Epidemiologia molecular do vírus da Hepatite C: análise comparativa de diferentes regiões subgenômicas aplicadas a estudos de associação genética

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A análise morfométrica para avaliar a taxa de progressão da fibrose hepática na hepatite C crônica após o transplante renal: uma série de casos

The real role of renal transplantation in hepatic fi brosis progression caused by hepatitis C virus is still unpredictable. Histological evaluation of the liver is the best form to estimate fi brosis evolution, although semiquantitative analysis carries important limitations. Objective: To apply a morphometric quantitative assay on hepatic fi brosis progression in renal recipients with hepatits C. Methods: Thirty patients were initially evaluated, but only seven were included. They underwent the fi rst biopsy near the transplantation date and the second biopsy at least 4 years later. The immunosuppressant therapy adopted in all cases was azatioprine and micofenolate. Fibrosis progression rate (FPR) was calculated before and after the surgery date in each patient according to Metavir score and morphometric analysis. Results: The FPR calculated by Metavir score showed no statistical difference between pre- and post-transplantation (p=0.9). The FPR calculated by the morphometric analysis was 0.58 ± 0.78 before transplantation and 3.0 ± 3.3 after the surgery, with statistical signi- fi cance between these values (p=0.0026). Conclusion: In the sample assessed, the progression of hepatic fi brosis was documented and quantifi ed only by the morphometric analysis, which is as a promising approach to histological evaluation of these patients.

Investigação do Ângulo de Fase como preditor da resposta virológica em pacientes portadores de hepatite C

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Prevalence of hepatitis B virus infection and carriage after nineteen years of vaccination program in the Western Brazilian Amazon

Introduction: Reductions in the prevalence of hepatitis B virus (HBV) infection and carriage, decreases in liver cancer incidence, and changes in patterns of liver dysfunctions are described after hepatitis B vaccination. Methods: We conducted a population-based seroprevalence study aimed at estimating the HBV prevalence and risk of infection in the rural area of Labrea following nineteen years of HBV vaccination. Results: Half of the subjects showed total anti-HBc of 52.1% (95% CI 49.6-54.7). The HBsAg prevalence was 6.2% (95% CI 5.1-7.6). Multivariate analysis showed an inverse association between HBV infection and vaccination (OR 0.62; 95% CI 0.44-0.87). HBsAg remained independently associated with past hepatitis (OR 2.44; 95% CI 1.52-3.89) and inversely to vaccination (OR 0.43; 95% CI 0.27-0.69). The prevalence of HBeAg among HBsAg-positive individuals was 20.4% (95% CI 12.8-30.1), with the positive subjects having a median age of 11 years (1-46) p=0.0003. Conclusions: We demonstrate that HBV infection is still an important public health issue and that HBV vaccination could have had better impact on HBV epidemiology. If we extrapolate these findings to other rural areas in the Brazilian Amazon, we can predict that the sources of chronic infected patients remain a challenge. Future studies are needed regarding clinical aspects, molecular epidemiology, surveillance of acute cases, and risk groups.

Hepatitis B virus infection in Haemodialysis Centres from Santa Catarina State, Southern Brazil. Predictive risk factors for infection and molecular epidemiology

