The Thr205 phosphorylation site within respiratory syncytial virus matrix (M) protein modulates M oligomerization and virus production.

UNLABELLED: Human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and the elderly worldwide; however, there is no licensed RSV vaccine or effective drug treatment available. The RSV matrix (M) protein plays key roles in virus assembly and budding, but the protein interactions that govern budding of infectious virus are not known. In this study, we focus on M protein and identify a key phosphorylation site (Thr205) in M that is critical for RSV infectious virus production. Recombinant virus with a nonphosphorylatable alanine (Ala) residue at the site was markedly attenuated, whereas virus with a phosphomimetic aspartate (Asp) resulted in a nonviable virus which could only be recovered with an additional mutation in M (serine to asparagine at position 220), strongly implying that Thr205 is critical for viral infectivity. Experiments in vitro showed that mutation of Thr205 does not affect M stability or the ability to form dimers but implicate an effect on higher-order oligomer assembly. In transfected and infected cells, Asp substitution of Thr205 appeared to impair M oligomerization; typical filamentous structures still formed at the plasma membrane, but M assembly during the ensuing elongation process seemed to be impaired, resulting in shorter and more branched filaments as observed using electron microscopy (EM). Our data thus imply for the first time that M oligomerization, regulated by a negative charge at Thr205, may be critical to production of infectious RSV. IMPORTANCE: We show here for the first time that RSV M's role in virus assembly/release is strongly dependent on threonine 205 (Thr205), a consensus site for CK2, which appears to play a key regulatory role in modulating M oligomerization and association with virus filaments. Our analysis indicates that T205 mutations do not impair M dimerization or viruslike filament formation per se but rather the ability of M to assemble in ordered fashion on the viral filaments themselves. This appears to impact in turn upon the infectivity of released virus rather than on virus production or release itself. Thus, M oligomerization would appear to be a target of interest for the development of anti-RSV agents; further, the recombinant T205-substituted mutant viruses described here would appear to be the first RSV mutants affected in viral maturation to our knowledge and hence of considerable interest for vaccine approaches in the future.

Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis C.

As acute hepatitis C virus (HCV) infection is clinically inapparent in most cases, the immunologic correlates of recovery are not well defined. The cellular immune response is thought to contribute to the elimination of HCV-infected cells and a strong HCV-specific T-helper-cell (Th) response is associated with recovery from acute hepatitis C (ref. 2). However, diagnosis of resolved hepatitis C is based at present on the detection of HCV-specific antibodies and the absence of detectable HCV RNA, and detailed comparison of the humoral and cellular immune response has been hampered by the fact that patient cohorts as well as HCV strains are usually heterogeneous and that clinical data from acute-phase and long-term follow-up after infection generally are not available. We studied a cohort of women accidentally exposed to the same HCV strain of known sequence and found that circulating HCV-specific antibodies were undetectable in many patients 18-20 years after recovery, whereas HCV-specific helper and cytotoxic T-cell responses with an interferon (IFN)-gamma-producing (Tc1) phenotype persisted. The data indicate these HCV-specific CD4 + and CD8+ T cells are biomarkers for a prior HCV exposure and recovery. Because of undetectable antibodies against HCV, the incidence of self-limited HCV infections and recovery may be underestimated in the general population.

High-level transient expression of influenza virus proteins from a series of SV40 late and early replacement vectors.

We have constructed a collection of simian virus 40 (SV40) plasmid vectors useful for transient or constitutive expression of cDNA or genomic DNA in animal cells. Most vectors contain several unique restriction sites downstream from the SV40 late or early promoter, and are available with or without the virus-specific splicing signals. The use of these vectors for transient expression in monkey cells of X47 (H3N2) influenza hemagglutinin (HA) and matrix protein (M1) was demonstrated. Membrane-bound (HAm) as well as secreted forms of the HA glycoprotein lacking the sequence of the C-terminal anchor (HA-) have been obtained. Depending on the insert, the type of vector and the amount of transfected DNA, HA levels in COS cells [Gething and Sambrook, Nature 293 (1981) 620-625] transfected with late replacement SV40 vectors vary from 10(9) (HAm) to 10(8) (HA-) molecules per transfected cell. The maximum expression levels with early replacement vectors in COS cells are at least 50 times lower. In addition to the optimalization and the characterization of the expression of each vector-coded influenza protein, cotransfections, including vectors expressing HAm, neuraminidase (NA) and M1, were undertaken. The latter experiments did not result in a measureable amount of HAm or NA in the cell culture medium, suggesting that expression of these three structural viral proteins does not result in budding of (empty) influenza particles from the cell surface.

