Molecular characterization of hepatitis B virus X gene in chronic hepatitis B patients

Background: HBV-X protein is associated with the pathogenesis of HBV related diseases, specially in hepatocellular carcinomas of chronic patients. Genetic variability of the X gene includes genotypic specific variations and mutations emerging during chronic infection. Its coding sequence overlaps important regions for virus replication, including the basal core promoter. Differences in the X gene may have implications in biological functions of the protein and thus, affect the evolution of the disease. There are controversial results about the consequences of mutations in this region and their relationship with pathogenesis. The purpose of this work was to describe the diversity of HBV-X gene in chronic hepatitis patients infected with different genotypes, according to liver disease. Methods. HBV-X gene was sequenced from chronic hepatitis B patient samples, analyzed by phylogeny and genotyped. Nucleotide and aminoacid diversity was determined calculating intragenetic distances. Mutations at 127, 130 and 131 aminoacids were considered in relation to liver disease. Results: The most prevalent genotype detected in this cohort was F (F1 and F4), followed by D and A. Most of the samples corresponding to genotypes A and F1 were HBeAg(+) and for genotypes D and F4, HBeAg(-) samples were represented in a higher percentage. Intragenetic distance values were higher in HBeAg(-) than in positive samples for all genotypes, and lower in overlapped regions, compared to single codification ones. Nucleotide and aminoacid diversities were higher in HBeAg(-), than in HBeAg(+) samples. Conclusions: Independently of the infecting genotypes, mutations at any of 127, 130 and/or 131 aminoacid positions and HBeAg(-) status were associated with mild liver disease in this cohort.Fil: Barbini, Luciana Fernanda. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tadey, Luciana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fernandez, Silvina. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Bouzas, Belén. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Campos, Rodolfo Hector. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Molecular and biological characterization of hepatitis B virus subgenotype F1b clusters: Unraveling its role in hepatocarcinogenesis

Hepatitis B virus (HBV) subgenotype F1b infection has been associated with the early occurrence of hepatocellular carcinoma in chronically infected patients from Alaska and Peru. In Argentina, however, despite the high prevalence of subgenotype F1b infection, this relationship has not been described. To unravel the observed differences in the progression of the infection, an in-depth molecular and biological characterization of the subgenotype F1b was performed. Phylogenetic analysis of subgenotype F1b full-length genomes revealed the existence of two highly supported clusters. One of the clusters, designated as gtF1b Basal included sequences mostly from Alaska, Peru and Chile, while the other, called gtF1b Cosmopolitan, contained samples mainly from Argentina and Chile. The clusters were characterized by a differential signature pattern of eight nucleotides distributed throughout the genome. In vitro characterization of representative clones from each cluster revealed major differences in viral RNA levels, virion secretion, antigen expression levels, as well as in the localization of the antigens. Interestingly, a differential regulation in the expression of genes associated with tumorigenesis was also identified. In conclusion, this study provides new insights into the molecular and biological characteristics of the subgenotype F1b clusters and contributes to unravel the different clinical outcomes of subgenotype F1b chronic infections.Fil: Elizalde, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Mojsiejczuk, Laura Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Virología; ArgentinaFil: Speroni, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Bouzas, Belén. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Tadey, Luciana. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mammana, Lilia. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Campos, Rodolfo Hector. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Flichman, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentin

Quantitative HBsAg an unreliable marker for diagnosis and disease progression in genotype F chronic HBeAg-negative infections

Quantitative hepatitis B surface antigen (qHBsAg) has been proposed as a biomarker to distinguish HBeAg-negative chronic infections (ENI) from HBeAg-negative chronic hepatitis (ENH), identify patients prone to achieving sustained HBsAg loss, and predict the risk of liver disease progression. There is evidence that qHBsAg varies among genotypes, however there is a paucity of data on genotype F. The aim of this study was to investigate the performance of qHBsAg in the diagnosis and evolution of genotype F chronic HBeAg-negative infections. HBV-DNA and HBsAg levels from 153 patients with ENI were correlated with the genotype. Liver disease progression was assessed by abdominal ultrasound and a transient elastography. The qHBsAg levels were significantly different among genotypes (p 3.0 log10 IU/ml, no cases of advanced liver disease were observed at the end of follow-up. This study provides new insights into the impact of HBV genotypes, in particular GTF, on serum HBsAg levels, emphasizing the need to implement a genotype-specific cut-off to achieve diagnostic certainty in the identification of ENI and the risk of liver disease progression. Regardless of HBV genotype, qHBsAg has been shown to be a powerful and reliable biomarker for predicting HBsAg loss.Fil: Fainboim, Hugo. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Di Benedetto, Nicolas. Hospital Arrecifes; ArgentinaFil: Paz, Silvia. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Mendizabal, Manuel. Universidad Austral; ArgentinaFil: Campuzano, Soledad. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Elizalde, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Tadey, Luciana. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Deluchi, Gabriel. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Bouzas, María Belén. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Mammana, Lilia. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Flichman, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentin

Molecular characterization of hepatitis B virus X gene in chronic hepatitis B patients

