infant was infected with two mutant genomes whereas the mother had a unique variant. The prevalent strain detected in the baby was not dominant in the mother at delivery and the pattern of quasispecies in the infant at birth was not the same as her mother, suggesting that the infant acquired the infection in utero. Changes in the dominant strain and evolution of the pattern of quasispecies in the infant from the 10th month of age were possibly due to the immune selection of escape mutants.

New infection with heterotypic hepatitis C virus in a patient with long-term hepatitis C virus eradication.

BACKGROUND: Experimental hepatitis C virus infection in chimpanzees has shown that natural hepatitis C virus infection does not induce protective immunity and reinfection can occur in seroconverted animals. AIM: To study the clinical, virological and histological outcome of a new infection sustained by a different hepatitis C virus strain after a primary infection with eradication of the original virus. PATIENTS AND METHODS: A young Italian man with chronic hepatitis C virus type 4 hepatitis was treated with Interferon therapy and achieved a sustained biochemical and virological response. After long follow-up, an asymptomatic flare-up of alanine transaminase occurred. This alanine transaminase increase was associated with serum hepatitis C virus RNA positivity and a low viral load, and the infecting hepatitis C virus genotype was type 3. The clinical and virological course of this new infection is described. RESULTS AND CONCLUSIONS: This report shows that there is no protective immunity against hepatitis C virus type 3 after infection by hepatitis C virus type 4 strain.

Effect of increasing dose of interferon on the evolution of hepatitis C virus 1b quasispecies.

The effects of interferon therapy on hepatitis C virus (HCV) genome are still controversial in terms of biological and clinical significance. Changes in the quasispecies of the hypervariable (HVR) and non-structural 5A (NS5A) regions of HCV 1b were evaluated in nine patients treated with increasing doses of interferon and five untreated controls. HCV quasispecies were analyzed in HVR and NS5A by single-strand conformation polymorphism assay. The HVR quasispecies varied over time both in treated and untreated patients. However, at least one persistent strain was present in all patients. With low doses of interferon, variations in HVR complexity were found in seven of nine patients and in four patients new variants became detectable. A reduction in the heterogeneity of the HVR quasispecies was observed after increase of the interferon dose. In contrast, NS5A profiles remained unmodified in all but three cases in which direct sequencing showed no changes in amino acid sequences of the predominant strain. The results suggest that interferon sensitivity of some HCV strains may be dose dependent. The homogeneity of NS5A pattern populations during treatment suggests that interferon exerts much less pressure on this region.

Comparison of serum and liver hepatitis C virus quasispecies in HCV-related hepatocellular carcinoma.

BACKGROUND/AIMS: The hepatitis C virus (HCV) genome consists of quasispecies populations of heterogeneous variants, especially in the hypervariable region. To assess the profiles of viral quasispecies in HCV-related hepatocellular carcinoma, we studied the viral population patterns in serum and liver tissues of 13 HCV-positive patients with hepatocellular carcinoma developed on cirrhotic and non-cirrhotic livers (5 and 8 cases, respectively). METHODS: HCV genome heterogeneity was analyzed by polymerase chain reaction-mediated single-strand conformation polymorphism analysis, which showed multiple DNA bands representing different hypervariable region sequences. RESULTS: The HCV populations were different between tumorous and nontumorous tissues in 3/5 hepatocellular carcinomas with cirrhosis and in 6/8 without cirrhosis. At least one or more than one common band was detected in both compartments in all but one case. No significant differences in the complexity of HCV quasispecies were found in hepatocellular carcinoma with or without underlying cirrhosis. Comparison of the HCV quasispecies profiles in serum and liver tissues showed a different distribution of HCV variants between these two compartments in 6/7 patients. In four cases, both common and compartmentalized sequences were detected, whereas in two cases, both without cirrhosis, the HCV population in serum was completely different from that found in the liver. CONCLUSIONS: These results suggest that the complexity of HCV populations is influenced by the presence of hepatocellular carcinoma rather than by the severity of the underlying chronic liver disease. The different quasispecies patterns found in serum and liver may reflect different biological properties of circulating and intrahepatic HCV particles or the existence of extrahepatic sites of replication.

Complete eradication of hepatitis C virus after interferon treatment for chronic hepatitis C.