Abstract Background Patients under haemodialysis are considered at high risk to acquire hepatitis B virus (HBV) infection. Since few data are reported from Brazil, our aim was to assess the frequency and risk factors for HBV infection in haemodialysis patients from 22 Dialysis Centres from Santa Catarina State, south of Brazil. Methods This study includes 813 patients, 149 haemodialysis workers and 772 healthy controls matched by sex and age. Serum samples were assayed for HBV markers and viraemia was detected by nested PCR. HBV was genotyped by partial S gene sequencing. Univariate and multivariate statistical analyses with stepwise logistic regression analysis were carried out to analyse the relationship between HBV infection and the characteristics of patients and their Dialysis Units. Results Frequency of HBV infection was 10.0%, 2.7% and 2.7% among patients, haemodialysis workers and controls, respectively. Amidst patients, the most frequent HBV genotypes were A (30.6%), D (57.1%) and F (12.2%). Univariate analysis showed association between HBV infection and total time in haemodialysis, type of dialysis equipment, hygiene and sterilization of equipment, number of times reusing the dialysis lines and filters, number of patients per care-worker and current HCV infection. The logistic regression model showed that total time in haemodialysis, number of times of reusing the dialysis lines and filters, and number of patients per worker were significantly related to HBV infection. Conclusions Frequency of HBV infection among haemodialysis patients at Santa Catarina state is very high. The most frequent HBV genotypes were A, D and F. The risk for a patient to become HBV positive increase 1.47 times each month of haemodialysis; 1.96 times if the dialysis unit reuses the lines and filters ≥ 10 times compared with haemodialysis units which reuse < 10 times; 3.42 times if the number of patients per worker is more than five. Sequence similarity among the HBV S gene from isolates of different patients pointed out to nosocomial transmission.

Characterization of Hepatitis B virus (HBV) genotypes in patients from Rondônia, Brazil

Abstract Background Hepatitis B virus (HBV) can be classified into nine genotypes (A-I) defined by sequence divergence of more than 8% based on the complete genome. This study aims to identify the genotypic distribution of HBV in 40 HBsAg-positive patients from Rondônia, Brazil. A fragment of 1306 bp partially comprising surface and polymerase overlapping genes was amplified by PCR. Amplified DNA was purified and sequenced. Amplified DNA was purified and sequenced on an ABI PRISM® 377 Automatic Sequencer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were aligned with reference sequences obtained from the GenBank using Clustal X software and then edited with Se-Al software. Phylogenetic analyses were conducted by the Markov Chain Monte Carlo (MCMC) approach using BEAST v.1.5.3. Results The subgenotypes distribution was A1 (37.1%), D3 (22.8%), F2a (20.0%), D4 (17.1%) and D2 (2.8%). Conclusions These results for the first HBV genotypic characterization in Rondônia state are consistent with other studies in Brazil, showing the presence of several HBV genotypes that reflects the mixed origin of the population, involving descendants from Native Americans, Europeans, and Africans.

New insights regarding HCV-NS5A structure/function and indication of genotypic differences

Abstract Background HCV is prevalent throughout the world. It is a major cause of chronic liver disease. There is no effective vaccine and the most common therapy, based on Peginterferon, has a success rate of ~50%. The mechanisms underlying viral resistance have not been elucidated but it has been suggested that both host and virus contribute to therapy outcome. Non-structural 5A (NS5A) protein, a critical virus component, is involved in cellular and viral processes. Methods The present study analyzed structural and functional features of 345 sequences of HCV-NS5A genotypes 1 or 3, using in silico tools. Results There was residue type composition and secondary structure differences between the genotypes. In addition, second structural variance were statistical different for each response group in genotype 3. A motif search indicated conserved glycosylation, phosphorylation and myristoylation sites that could be important in structural stabilization and function. Furthermore, a highly conserved integrin ligation site was identified, and could be linked to nuclear forms of NS5A. ProtFun indicated NS5A to have diverse enzymatic and nonenzymatic activities, participating in a great range of cell functions, with statistical difference between genotypes. Conclusion This study presents new insights into the HCV-NS5A. It is the first study that using bioinformatics tools, suggests differences between genotypes and response to therapy that can be related to NS5A protein features. Therefore, it emphasizes the importance of using bioinformatics tools in viral studies. Data acquired herein will aid in clarifying the structure/function of this protein and in the development of antiviral agents.

Genomic mosaicism in two strains of Dengue virus type 3

Recombination is a significant factor driving genomic evolution, but it is not well understood in Dengue virus. We used phylogenetic methods to search for recombination in 636 Dengue virus type 3 (DENV-3) genomes and unveiled complex recombination patterns in two strains, which appear to be the outcome of recombination between genotype II and genotype I parental DENV-3 lineages. Our findings of genomic mosaic structures suggest that strand switching during RNA synthesis may be involved in the generation of genetic diversity in dengue viruses.

