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.

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

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-Hodgkin's 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.

Hepatitis C virus quasispecies in the natural course of HCV-related disease in patients with haemophilia.

Patients with haemophilia show high prevalence of hepatitis C infection but low rate of progressive liver disease when they are not co-infected with HIV. The balance between host immune system and hepatitis C virus (HCV) variability seems to play a major role in the evolution of the HCV-related disease. To address this point we have studied, in a group of selected patients with haemophilia, the composition and in some cases the evolution, of the highest variable envelope gene within the hypervariable region 1 (HVR1) of the HCV, which is the region more directly exposed to the host immune response. Five of 12 patients show a very high homogeneity of the HVR1 and four of those had severe progressive liver disease. These results seem to confirm the major role of the immunity in driving the variability of the HCV rather than the high degree of different HCV strains to which haemophiliacs have been in touch with, during their long-term replacement therapy. Our results seem in keeping with other studies on different type of patients, where a low degree of quasispecies variability has been demonstrated in relationship with the progression and the severity of their liver disease.

A 385 insertion in the hypervariable region 1 of hepatitis C virus E2 envelope protein is found in some patients with mixed cryoglobulinemia type 2.

Chronic hepatitis C virus (HCV) infection has been associated with development of mixed cryoglobulinemia type 2 (MC2), a lymphoproliferative disorder characterized by B cell monoclonal expansion and immunoglobulin M/k cryoprecipitable immunoglobulin production. A short sequence (codons 384-410) of the HCV E2 protein, which has the potential to promote B cell proliferation, was investigated in 21 patients with HCV-related MC2 and in a control group of 20 HCV carriers without MC2. In 6 of the 21 (29%) patients with MC2, all the clones isolated from plasma, peripheral blood mononuclear cells, and liver showed sequence length variation compared with the hypervariable region 1 (HVR1) consensus sequence; 5 patients had an insertion at codon 385, and 1 patient had a deletion at codon 384. Inserted residues at position 385 were different within and between patients. No such mutations were observed in any of the HVR1 clones from control patients without MC2, and the difference between the 2 groups was statistically significant (P =.02). Analysis of 1345 HVR1 sequences obtained from GenBank strongly supported the conclusion that the observed insertions and deletion represent a rare event in HCV-infected patients, suggesting that they are significantly associated with MC2. The physical and chemical profiles of the 385 inserted residues detected in the MC2 patients were consistent with the possibility that these mutations, which occurred in a region containing immunodominant epitopes for neutralizing antibodies and binding sites for B lymphocytes, may be selected by functional constraints for interaction with host cells.

Two PKR inhibitor HCV proteins correlate with early but not sustained response to interferon.

BACKGROUND & AIMS: The NS5A and the E2 proteins of hepatitis C virus (HCV)-1b can bind and inhibit in vitro the interferon (IFN)-induced cellular kinase PKR. The role of such interaction in modulating the antiviral effect of IFN is still controversial. We have analyzed the E2 and the NS5A sequences in HCV-1b-infected patients treated with IFN to assess whether and how different combinations of wild-type and mutant proteins correlated with early and long-term virological response. METHODS: In 30 patients, sequences of pretreatment and on-treatment E2-PePHD and NS5A-PKR binding domain (including the putative ISDR) were analyzed in parallel by sequencing cDNA-polymerase chain reaction products and up to 25 independent clones. RESULTS: The E2-PePHD sequence was highly conserved with a homogeneous quasispecies and was identical in 29 of 30 cases with no association with the pattern of response and no evidence of evolution during therapy. Patients with a mutated NS5A-ISDR had a higher rate of early virological response (67%) than cases with wild-type ISDR (17%). This association was lost in long-term responders (33% vs. 17%). CONCLUSIONS: Although the highly conserved E2-PePHD motif might contribute to reduce IFN responsiveness, variations within this region do not seem to play a role in modulating IFN sensitivity. The NS5A-ISDR sequence influenced the early, but not the sustained response, to IFN, suggesting that other factors may be more important for the long-term outcome of therapy.