Antibodies to hepatitis C virus envelope proteins correlate with hepatitis C viraemia after liver transplantation.

BACKGROUND: Liver transplant recipients for hepatitis C virus (HCV)-related cirrhosis usually remain anti-HCV-seropositive after transplantation. The aim of this study was to characterize, longitudinally, the profile of HCV-specific antibodies and cryoglobulins in liver transplant recipients with recurrent HCV infection. METHODS: Serial serum samples were collected prospectively before, at 1 month after, and at 12 months after transplantation for HCV cirrhosis in 30 patients infected with genotype 1. The antibodies against HCV envelope proteins (E1 and E2) were quantitated by enzyme-linked immunosorbent assay and antibodies against core, E2/hypervariable region I (HVRI), NS3, NS4, and NS5A antigens by a line immunoassay. Sera were also tested for cryoglobulins. RESULTS: The titer of each anti-HCV antibody had fallen at 1 month after transplantation (P<0.05) with the exception of anti-E1 levels, which had risen in 16 patients with acute hepatitis C at that time (P=0.01). Anti-E1 and anti-E2 titers, but not antibodies against other HCV antigens, increased to pre-transplantation levels or higher at 12 months, which correlated with serum HCV RNA levels. Cryoglobulinemia was present in nine patients after transplantation (30%) and was associated with lower anti-E1 levels (P=0.04) and more severe graft damage. CONCLUSIONS: The early increase in antibodies to HCV envelope proteins in correlation with viremia suggests that the envelope-specific humoral immune response may be directly stimulated by HCV replication. Anti-E1 levels may be a useful marker in monitoring patients with recurrent HCV infection.

Seroepidemiological survey of hepatitis C virus among commercial sex workers and pregnant women in Kinshasa, Democratic Republic of Congo.

BACKGROUND: Studies conducted mainly in industrialized countries have shown that the transmission of hepatitis C virus (HCV) is mainly parenteral, and have emphasized the role of nosocomial transmission. In Equatorial Africa, the respective contributions of parenteral and non-parenteral routes of transmission are unknown. The potential role of sexual transmission in this area of high HCV endemicity, where sexually transmitted infections (STI) are frequent, is suggested by the fact that HCV infection is rare in infants and young adolescents, but increases thereafter with age. The present study, conducted in Democratic Republic of Congo, was designed to determine the prevalence of HCV infection and associated sexual risk factors in two female populations with different sexual behaviour. METHODS: Cross-sectional studies conducted among commercial sex workers (CSW; n = 1144) and pregnant women (n = 1092) in the late 1980s in Kinshasa showed a high frequency of at-risk sexual behaviour, STI and human immunodeficiency virus (HIV) infection, particularly among CSW. We screened samples collected during these epidemiological studies for antibodies to HCV using a second-generation ELISA with confirmation by a third-generation LIA. We also assessed sociodemographic variables, medical history, STI markers and sexual behaviour, and their potential association with HCV infection. RESULTS: The overall prevalence of anti-HCV was 6.6% (95% CI : 5.2-8.2) among CSW and 4.3% (95% CI : 3.2-5.7) among pregnant women (age-adjusted OR = 1.5, 95% CI : 1.0-2.1, P = 0.05). Multivariate analysis showed that the presence of anti-HCV among CSW was independently associated with a previous history of blood transfusion (P < 0.001), age >30 years (P < 0.001) and the presence of at least one biological marker of STI (P < 0.03). No such links were found among pregnant women (although the history of blood transfusions was not investigated in this group). Anti-HCV was not associated with sociodemographic variables or sexual behaviour in either group, or with individual markers of STI. Despite the high-risk sexual behaviour and the higher prevalence of STI in CSW, the difference in HCV seroprevalence between CSW and pregnant women (6.6% versus 4.3%) was small, particularly when compared with the difference in the seroprevalence of HIV (34.1% versus 2.8%). CONCLUSION: The role of sexual transmission in the spread of HCV seems to be limited. Parenteral transmission (including blood transfusion and injections), possibly related to the treatment of STI, probably plays a major role.