Untersuchungen über Wechselwirkungen zwischen Proteinen der Leber und dem großen Hüllprotein des Hepatitis-B-Virus

Zusammenfassung:Im Infektionszyklus des Hepatitis-B-Virus spielt das große L-Hüllprotein mit seiner einzigartigen PräS1-Domäne eine zentrale Rolle. Es vermittelt die Bindung und Aufnahme in die Leberzelle, die Verpackung der Nukleokapside in die Virushülle, die Regulation der cccDNA-Amplifikation und eine transkriptionelle Aktivierung in der Wirtszelle. Zur Erfüllung seiner vielfältigen Aufgaben benötigt das L-Protein Unterstützung durch Wirtzellfaktoren, von denen einige im Rahmen dieser Untersuchung durch Verwendung von PräS1-Konstrukten als Fängerproteine im Hefe-Zwei-Hybrid-System identifiziert wurden. Mehrere Klone, die im Hefe-Zwei-Hybrid-Test mit dem C-terminalen PräS1-Fängerprotein (Aminosäure 44-108) isoliert worden waren, enthielten Teile der cDNA von gamma2-Adaptin, einem mutmaßlichen Mitglied der Clathrin-Adaptor-Proteine. Diese sind für intrazelluläre Membrantransportprozesse mittels clathrinumhüllter Vesikel verantwortlich. Unter den interagierenden Klonen, die mit dem N-terminalen Konstrukt des L-Proteins (Aminosäure 1-70) isoliert worden waren, befand sich überproportional häufig eine cDNA, die der schweren Kette H4 der Inter-Alpha-Trypsin-Inhibitor-Familie homolog war. H4 besitzt vermutlich bei der 'Akute-Phase-Reaktion', die Entzündungen folgt, und bei der Stabilisierung der extrazellulären Matrix physiologische Bedeutung. Weitere Klone kodierten für die Serinprotease C1r. Diese ist Bestandteil des C1-Komplex, der ersten Komponente des klassischen Komplementsystems. Die Spezifität der Bindung zwischen den positiven Klonen und der PräS1-Domäne wurde in weiteren biochemischen Interaktionstests bestätigt, sodaß H4, C1r und gamma2-Adaptin als Wirtszellfaktoren in der Physiologie des Hepatitis-B-Virus wahrscheinlich eine Rolle spielen.Abstract:Little is known about host cell factors necessary for hepatitis B virus assembly and infectivity. Central to virogenesis is the large L envelope protein that mediates hepatocyte receptor binding, envelopment of viral capsids, regulation of supercoiled DNA amplification and transcriptional transactivation. To assess its multiple functions and host-protein assistance involved, we here initiated a yeast two-hybrid screen using the L-specific preS1 domain as bait to screen a human liver cDNA library for L-interacting proteins. One of the most prominent cDNAs interacting with aminoacid sequence 44-108 of L-protein encodes for gamma2-adaptin, a novel clathrin adaptor-related protein responsible for protein sorting and trafficking. Among the clones interacting with the N-terminal construct of L-protein (aminoacid sequence 1-70), a frequently isolated cDNA corresponds to the gene for inter-alpha-trypsin family heavy chain H4, likely to be involved in acute inflammatory phase response and stabilization of extracellular matrices. Some other interacting clones were found to carry the cDNA for the serine protease C1r, a subunit of the C1 complex which initiates the classical complement cascade. The specificity of the interaction between the positive clones and the preS1 domain was further confirmed in independent biochemical experiments. Taken together, the results suggest a role for H4, C1r and gamma2-adaptin as host-cell factors in L-mediated process of viral biogenesis and/or pathogenesis.