The molecular basis for responsiveness to anti-viral therapy in hepatitis C.

Hepatitis C virus (HCV) infection is an important clinical problem, with a world-wide prevalence of approximately 1-2%. HCV infection is associated with an increased risk for the development of severe liver disease. HCV is inherently resistant to anti-viral therapy with interferon (IFN). The virus circulates in infected individuals as a mixture of related, yet genetically distinct variants, or quasispecies. Many studies have implicated HCV quasispecies in IFN responsiveness. Effective containment of HCV quasispecies mutation and selection through more aggressive therapy (e.g. daily induction), combination therapy (e.g. IFN plus ribavirin), or longer lasting therapy (e.g. pegylated IFN) is required for IFN responsiveness. Recently, several HCV proteins including the non-structural 5A and envelope gene 2-glycoprotein have been implicated in HCV anti-viral resistance. It is likely that multiple HCV genes disrupt IFN-induced anti-viral responses at many levels and that these virus-host cell interactions are associated with IFN resistance. Characterisation of HCV-encoded mechanisms of anti-viral resistance has important implications for the development of new anti-virals.

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

Resistance of the hepatitis C virus (HCV) to interferon-alpha (IFN-alpha) 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-alpha 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.

Effect of retreatment with interferon alone or interferon plus ribavirin on hepatitis C virus quasispecies diversification in nonresponder patients with chronic hepatitis C.

Alpha interferon (IFN-alpha) treatment is effective on a long-term basis in only 15 to 25% of patients with chronic hepatitis C. The results of recent trials indicate that response rates can be significantly increased when IFN-alpha is given in combination with ribavirin. However, a large number of patients do not respond even to combination therapy. Nonresponsiveness to IFN is characterized by evolution of the hepatitis C virus (HCV) quasispecies. Little is known about the changes occurring within the HCV genomes when nonresponder patients are retreated with IFN or with IFN plus ribavirin. In the present study we have examined the genetic divergence of HCV quasispecies during unsuccessful retreatment with IFN or IFN plus ribavirin. Fifteen nonresponder patients with HCV-1 (4 patients with HCV-1a and 11 patients with HCV-1b) infection were studied while being retreated for 2 months (phase 1) with IFN-alpha (6 MU given three times a week), followed by IFN plus ribavirin or IFN alone for an additional 6 months (phase 2). HCV quasispecies diversification in the E2 hypervariable region-1 (HVR1) and in the putative NS5A IFN sensitivity determining region (ISDR) were analyzed for phase 1 and phase 2 by using the heteroduplex tracking assay and clonal frequency analysis techniques. A major finding of this study was the relatively rapid evolution of the HCV quasispecies observed in both treatment groups during the early phase 1 compared to the late phase 2 of treatment. The rate of quasispecies diversification in HVR1 was significantly higher during phase 1 versus phase 2 both in patients who received IFN plus ribavirin (P = 0.017) and in patients who received IFN alone (P = 0. 05). A trend toward higher rates of quasispecies evolution in the ISDR was also observed during phase 1 in both groups, although the results did not reach statistical significance. However, the NS5A quasispecies appeared to be rather homogeneous and stable in most nonresponder patients, suggesting the presence of a single well-fit major variant, resistant to antiviral treatment, in agreement with published data which have identified an IFN sensitivity determinant region within the NS5A. During the entire 8 months of retreatment, there was no difference in the rate of fixation of mutation between patients who received combination therapy and patients who were treated with IFN alone, suggesting that ribavirin had no major effects on the evolution of the HCV quasispecies after the initial 2 months of IFN therapy.

[Environmental lead exposure in the Venetian population from 1976-1992]. / Esposizione ambientale al piombo nella popolazione dell'area veneziana dal 1976 al 1992.