Prevalence and clinical expression of HCV-genotypes in haemodialysis-patients of two geographically remote countries: Belgium and Saudi-Arabia.

Hepatitis C virus is the leading cause of acute and chronic liver disease in hemodialysis patients. There are at least six major HCV-genotypes, with a well documented geographical distribution in the general population. Moreover, HCV-genotype is one of the major determinants of the therapeutic response to Interferon Alpha in affected patients. Since the therapeutic outcome in HCV-positive hemodialysis patients, especially with regard to the different HCV-genotypes, is of interest, a multicentre epidemiologic study was performed in HCV-antibody positive hemodialysis patients of two geographically remote countries, i.e. in Flanders (Belgium) and in Saudi-Arabia. 184 chronic hemodialysis patients, with a positive second or third generation Elisa assay for HCV, were tested for HCV-viremia and HCV-genotype, using a 5' untranslated region (UR) nested PCR for the detection of HCV-RNA and subsequently type-specific probes to hybridize with HCV-RNA (Inno-Lipa). Additionally, clinical data were collected by means of a standardized questionnaire, thoroughly completed by the nephrologist in charge of each respective patient. Viremia was present in 79% of the patients (146 out of 184). The prevalence of HCV-genotypes differed significantly between Belgian and Saudi-Arabian dialysis-patients. In Belgian dialysis patients HCV-genotype 1b was most prevalent (i.e. 62%), while in Saudi-Arabian patients HCV-genotypes 4, 1b, and la were present in respectively 36,4%, 31,7%, and 25,8% of the HCV-PCR positive patients. Although there were significant differences between Belgian and Saudi-Arabian dialysis patients, no clinical data showed any significant correlation with the HCV-genotype. Transaminases, determined over a six months period, showed normal average values. Doubling of the transaminases, in at least one out of six measurements over a six monthly period, occurred only in 14% (alanine aminotransferase, ALT) and 10% (aspartate aminotransferase, AST) of the patients. In Belgian dialysis patients, HCV-genotype 4 (or HCV-genotype 5) significantly correlated with a more recent start of dialysis treatment. We conclude that there is a significant different geographical prevalence of HCV-genotypes in HCV-affected hemodialysis patients. None of the different HCV-genotypes shows any particular clinical expression. Transaminases are not a sensitive marker for ongoing HCV-replication in hemodialysis patients. In Belgian dialysis patients, a changing pattern of HCV-infection is suggested, with an increasing prevalence of HCV-genotype 4 (or HCV-genotype 5) in more recent years. These data suggest possible implications for the therapeutic strategy in dialysis patients.

HCV Genotyping by the Line Probe Assay INNO-LiPA HCV II.

Hepatitis C viruses (HCVs) constitute a highly variable genus within the Flaviviridae, with closest homology to the hepatitis G and GB viruses, and Pestiviruses. The positive-stranded RNA genome encodes a polyprotein which is co- and posttranslationally cleaved into at least nine proteins. Core, E1, and E2 (the structural proteins) and NS2, NS3, NS4A, NS4B, NS5A, and NS5B (the nonstructural [NS] proteins). A theoretical protein of 7 kDa (tp7) may be processed from the carboxy terminal E2 region (1).

Confirmation of HCV Antibodies by the Line Immunoassay INNO-LIA HCV Ab III.