BACKGROUND: Alpha-interferon therapy can lead to a persistent biochemical response, but discordant opinions have been expressed on the definition of sustained response and on the real possibility of complete eradication of hepatitis C virus (HCV). AIMS: To define the clinical, virological and histologic profiles of the patients with sustained response. PATIENTS: Twenty-eight patients with three different biochemical and virological patterns of response to interferon therapy (16 sustained responders, 6 responders with relapse and 6 non responders) were studied for a follow-up period of 36 months. METHODS: HCV-RNA sequences were investigated in serum, peripheral blood mononuclear cells and in liver tissue by means of reverse transcriptase-polymerase chain reaction, targeted to the 5' non coding region. Viral load in serum was quantified by branched-DNA signal amplification. HCV genotypes were evaluated using a line probe assay. RESULTS: All sustained responders showed persistent normal ALT values and loss of serum HCV-RNA during the treatment and in the entire follow-up period. The HCV clearance was also demonstrated in peripheral blood mononuclear cells and in liver tissue. Pre-treatment HCV-RNA quantitation showed that sustained responders had a significantly lower viral load compared to relapsers and non responders (p = 0.005). HCV genotyping showed that patients infected by genotypes 2a, 3a were more likely to achieve a sustained response. Interestingly, a prolonged response was also observed in the only three patients with pre-treatment detectable viral load infected by genotype 3a and in patients with genotype 1b and low viraemia levels. To assess the histologic outcome following HCV eradication, all sustained responders underwent a second liver biopsy in the follow-up period (6-18 months). Periportal necrosis and portal inflammation were significantly improved. CONCLUSIONS: Our results suggest that persistent loss of HCV-RNA in serum, peripheral blood mononuclear cells and liver as well as histologic improvement are consistent with the complete HCV eradication even from intracellular compartments and from potential extra-hepatic sites of viral persistence. Moreover, pre-treatment viral load, HCV infecting genotypes and histologic features may influence the clinical outcome of hepatitis C and the response to interferon therapy.

Structure and expression of the cyclin A gene in human primary liver cancer. Correlation with flow cytometric parameters.

BACKGROUND/AIMS: The cyclin A gene plays an important role in both the S and G2-M phases of the cell cycle, and has been identified at a site of hepatitis B virus DNA integration in a human liver cancer. We analyzed tumorous and non-tumorous samples from patients with primary liver cancer to determine whether a) the cyclin A gene is rearranged in liver tumors and b) the cyclin A transcript level correlates with the percentage of proliferating cells. METHODS: Samples from 43 patients were analyzed by Southern blot. Cyclin A RNA accumulation was evaluated in 18 cases by slot blot and correlated with the percentage of cells in S plus G2-M phases defined by flow cytometry. RESULTS: No rearrangement of the cyclin A gene was found in tumorous compared to non-tumorous tissue. A very strong positive correlation was found between the cyclin A RNA level and the cumulative percentage of cells in S plus G2-M phases (r = 0.99; p < 0.0001). CONCLUSIONS: This in vivo study shows that the expression of cyclin A RNA correlates with the percentage of proliferating cells in primary liver cancer. Thus, cyclin A is a new potential liver tumor cell proliferation index.

HCV-associated liver cancer without cirrhosis.

Chronic infection with hepatitis C virus (HCV) is regarded as a risk factor for hepatocellular cancer, mostly in patients with liver cirrhosis. We looked for HCV genomes in the livers of patients with hepatocellular cancer who did not have cirrhosis to see whether HCV was directly oncogenic. Cancerous and non-cancerous liver tissue, and serum samples from 19 patients negative for hepatitis B surface antigen were analysed by polymerase chain reaction for the presence of HCV genome, HCV replication, HCV genotyping, and HBV genome. 13 of 19 patients were HCV RNA-positive in cancerous and non-cancerous liver tissue; 8 of 17 tested were anti-HCV positive. Among the 13 HCV RNA-positive patients, 11 had genotype 1b and 2 had genotype 2a. 7 of 13 serum samples were HCV RNA positive. 7 of 19 patients were HBV DNA positive in cancerous and non-cancerous liver tissue, 5 of them anti-HBc positive. 4 patients were both HCV RNA and HBV DNA positive and 3 were both HCV RNA and HBV DNA negative. Our results provide evidence for the association of HCV, mostly genotype 1b, with hepatocellular cancer without the intermediate step of cirrhosis.

