Martina Gerotto

Genotypes of hepatitis C virus in Italian patients with chronic hepatitis C

Abstract To assess the prevalence of different hepatitis C virus genotypes in an European population of patients with chronic hepatitis C, 79 consecutive Italian patients were studied. After having cloned and sequenced part of the 5′ untranslated region of the virus in 21 patients, oligonucleotide probes were synthesized to be used in a more rapid dot-blot hydridization test. Using this method, 42% of patients were found infected by HCV type 1, 45% by HCV type 2 and 4% by HCV type 3, while seven patients remained unclassified. Patients infected by HCV type 3 were significantly younger and had a milder form of liver disease, compared to those infected by HCV type 1 or HCV type 2. Patients with HCV type 2 or HCV type 3 responded to interferon therapy much better than those with HCV type 1. These results provide information on the prevalence of different HCV genotypes in our region, and indicate the usefulness of the dot-blot hybridization procedure for rapid screening of HCV genotypes.

Hepatitis C genotypes in patients with dual hepatitis B and C virus infection

In patients with chronic hepatitis B and C virus (HBV, HCV) infection, an inverse relationship in the replicative activity of the two viruses has been reported. In the present study the genotype of HCV was evaluated in 34 consecutive cases found with hepatitis B surface antigen (HBsAg) and anti‐HCV in the serum, in order to identify its possible influence in determining the pattern of HBV/HCV interaction. Nineteen patients were HCV‐RNA positive and could be genotyped: 8 were infected by HCV‐1 (3 by HCV‐1a and 5 by HCV‐1b), 10 by HCV‐2, and only 1 by HCV‐3. Among these, 3 were HBV‐DNA positive, compared to 10 of 15 HCV‐RNA‐negative patients (P = 0.003), and all 3 were coinfected with HCV‐2.

Evidence for sequence selection within the non‐structural 5A gene of hepatitis C virus type 1b during unsuccessful treatment with interferon‐α

Resistance of the hepatitis C virus (HCV) to interferon‐α (IFN‐α) therapy in patients with hepatitis C may be genetically controlled by an IFN sensitivity‐determining region (ISDR) within the non‐structural 5A (NS5A) gene. To assess whether HCV 1b strains carrying a ‘resistant’ type of ISDR are selected during unsuccessful IFN therapy, we analysed the evolution of the NS5A quasispecies, as detected by the clonal frequency analysis technique, and of the ISDR sequence by nucleotide sequence determination, in 11 patients showing no virological response during two consecutive cycles of IFN‐α therapy. IFN‐resistant patients had a homogeneous ISDR quasispecies with sequences identical to those described as ‘resistant‐’ or ‘intermediate‐’ type ISDR. After retreatment with IFN, further selection towards a homogeneous viral population was observed and 10 out of 11 patients had only one variant of HCV with no or just one single amino acid mutation within the ISDR sequence. Treatment and retreatment with IFN was associated in our non‐responder patients with evolution of the ISDR quasispecies towards a rather homogeneous viral population carrying a conserved or minimally mutated ISDR motif, supporting the idea that this motif may be relevant for IFN resistance in HCV 1b‐infected individuals.

Microsomal triglyceride transfer protein polymorphism (−493G/T) is associated with hepatic steatosis in patients with chronic hepatitis C

Background: Hepatic steatosis may promote progression of chronic hepatitis C (CHC). Microsomal triglyceride transfer protein (MTP) is required for assembly and secretion of ApoB lipoprotein and is implicated in hepatitis C virus (HCV)‐related steatosis. The MTP −493G/T polymorphism may promote liver fat accumulation, but its role in HCV‐related steatosis is still unclear.

Impact of NS5A Sequences of Hepatitis C Virus Genotype 1a on Early Viral Kinetics during Treatment with Peginterferon-α2a plus Ribavirin

UNLABELLED BACKGROUND. Hepatitis C virus (HCV) genotype 1 is the most prevalent genotype in Western countries, and treatment with pegylated interferon (pegIFN) plus ribavirin fails in 50%-60% of patients. Genetic variability within the NS5A dsRNA-dependent protein kinase binding domain (PKRbd) of HCV-1b has been associated with responsiveness to IFN- alpha . Little is known about NS5A sequences of HCV-1a. We investigated whether genetic variability of HCV-1a NS5A correlates with the early HCV kinetics during treatment. METHODS Twenty-four treatment-naive, HCV-1a-infected patients were treated with standard doses of pegIFN- alpha 2a plus ribavirin. HCV viremia was quantitated at days 0, 1, 2, and 3 and weeks 1, 2, 4, 8, and 12 of treatment. According to HCV kinetics, patients were classified as early rapid responders, early moderate responders, or early slow responders. The full-length HCV NS5A was sequenced at baseline and at week 1. RESULTS At baseline, variability of the NS5A C terminus (concentrated in the PKRbd) is associated with interferon efficacy but not with the second phase of the early viral decline that has been associated with a sustained virologic response. Comparisons between baseline and week-1 full-length sequences did not show significant increases in mutations. CONCLUSIONS Genetic variability of HCV-1a NS5A does not predict responsiveness to IFN- alpha . Differences in early kinetics during combination therapy are not due to selection of IFN-resistant HCV strains.