HCVs constitute a genus within the Flaviviridae, with closest homology to the hepatitis G and GB viruses, and Pestiviruses. The positive-stranded RNA genome encodes at least nine proteins. Core, El, and E2 constitute the structural proteins; NS2, NS3, NS4A, NS4B, NS5A, and NS5B are nonstructural (NS) proteins. HCV isolates display high levels of sequence heterogeneity allowing classification into at least 11 types and 90 subtypes (1). HCV infection of the human liver is often clinically benign, with mild icterus in the acute phase, the disease may even go unnoticed in some cases of acute resolving hepatitis C. In the majority (>70%) of cases, however, HCV infection leads to chronic persistent or active infection, often with complications of liver cirrhosis and auto-immune disorders. Hepatocellular carcinoma may occur after about 20-35 yr (2); sometimes even without the intermediate phase of cirrhosis. No prophylaxis is available today and treatment with interferon-alpha (IFN-α) only leads to long-term resolution in about 4-36% of treated cases, depending on the HCV genotype (1).

[Update on hepatitis C virus: its variability and the implications]. / Mise au point sur le virus de l'hépatite C: variabilité et implications.

Hepatitis C virus (HCV) is the main etiologic factor of post-transfusional and sporadic hepatitis, called non-A non-B in the past. These infections are characterized by a very high number of chronic carriers always with a persistent viral increase, but often at a slow pace. The seriousness of liver disease differs from one individual to another, varying from an asymptomatic form with minor or no liver injuries, to cirrhosis and hepatocellular carcinoma. Physiopathological mechanisms involved in liver injuries are still poorly understood. The direct role of immune response and of possible genetic factors is still under study. This review aims at summing up the discovery of HCV, its structure, and its variability in the various genome regions in the same individual and from one individual to another. The different methods and techniques to analyze this variability are also reviewed, as well as the various suggested ways of classifying the different types. The geographical distribution and both clinical and biological consequences of this variability are also discussed.

High prevalence of hepatitis C infection among Brazilian hemodialysis patients in Rio de Janeiro: a one-year follow-up study.

Nearly 400 hemodialysis patients treated at 5 different hemodialysis units in Rio de Janeiro were tested for one year for the presence of hepatitis C and B markers. During the same period, samples were also obtained from 35 continuous ambulatory peritoneal dialysis (CAPD) patients and from 242 health care workers. Depending on the hemodialysis unit studied, anti-HCV prevalence rates ranging from 47% to 82% (mean 65%) were detected. CAPD patients showed a lower prevalence of 17%. The prevalence of antibodies against hepatitis C virus (anti-HCV) among health care workers was 2.9%. We observed a hepatitis C attack rate of 11.5% per year in the anti-HCV-negative hemodialysis patient population. An average of 9.4% of the hemodialysis patients were chronic carriers of hepatitis B virus (HBV) (range 1.8% - 20.4%), while 48.9% showed markers of previous HBV infection. The HBV attack rate was 4.5% per year (range 0% - 6%). These results indicate an alarming high prevalence of anti-HCV among hemodialysis patients of this studied region.

Recombinant secreted haemagglutinin protects mice against a lethal challenge of influenza virus.

Balb/c mice were immunized with 2 x 2 micrograms of purified recombinant secreted haemagglutinin, derived from the A/Victoria/3/75 (H3N2) virus. In the first immunization, Ribi adjuvant was used, while for the booster injection a monophosphoryl lipid A/muramyl dipeptide combination was chosen. Mice immunized in this way were 90-100% protected against a challenge with 20 LD50 of mouse-adapted, homologous virus (strain X47). Bromelain-solubilized haemagglutinin gave only 70% protection under comparable conditions.

Cloning and phylogenetic analysis of the core, E2, and NS3/NS4 regions of the hepatitis C virus type 5a.