The interaction between hepatitis B virus and hepatitis C virus in acute and chronic liver disease.

Infections by the hepatitis B or C virus are extremely common causes of acute and chronic liver disease, and coexistence of the two viruses in the same patient is not rare. Evidence has been found that such interaction may play an important role in fulminant hepatitis and in the development of hepatocellular carcinoma in cirrhotic patients. Liver disease activity and prognosis have been reported to be generally more serious in the presence of double infection, although an inverse relationship in the replicative levels of the two agents has been noted, suggesting viral interference, particularly in cases of chronic hepatitis. Thus, the two viruses seem to inhibit each other at the molecular level, while cytopathic effects appear to be enhanced. Further studies are needed to explain the mechanisms of these apparently contrasting effects.

Lowering effects of a preparation containing fibres and lactulose on glucose and insulin levels in obesity.

The short-term metabolic effects of a dietary supplementation of biscuits containing raw fibre and lactulose (Fiberlac, Bracco) on circadian glucose, insulin and amino acid concentrations were studied in 10 obese patients in a crossover comparison. The biscuits (3 at breakfast, 4 at lunch and 4 at evening meal; approximately 10 g total dietary fibre, 2 g raw fibre, 8.25 g lactulose) were randomly substituted for an equicaloric part of a diet containing 20-22 kcal per kg ideal body weight under strict medical surveillance. Blood glucose in response to meal, as well as mean concentration throughout the day was lower during fibre supplementation. Also mean insulin was halved, and the insulin response to meals was blunted by 100-250 pmol/L. The plasma amino acid response to meals was increased, possibly in relation to decreased insulinemia. The data show a remarkable metabolic effect of the preparation in obese patients, without any further dietary restriction. The clinical effects and compliance remain to be determined in long-term studies, and in other states of glucose-intolerance, e.g. liver cirrhosis.

Different in situ expression of insulin-like growth factor type II in hepatocellular carcinoma. An in situ hybridization and immunohistochemical study.

Reexpression of the insulin-like growth factor type II (IGF-II) gene has recently been described in hepatocellular carcinoma (HCC). In this study, we used a nonisotopic in situ hybridization method to analyze the expression of IGF-II mRNA in a series of 28 HCCs arising on cirrhotic and noncirrhotic livers. An immunohistochemical method was used to detect IGF-II peptide. Hepatitis B virus (HBV) status and the histological differentiation degree were also evaluated. Increased expression of IGF-II mRNA was found in 4 of 28 HCCs, and 7 of 17 cirrhotic patients showed IGF-II mRNA in the cirrhotic nodules surrounding the HCC. A slightly higher rate of positivity for IGF-II mRNA was found in the HBV-negative patients than in HBV-positive ones. Positive immunostaining for the IGF-II peptide in the HCC and/or in surrounding cirrhotic nodules was found in 10 of 28 cases. The normal hepatocytes of the noncirrhotic patients were always negative for IGF-II peptide and mRNA. The similarities between our results and those from experimental models in woodchucks seem to support the concept that heterogeneous phenotypic groups could exist in human HCCs.

Low frequency of allelic loss in the cyclin A gene in human hepatocellular carcinomas: a study based on PCR.

The cyclin A gene was first identified at a site of hepatitis B virus DNA integration in a primary liver cancer (PLC). It has now been mapped to 4q27, in the proximity of a chromosomal locus (4q32) which is frequently rearranged and deleted in PLC. We took advantage of the TaqI polymorphism recently described in the cyclin A gene to search for allelic loss in this gene by means of PCR. Tumorous and non-tumorous tissue from 50 patients with PLC was analyzed: 27 samples (54%) were homozygous for the A1 type allele (i.e. the allele bearing the TaqI restriction site), three (6%) were homozygous for the A2 type allele (without the TaqI site) and 20 (40%) were heterozygous. Comparison of tumorous and non-tumorous patterns showed that only one (5%) tumorous tissue out of 20 heterozygous patients was homozygous, indicating the loss of an allele at this locus. We conclude that allelic loss in the cyclin A gene is a rare event in patients with PLC, and that PCR is rapid and reliable for the detection of allelic loss in the cyclin A gene when only small amounts of DNA are available.