Endogenous Interferon-α Concentration and Outcome of Interferon Treatment in Patients with Chronic Hepatitis C

To verify its value with regard to the outcomeof therapy in chronic hepatitis C, seruminterferon-α (IFN) was measured by ELISA in 70patients (43 male, 27 female) with chronic hepatitis C,treated with IFN 9 MU/week subcutaneously for up to oneyear. Serum IFN was detectable in 49/70 patients, 16 ofwhom had IFN values >125 pg/ml. Only 1/22 patientswho 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 didnot respond or who relapsed (χ 6.1, P < 0.02). Atmultivariate analysis the predictive value of serum IFN was independent of age, sex, presence ofcirrhosis, infection by genotype 1b (improvement χ7.1, P < 0.01). In patients with chronic hepatitis C,measurement of serum IFN at baseline might be useful for the selection of patients with higherprobability of long-term response.

Detailed analysis of the E2–IgM complex in hepatitis C-related type II mixed cryoglobulinaemia

Summary.  Hepatitis C virus (HCV) plays a major role in the induction of type II mixed cryoglobulinaemia (MCII). The role of HCV proteins and virus–host interaction in the pathogenesis of MC remains to be defined. To address this issue, we have characterized, in detail, the monoclonal IgM and the viral component of circulating immune complexes in eight patients with HCV‐associated MCII. The proportion of HCV‐RNA compartmentalized in the cryoprecipitate (CP) varied greatly (10–80% of total HCV‐RNA). The complementary determining region (CDR)3 sequences of monoclonal immunoglobulin M (IgM) VH and VK genes were highly homologous to rheumatoid factor and to antibodies against HCV‐E2. Furthermore, the CDR3 sequences in some of our MCII patients were highly similar to those described in HCV‐positive patients with non‐Hodgkins lymphoma (NHL). From these results, it appears that, as in the case of NHL, the IgM‐rheumatoid factor (RF) production in MCII patients is antigen driven, namely by E2. However, the limited number of mutations in VH and VK genes with respect to the germline and their distribution showed that the B‐cell response in these cases was prevented from undergoing affinity maturation. Furthermore, in patients with monoclonal IgM and definite compartmentalization of HCV in either CP or supernatant, a highly homogeneous E2‐hypervariable region (HVR)1 sequence distribution was found (90–100% identical clones), a feature of the quasispecies frequently associated with an impaired humoral immune response to HCV. These findings suggest that in patients with HCV‐associated MCII, maturation of monoclonal B lymphocytes may be blocked in a primitive stage preventing serious damaging effects because of the auto‐reactivity of their secreted immunoglobulins.

Evolution of hepatitis C virus quasispecies in children with chronic hepatitis C.

Background:Hepatitis C virus (HCV) circulates as a mixture of different but closely related genomes: this quasispecies nature could be essential for virus persistence and could induce resistance to interferon therapy. Since little is known on the behavior of HCV quasispecies in children and adolescents with chronic hepatitis C, we analyzed the virus population in six untreated children during a 5-year follow-up.Methods:Six children aged 1–8 years, infected early in life with HCV, were included in the study. From each of them, 2 or 3 sequential serum samples obtained over a 5-year follow-up period were examined. The HCV quasispecies heterogeneity and diversity in the E2 hypervariable region-1 (HVR-1) were analyzed among samples by the heteroduplex mobility assay, and the distance between variants was estimated by the heteroduplex mobility ratio (HMR).Results:The HCV population was initially highly homogeneous in all six children. During follow-up, diversification of HVR-1 leading to a more complex viral population occurred in all cases, and was particularly evident in the three older children (HMR: 0.82–0.54). Changes in the HVR-1 sequence occurred without relation to the profile of ALT and HCV-RNA levels.Conclusions:HCV quasispecies diversification is a common event during chronic hepatitis C in childhood. Host and environmental pressure could be major determinants. The increasing viral heterogeneity could impair the response to antiviral therapy, thus indicating a rationale for early antiviral treatment in children with chronic hepatitis C.

Characteristics of hepatitis C virus before and after interferon treatment in patients with ongoing viraemia but sustained biochemical response.

In hepatitis C virus (HCV) infection, persistent viraemia can occur after successful biochemical response to interferon treatment. To assess whether this unusual profile might be due to trivial amounts of remaining virus or to the emergence of less pathogenic HCV strains, pre‐ and posttreatment sera from 27 patients who remained with HCV‐RNA, despite sustained transaminase normalisation after interferon therapy, were investigated. All but one had infection by genotype 2 (P < 0.0001), and levels of HCV‐RNA were not decreased after therapy. Sequence comparison of the 5′ untranslated region revealed mixed viral populations and “not compensatory” nucleotide transitions localised at the stem level of the secondary structure of this region in samples taken before and after treatment. Neither quantitative nor qualitative viral changes, at least for the 5′ untranslated region, are responsible for interferon‐induced biochemical remission in these patients typically infected by genotype 2. J. Med. Virol. 54:7–11, 1998.