By means of the Line Probe Assay, a hepatitis C virus type 5a infected serum from a Belgian patient was selected. The complete core region (573 bp), the carboxyterminal part of E1 and the aminoterminal part of E2/NS1 (661 bp), and an epitope containing region in NS3-NS4 (1452 bp) were cloned and sequenced. The deduced amino acid sequence revealed type-specific variations in regions of core and NS4 which were previously recognized as evoking a type-specific antibody response. In addition, the aminoterminal region of E2 showed high variability when compared with sequences of type 1, 2, and 3. Phylogenetic analysis showed separate branching of this isolate. The analysis in the core region was not conclusive for some of the strains included and should therefore be carried out in combination with the NS5B region.

Classification of hepatitis C viruses based on phylogenetic analysis of the envelope 1 and nonstructural 5B regions and identification of five additional subtypes.

Genotyping of hepatitis C virus-positive sera by means of a line probe assay indicated that < 3% of European samples, but up to 30% of Gabonese sera, could not be classified as either 1a, 1b, 2a, 2b, 3a, 3b, 4c, 5a, or 6a. Such samples were analyzed in the 5' untranslated region and in the nonstructural 5 (NS5) region. Classification based on phylogenetic analysis of the commonly used 222-bp-long NS5B region was possible for most but not all of the selected sera. Therefore, the core/envelope 1 region (579 bp) and a larger NS5B (340 bp) region were also analyzed. Only the phylogenetic analysis of the 340-bp NS5B region of these newly identified and published isolates provided unambiguous classification into types and subtypes. Furthermore, unequivocal evidence for four subtypes in type 2 and eight subtypes in type 4 was provided. A specific recognition sequence in the 5' untranslated region was observed for every newly identified subtype. Based on 1830 pair-wise comparisons in NS5B, isolates belonging to the same subtype showed evolutionary distances of < 0.127 and isolates of the same type exhibited evolutionary distances of < 0.328. These phylogenetic border distances can be conveniently used for classification of hepatitis C virus isolates into types and subtypes.

Analysis of the putative E1 envelope and NS4a epitope regions of HCV type 3.

Using a Line Probe Assay, type 3 HCV genotype-infected sera were selected from Brazilian blood donors. The partial nucleotide sequences of the core/E1 and NS4a epitope-containing regions and the NS5b typing region were determined. The E1 region had a nucleic acid homology of only 61 to 65% with the type 1 prototype genomes, and 56 to 58% homology with the type 2 prototype HCV genomes. Similar homologies were also found for the NS4a epitope region and for NS5. Furthermore, the deduced amino acid sequence of type 3 NS4a was used to generate synthetic peptides which were strongly reactive with human HCV-infected sera which were previously determined as anti-NS4 negative, indicating that a type-specific antibody response to the NS4a protein may exist.

Expression of noncovalent hepatitis C virus envelope E1-E2 complexes is not required for the induction of antibodies with neutralizing properties following DNA immunization.

Interactive glycoproteins present on the surface of viral particles represent the main target of neutralizing antibodies. The ability of DNA vaccination to induce antibodies directed at such structures was investigated by using eight different expression plasmids engineered either to favor or to prevent interaction between the hepatitis C virus (HCV) envelope glycoproteins E1 and E2. Independently of the injection route (intramuscular or intraepidermal), plasmids expressing antigens capable of forming heterodimers presumed to be the prebudding form of the HCV envelope protein complex failed to induce any significant, stable antibodies following injection in mice. In sharp contrast, high titers of antibodies directed at both conformational and linear determinants were induced by using plasmids expressing severely truncated antigens that have lost the ability to form native complexes. In addition, only a truncated form of E2 induced antibodies reacting against the hypervariable region 1 of E2 (specifically with the C-terminal part of it) known to contain a neutralization site. When injected intraepidermally into small primates, the truncated E2-encoding plasmid induced antibodies able to neutralize in vitro the binding of a purified E2 protein onto susceptible cells. Because such antibodies have been associated with viral clearance in both humans and chimpanzees, these findings may have important implications for the development of protective immunity against HCV.

Characterization of small hepatitis B surface antigen epitopes involved in binding to human annexin V.