Regulation und funktionelle Bedeutung der zellulären und humoralen Immunantwort in der Pathogenese der chronischen Hepatitis C Virusinfektion

Patienten mit akuter, selbstlimitierender HCV-Infektion und solche mit IFN-a Therapieresponse (SCR) zeigten eine hochregulierte NS3-spezifische T-Helferzellantwort.Über die funktionelle Bedeutung der T-Helferzellantwort und der Antikörpersynthese besteht jedoch noch Unklarheit.In dieser Studie wurde die proliferative Antwort der PBMC von Patienten mit anhaltender Therapieresponse (SCR; n=8), Non-Respondern (NR; n=13) und unbehandelten HCV-Patienten(UTR; n=10) sowie gesunden Kontrollen (HC; n=5) auf die rekombinanten HCV-Antigene Core, Helicase, NS3, NS4 und NS5-4 bestimmt und ihre Sekretion von IFN-.

Accuracy of liver stiffness measurement using fibroscan ® to predict the response to antiviral therapy in patients with chronic hepatitis c viral infection

Introduction: Antiviral therapy can prevent disease progression in patients with chronic hepatitis C . Transient Elastografy (TE; Fibroscan) is an accurate surrogate marker to liver fibrosis, by measuring liver stiffness (LS). LS decrease has been associated with sustained virologic response (SVR). Aim: to assess the changes of LS measurments in CHC patients during and one year after Interferon (IFN)-based antiviral therapy (IFN/ribavirin) or (telaprevir+IFN/ribavirin). Methods: consecutive 69 CHC patients (53.6% females, mean age 57.9 ± 11.4) who underwent antiviral therapy for at least 20 weeks were enrolled. LS was measured using FibroScan at baseline, after three months, at the end of treatment and one year after treatment discontinuation. Fibrosis was graded using METAVIR score. Results: twenty patients treated with triple therapy and 49 with IFN/ribavirin. Fifty patients had SVR and 19 were non-responders. SVR patients: F0-F1, F2 and F3 patients (39.1%, 7.2% and 17.4%; respectively) showed no significant LS decrease (P= 0.186, 0.068 and 0.075; respectively). Conversely, in F4 patients (36.2%) LS was significantly decreased (P=0.015) after one year of treatment completion. In all patients with no SVR, no significant decrease in LS was observed. Interestingly, all Patients with F4 fibrosis (even non-responders) showed an initial significant decrease in LS (P=0.024) at 3 months after the start of treatment. However, this decrease was not predictive of SVR; area under the ROC curve 0.369 (CI %: 0.145-0.592) P= 0.265. Conclusion: Our study showed that initial decrease in LSM, especially in patients with higher baseline fibrosis score is unlikely to predict an SVR. In addition no significant association was found between clinical or virological parameters and fibrosis improvement. Further studies are needed to delineate the most appropriate clinical scenarios for the LSM by Fibroscan in chronic hepatitis C and its role in monitoring the response to antiviral treatment.

Serum anti-interferon alpha antibodies in chronic hepatitis C patients treated with pegylated interferon alpha containing therapy

The development of anti-IFNα antibodies is an occurrence described in chronic hepatitis C patients during treatment with Interferonα/PEG-Interferonα. However, its relevance, especially in difficult-to treat patients, has not been defined. Methods: We retrospectively measured the serum levels of anti-IFNα antibodies (baseline and week 12) and IFNα levels (week 12) by ELISA in 76 previous non-responders, and in 14 naive patients treated with Pegylated-IFNα and Ribavirin. A group of 57 healthy donors (HD) was also assessed as control. Positivity to anti-IFNα antibodies was established on the values of HD. Results: Baseline anti-IFNα antibodies were detected in 15.5% of patients and in 7% of HD, with significantly higher concentrations in patients than HD (181.5±389.9 vs 95.9±143.0 ng mL−1, p=0.0023). All positive patients were IFNα-experienced. At week 12, the prevalence of positivity increased to 22.3 and 28.5% in experienced and naïve patients, respectively, and the levels of anti-IFNα antibodies did not differ between the two groups (391±792.3 vs 384.7±662.6 ng mL−1, respectively). IFNα concentrations were significantly lower in antibody-positive patients than in antibody-negatives (988.2±1402 vs 3462±830.8 pg mL−1, p≤0.0001) and the levels of antibodies and IFNα were inversely correlated (r=-0.405, p=0.0001). The antibody-positive population clustered in null responders (67%) and 19/21 patients (90%) did not achieve SVR. Conclusions: The development of anti-IFNα antibodies is a non-negligible occurrence in patients treated with PEG-IFNα, is stable over time, and has a relevant clinical impact when associated with low levels of circulating PEG-IFNα. It should be considered in patients undergoing treatments including PEG-IFNα.