Blood lead levels observed in the general population of Venice and the surrounding area are reported for the period between 1976 and 1992. A time dependent decrease of blood lead levels is evident and parallels the step wise decrease of lead levels in gasoline which took place between 1981 and 1991. The observed lowering time trend of blood lead levels could possibly be ascribed, perhaps not negligibly, to technological improvements, the development of new analytical procedures and the continuous practice of quality control.

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.

Coinfection by hepatitis B virus and hepatitis C virus.

Coinfection by hepatotropic viruses can occur due to the fact that hepatitis B virus (HBV) and hepatitis C virus (HCV) share similar routes of transmission. Different clinical features of liver disease can be observed in infected patients, ranging from fulminant, acute and chronic hepatitis to hepatocellular carcinoma (HCC). The relative role of the infecting viruses in determining the final clinical picture is not yet well defined. Several reports indicate that clinical and pathological severity of liver disease among coinfected patients is increased and in patients with HCC, co-occurrence of both viruses is a common event. The potential mechanism of tumour development still remains speculative, although direct and indirect roles for both HBV and HCV have been proposed. At the molecular level, reciprocal interference of virus replication has been repeatedly described and the extent of interference is influenced by the infecting HCV genotype, genotype 1 of HCV having more efficient inhibitory activity on HBV than genotype 2. Sequence similarities between an arginine-rich nucleocapsid motif of both viruses could support these clinical observations. Concerning response rates to interferon therapy, no satisfactory results have been achieved to date, although identification of effective therapeutic schemes, based on virological status of both viruses are warranted.

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.

Determination of Cd, Co, Cu, Mn, Ni, Pb, and Zn by Inductively Coupled Plasma Mass Spectroscopy or Flame Atomic Absorption Spectrometry after On-line Preconcentration and Solvent Extraction by Flow Injection System

The concentrations of Cd, Co, Cu, Mn, Ni, Pb, and Zn in natural and sea waters are too low to be directly determined with by flame atomic absorption spectrometry (FAAS) or graphite furnace atomic absorption spectrometry (GFAAS). Specific sample preparations are requested that make possible the determination of these analytes by preconcentration or extraction. These techniques are affected by severe problems of sample contamination. In this work Cd, Co, Cu, Mn, Ni, Pb, and Zn were determined by inductively coupled plasma mass spectroscopy (ICP-MS) or by atomic absorption spectrometry, in fresh and seawater samples, after on-line preconcentration and following solvent elution with a flow injection system. Bonded silica with octadecyl functional group C18, packed in a microcolumn of 100-μl capacity, was used to collect diethyldithiocarbamate complexes of the heavy metals in aqueous solutions. The metals are complexed with a chelating agent, adsorbed on the C18 column, and eluted with methanol directly in the flow injection system. The methanolic stream can be addressed to FAAS for direct determination of Cu, Ni, and Zn, or collected in a vial for successive analysis by GFAAS. The eluted samples can be also dried in a vacuum container and restored to a little volume with concentrated HNO3 and Milli-Q water for analysis by ICP-MS or GFAAS.

Discordant results from hepatitis C virus genotyping by procedures based on amplification of different genomic regions.

We compared the results of genotyping hepatitis C virus (HCV) either by PCR amplification of the core region or by hybridization of PCR-amplified products of the 5' untranslated region (5'UTR assay). Serum samples from 144 Italian anti-HCV-positive patients (106 drug abusers and 38 patients with chronic viral liver disease but no history of drug abuse) were studied. The original core region assay described by Okamoto et al. (H. Y. Okamoto, Y. Sugiyama, S. Okada, K. Kurai, Y. Akahane, Y. Sugai, T. Tanaka, K. Sato, F. Tsuda, Y. Miyakawa, and M. Mayumi, J. Gen. Virol. 73:673-679, 1992) allowed genotyping of 75 of 144 samples. A modified version of Widell et al. (A. Widell, S. Shev, S. Månsson, Y.-Y. Zhang, U. Foberg, G. Norkrans, A. Frydén, O. Weiland, J. Kurkus, and E. Nordenfelt, J. Med. Virol. 44:272-279, 1994) allowed genotyping of 11 of 79 samples (50 of 79 samples remained unclassified by the method of Okamoto et al. In contrast, all 144 samples were genotyped by the 5'UTR assay. Forty-six of 75 (61 percent) of the samples genotyped by the method of Okamoto et al. and 10 of 11 (91 percent) of the samples genotyped by the method of Widell et al. had results consistent with those obtained by the 5'UTR assay. According to the results of direct sequencing, the method of Okamoto et al. erroneously classified seven samples as having mixed infections. In conclusion, HCV genotyping seems more reliable when it is performed by the 5'UTR assay than by either of two core region assays. The major advantage provided by the 5'UTR assay is a much lower proportion of negative or indeterminate results in younger patients with histories of drug abuse or infection by genotypes other than HCV type 1.