Previously, we have shown that small hepatitis B surface antigen (SHBsAg) binds specifically to human annexin V (hAV) and that hAV plays a key role in the initial steps of hepatitis B virus (HBV) infection. We have also demonstrated the spontaneous development of anti-idiotypic antibodies (antibodies to HBsAg Ab2) in rabbits immunized with hAV. As Ab2 is able to inhibit the binding of hAV to SHBsAg, Ab2 might contain epitope(s) mimicking a region of hAV for binding to SHBsAg. Identification of this epitope will therefore reveal a SHBsAg sequence involved in hAV binding. Using a panel of synthetic peptides covering the region of SHBsAg located on the outer surface of the virus, binding studies showed that the region incorporating amino acids (aa) 125-131 of SHBsAg is important for binding to Ab2 and consequently also for binding to hAV. Further experiments revealed that not only this region, but also the region incorporating aa 158-169, is involved in the binding of SHBsAg to hAV. As these regions are located in the structural vicinity according to the topological model of HBsAg proposed by Chen et al., our findings suggest that these regions are parts of a conformational epitope of SHBsAg for binding to hAV. Because of the crucial role of hAV in HBV infection, further studies on the HBsAg epitopes for hAV binding may lead to the development of a new generation of vaccines or molecules for prevention and for treatment of patients with chronic hepatitis B.

Involvement of phosphatidylserine and non-phospholipid components of the hepatitis B virus envelope in human Annexin V binding and in HBV infection in vitro.

BACKGROUND/AIMS: We have previously demonstrated that human liver Annexin V (hAV), a Ca2+-dependent phospholipid binding protein, binds specifically to small HBsAg (SHBsAg). Because of the propensity of AV to bind phospholipids, we here examine the role of phospholipids, as component of the HBV envelope, in binding to hAV and in HBV infection. METHODS: The influence of phospholipids (phosphatidylserine and phosphatidylcholine) on the binding of hAV to SHBsAg or to anti-hAV monoclonals was determined by ELISA. Their influence on HBV infection was investigated using an in vitro HBV infection assay. RESULTS: Two monoclonals, specific against hAV, were able to block the binding of hAV to SHBsAg and recognized different epitopes of hAV. The binding of one of these monoclonals to hAV could be inhibited by phosphatidylserine, but not by phosphatidylcholine. Further experiments revealed that phosphatidylserine could also inhibit the binding of hAV to SHBsAg and could even prevent HBV infection in vitro. Phosphatidylcholine had no effect on the binding of hAV to SHBsAg and could not prevent HBV infection in vitro. However, since phosphatidylserine was not able to abolish the binding of the other blocking monoclonal to hAV, a non-phospholipid component of the HBV envelope must also be involved in hAV binding. CONCLUSIONS: These results indicate that phosphatidylserine and a non-phospholipid component of the HBV envelope are involved in hAV binding and in HBV infection.

Second-generation line probe assay for hepatitis C virus genotyping.

Because of the enormous variability of hepatitis C virus (HCV), the development of reliable genotyping assays is a formidable challenge. The optimal genotyping region appears to be the 5' untranslated region (UR) because of high conservation within, but considerable variability between, genotypes. In this study, 21 probes dispersed over seven variable 5' UR areas were applied to a line probe assay (LiPA) and used to analyze 506 HCV-infected sera from different geographical regions representing a multitude of subtypes. At least 31 different reactivity patterns emerged, with 404 (80%) of 506 distributed over 11 prototype patterns, in general corresponding to subtypes 1a, 1b, 2a/2c, 2b, 3a, 5a, and 6a and several type 4 subtypes. Subtyping specificity ranged from 97% in Hong Kong to 90% in Europe but was only 11% in West Africa, while typing specificity was always 100% when samples from Vietnam were excluded. In a second evaluation, the subtype prediction by LiPA of 448 GenBank 5' UR HCV sequences was scored. Of the 58 theoretically predicted patterns, 321 sequences (72%) were covered by the 11 prototype patterns. We concluded that (i) the selected probes detected the corresponding signature motifs in the seven variable regions with 100% reliability; (ii) these motifs allowed correct type interpretation of samples collected worldwide, with the exclusion of Vietnam, Thailand, or Vietnamese patients residing in European hospitals; and (iii) subtyping specificities vary according to geographical region, with 11 prototype subtyping patterns identifying the majority of samples from Europe and the Americas. These results indicate that the LiPA is a reliable assay applicable to routine typing and subtyping of HCV specimens.