Critical review of the use of erythropoietin in the treatment of anaemia during therapy for chronic hepatitis C

Combined pegylated interferon (PegIFN) and ribavirin represents the standard therapy for patients with chronic hepatitis C (CHC), which allows for sustained viral response (SVR) in up to 90% of patients depending on certain viral and host factors. Clinical studies have demonstrated the importance of adherence to therapy, that is, the ability of patients to tolerate and sustain a fully dosed therapy regimen. Adherence is markedly impaired by treatment-related adverse effects. In particular, haemolytic anaemia often requires dose reduction or termination of ribavirin treatment, which compromises treatment efficacy. Recent evidence points to a beneficial role of recombinant erythropoietin (EPO) in alleviating ribavirin-induced anaemia thereby improving quality of life, enabling higher ribavirin dosage and consequently improving SVR. However, no general consensus exists regarding the use of EPO for specific indications: its optimal dosing, treatment benefits and potential risks or cost efficiency. The Swiss Association for the Study of the Liver (SASL) has therefore organized an expert meeting to critically review and discuss the current evidence and to phrase recommendations for clinical practice. A consensus was reached recommending the use of EPO for patients infected with viral genotype 1 developing significant anaemia below 100 g/L haemoglobin and a haematocrit of <30% during standard therapy to improve quality of life and sustain optimal ribavirin dose. However, the evidence supporting its use in patients with pre-existing anaemia, non-1 viral genotypes, a former relapse or nonresponse, liver transplant recipients and cardiovascular or pulmonary disease is considered insufficient.

A genetic validation study reveals a role of vitamin D metabolism in the response to interferon-alfa-based therapy of chronic hepatitis C

Background To perform a comprehensive study on the relationship between vitamin D metabolism and the response to interferon-α-based therapy of chronic hepatitis C. Methodology/Principal Findings Associations between a functionally relevant polymorphism in the gene encoding the vitamin D 1α-hydroxylase (CYP27B1-1260 rs10877012) and the response to treatment with pegylated interferon-α (PEG-IFN-α) and ribavirin were determined in 701 patients with chronic hepatitis C. In addition, associations between serum concentrations of 25-hydroxyvitamin D3 (25[OH]D3) and treatment outcome were analysed. CYP27B1-1260 rs10877012 was found to be an independent predictor of sustained virologic response (SVR) in patients with poor-response IL28B genotypes (15% difference in SVR for rs10877012 genotype AA vs. CC, p = 0.02, OR = 1.52, 95% CI = 1.061–2.188), but not in patients with favourable IL28B genotype. Patients with chronic hepatitis C showed a high prevalence of vitamin D insufficiency (25[OH]D3<20 ng/mL) during all seasons, but 25(OH)D3 serum levels were not associated with treatment outcome. Conclusions/Significance Our study suggests a role of bioactive vitamin D (1,25[OH]2D3, calcitriol) in the response to treatment of chronic hepatitis C. However, serum concentration of the calcitriol precursor 25(OH)D3 is not a suitable predictor of treatment outcome.

The vitamin D receptor gene bAt (CCA) haplotype impairs the response to pegylated-interferon/ribavirin-based therapy in chronic hepatitis C patients

Chronic hepatitis C infection is a major cause of end-stage liver disease. Therapy outcome is influenced by 25-OH vitamin D deficiency. To further address this observation, our study investigates the impact of the vitamin D receptor (NR1I1) haplotype and combined effects of plasma vitamin D levels in a well-described cohort of hepatitis C patients.