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. Mean alanine aminotransferase (ALT) levels were similar between patients infected with HCV-1 and HCV-2. Among 7 patients with cirrhosis 5 were infected by HCV-2, while 6 of 12 of those without cirrhosis had HCV-1 infection. In conclusion, HBV replication was inhibited more efficiently by HCV-1 than by HCV-2. Cirrhosis was frequently found in patients with dual HBV and HCV-2 infection.

Comparison of genotyping and serotyping methods for the identification of hepatitis C virus types.

The usefulness of identification of hepatitis C virus (HCV) genotype has recently been investigated for the clinical management of patients infected by HCV. In the present study, the HCV genotype infecting 127 patients was determined by two different methods: HCV genotyping using a dot-blot assay with type-specific probes derived from the 5'-UTR of HCV genome and HCV serotyping using an ELISA system in which type-specific antibodies against the NS4 region were detected. Overall, a good correlation of the two methods was observed, the main discrepancy being 4 patients with sequence-confirmed HCV-2 (2 cases) and HCV-3 (2 cases) genotypes recognized as HCV-1 by serotyping. Mixed infections were not detected by either method. In 19 PCR negative sera, in which the HCV genotype could not be evaluated, no particular serotype profile was observed. In conclusion, the molecular and serological techniques are almost equivalent in determining the viral type, although in individual cases, especially in PCR negative patients, the clinical meaning of the serotyping result remains to be determined.

Analytical problems in mercury analysis of seafood.

It is generally accepted that seafood represents one of the major sources of mercury to man. In this work two interlaboratory proficiency tests are described for the analysis of mercury in seafood. Thirty-seven public control and food industries laboratories participated in the first test, while 29 participants were included in the second one. Moreover, in order to clarify whether sampling of different edible muscle tissues of the same fish could affect the analytical results, the top, the central and the bottom portion of 28 fishes were examined. The different portions of fish showed no significant difference in mercury concentrations. Two different wet digestion methods (microwave oven and reflux in quartz vessels) were also tested in the case of 11 fishes. A systematic difference was observed between the two sets of results obtained with these digestion methods.

Distribution of three major hepatitis C virus genotypes in Italy. A multicentre study of 495 patients with chronic hepatitis C.

Different genotypes of hepatitis C virus (HCV) have been shown to have distinct geographical distribution and to associate with variable clinical features. To evaluate their role in chronic hepatitis in Italian patients, we studied 495 consecutive cases with chronic hepatitis C seen in nine sentinel centres homogeneously distributed over Italy. HCV genotyping was carried out using a dot-blot hybridization assay with genotype-specific probes. Four hundred and eleven patients were viraemic and could be evaluated: 57% were found to be infected with HCV-1, 31% with HCV-2, 8% with HCV-3, 1% showed mixed infection and 3% were ascribed to HCV-2b or HCV-4 by direct sequencing. Geographical distribution showed discrete territorial variations. A history of drug addiction was commoner in patients infected with HCV-3. There were no significant differences in activity of liver disease among different HCV genotypes but the response to interferon therapy was reduced in patients infected with HCV-1 compared to HCV-2 or HCV-3.