Hepatitis C virus infection in the development of hepatocellular carcinoma in cirrhosis.

BACKGROUND/AIMS: The role of hepatitis C virus replication and different genotypes in the progression of cirrhosis to hepatocellular carcinoma is examined on the basis of a prospective follow-up of 1438 patients with histologically proven cirrhosis. METHODS: The presence of HCV RNA, anti-HCV and characterisation of virus genotypes were determined in 72 cases who developed hepatocellular carcinoma after a median follow-up of 5.3 years (range 1 to 16) and compared to 72 controls who had cirrhosis only, after a median follow-up of 4.8 years (range 1 to 16). Patients in the hepatocellular carcinoma group and controls were matched, one to one, for age, sex, nationality, HBsAg seropositivity, duration of follow-up and aetiology of cirrhosis. RESULTS: HCV RNA was detected in 31 of 72 (44%) patients who developed hepatocellular carcinoma, significantly more frequently than in 17 of 72 (23%) controls with cirrhosis (odds ratio 2.4, 95% confidence interval 1.2 to 5.0; p = 0.013). When cirrhosis of different aetiologies was analysed, hepatitis C virus replication was more frequently detected in patients developing hepatocellular carcinoma in association with cryptogenic cirrhosis (p = 0.007), alcoholic cirrhosis (p = 0.043) and hepatitis B virus seronegative cirrhosis (p = 0.05). Hepatitis C virus genotypes 1b and 4 were the most prevalent; they were found in 53% and 25%, respectively, of the patients studied, but were equally distributed between cirrhosis progressing to hepatocellular carcinoma and controls. CONCLUSIONS: Persistent hepatitis C virus replication is closely associated with hepatocellular carcinoma development in cirrhosis, and there is no preferential role of individual hepatitis C virus genotypes.

Evolution of hepatitis C virus genome in chronically infected patients receiving ribavirin monotherapy.

Recent results of clinical trials suggest that combination of interferon and ribavirin exhibits an enhanced antiviral effect in the treatment of chronic hepatitis C. To investigate the effect of ribavirin on hepatitis C virus (HCV) infection, we analysed the evolution of the genetic heterogeneity of HCV in relation to the anti-HCV humoral response in patients treated by ribavirin alone. The study population included 35 patients with liver biopsy proven chronic hepatitis C infected with HCV genotype 1. Among them, 26 were treated with ribavirin for at least 12 months and nine untreated patients served as a control group. Serum samples were analysed before and at 6 and 12 months of therapy. Three regions of the HCV genome, i.e. HVR1, a domain of NS5A including part of the interferon sensitivity determining region (ISDR), and a segment of NS5B, were amplified by RT-PCR using specific primers. The PCR products were then studied using single-strand conformation polymorphism (SSCP) analysis followed by either direct sequencing, or cloning and sequencing. In parallel, the humoral anti-E1 response was studied using an ELISA (Innotest HCV E1Ab, Innogenetics). The results of HCV genome analysis showed no significant effect on the amino acid sequence evolution of the HVR1, NS5A and NS5B regions of HCV. Analysis of a phylogenetic tree from the major quasispecies variants showed the absence of correlation with ribavirin response, and the absence of selection of viral strains during ribavirin treatment. A trend towards a decrease in the anti-E1 Ab response was also observed. Altogether these results suggest that ribavirin may not exhibit a direct antiviral effect, but may trigger a favourable response to interferon by modulating the immune response against HCV.