BACKGROUND: HBV-X protein is associated with the pathogenesis of HBV related diseases, specially in hepatocellular carcinomas of chronic patients. Genetic variability of the X gene includes genotypic specific variations and mutations emerging during chronic infection. Its coding sequence overlaps important regions for virus replication, including the basal core promoter. Differences in the X gene may have implications in biological functions of the protein and thus, affect the evolution of the disease. There are controversial results about the consequences of mutations in this region and their relationship with pathogenesis. The purpose of this work was to describe the diversity of HBV-X gene in chronic hepatitis patients infected with different genotypes, according to liver disease. METHODS: HBV-X gene was sequenced from chronic hepatitis B patient samples, analyzed by phylogeny and genotyped. Nucleotide and aminoacid diversity was determined calculating intragenetic distances. Mutations at 127, 130 and 131 aminoacids were considered in relation to liver disease. RESULTS: The most prevalent genotype detected in this cohort was F (F1 and F4), followed by D and A. Most of the samples corresponding to genotypes A and F1 were HBeAg(+) and for genotypes D and F4, HBeAg(−) samples were represented in a higher percentage. Intragenetic distance values were higher in HBeAg(−) than in positive samples for all genotypes, and lower in overlapped regions, compared to single codification ones. Nucleotide and aminoacid diversities were higher in HBeAg(−), than in HBeAg(+) samples. CONCLUSIONS: Independently of the infecting genotypes, mutations at any of 127, 130 and/or 131 aminoacid positions and HBeAg(−) status were associated with mild liver disease in this cohort

Role of viral load in Hepatitis B virus evolution in persistently normal ALT chronically infected patients

Chronic HBV infection has been associated with severe liver disease although most of them do not progress to this stage. Even though low replicative carriers form the largest group of HBV chronically infected patients, there is a paucity of longitudinal studies to evaluate the molecular evolution of the whole genome in this subset of patients. In this study, longitudinal samples from 10 patients with persistently normal ALT levels were collected. HBV full-length genome sequences were obtained from 3 samples per patient (baseline, 5 and 10-years of follow-up). Patients were grouped according to HBV-DNA level into  103 IU/ml (group B). The substitution rate was inversely related with HBV-DNA levels. Moreover, the rate in the 10-year follow-up was significantly higher in group A (6.9 × 10−4 ± 1.3 × 10−4) than group B (2.7 × 10−4 ± 7.4 × 10−5 substitution/site/year, p < .001). Most of the substitutions were in the Core region and the majority were non-synonymous changes. The rate of nucleotide substitution was inversely related to HBV-DNA levels, highlighting the role of viral load in the HBV intra-host dynamics, even in low replicative state patients. Moreover, the difference in the substitution rate between the analysed groups was mainly consequence of substitutions restricted to the Core region, particularly in the simple coding region and antigenic epitopes, which suggest that the immune pressure drives the different evolutionary behaviour of groups.Fil: Gauder, Catalina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Virología; ArgentinaFil: Mojsiejczuk, Laura Noelia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tadey, Luciana. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mammana, Lilia. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Bouzas, Maria Belen. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Campos, Rodolfo Hector. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Flichman, Diego Martin. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentin

Biological Characterization of Hepatitis B virus Genotypes: Their Role in Viral Replication and Antigen Expression

Hepatitis B virus (HBV) inter-host evolution has resulted in genomic diversification reflected in the existence of nine genotypes (A-I) and numerous subgenotypes. There is growing evidence that genotypes influence HBV natural history, clinical outcomes, and treatment response. However, the biological characteristics underlying these differences have not yet been established. By transfecting HuH-7 cells with unit-length constructs of genotypes A2, B2, C1, D1, and F1b, we identified major differences in HBV replicative capacity and antigen expression across genotypes. Genotypes B2 and F1b showed a 2-fold increase in cccDNA levels compared to the other genotypes (p<0.005). Genotype A2 expressed the lowest pgRNA levels, with a 70-fold decrease in relation to the other genotypes (p<0.0001), while genotype B2 showed the lowest Precore RNA levels, with a 100-fold reduction compared to genotype A2 (p<0.0001). The highest intracellular HBV DNA levels were observed for genotype B2 and the lowest for genotypes A2 and C1 (p<0.0001). Regarding antigen expression, genotype F1b secreted the highest HBsAg levels and genotype D1 the lowest (p<0.0001), while genotypes A2 and B2 showed the highest intracellular HBsAg levels (p<0.0001). Interestingly, genotype C1 secreted the highest HBeAg levels, while genotype A2 showed the highest intracellular levels (p<0.0001). Finally, the analysis of the intra/extracellular antigen ratios revealed that most genotypes retained intracellularly 5–20% of the antigens, except the genotype A2 that retained 50% of the total expressed antigens. In conclusion, this study provides new insights into the biological characteristics of HBV genotypes, being the first study to comparatively analyze European (A and D) and Asian (B and C) genotypes with the Latin American (F) genotype. The differences in HBV replication and antigen expression might contribute to understand the differential role of genotypes in pathogenesis.Fil: Elizalde, Maria Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Tadey, Luciana. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Mammana, Lilia. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Quarleri, Jorge Fabian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Campos, Rodolfo Hector. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Flichman, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentin

Relevance of HTLV-1 proviral load in asymptomatic and symptomatic patients living in endemic and non-endemic areas of Argentina.

HTLV-1 proviral load (pVL) in peripheral blood mononuclear cell (PBMCs) is proposed as a marker of disease progression but its role still remains controversial. The aim of this study was to evaluate the levels of HTLV-1 pVL in symptomatic patients and asymptomatic HTLV-1 carriers. In this cross-sectional study the pVL was measured by Real Time PCR in 102 asymptomatic carriers and 22 symptomatic patients (5ATLL, 15 TSP and 2 uveitis). We observed that the HTLV-1 pVL was significantly higher in symptomatic patients (median = 4.99 log10 HTLV-1 copies /106 PBMCs) compared to asymptomatic HTLV-1 carriers (median = 4.38 log10 HTLV-1 copies /106 PBMCs; p = 0.0030). A wide variation on the HTLV-1 pVL levels among asymptomatic HTLV-1 carriers was observed with some pVL as high as those observed in symptomatic patients. The asymptomatic HTLV-1 carriers were divided according to the place of birth and the highest levels of pVL were detected among patients from endemics areas from the North of Argentina. Our results reinforce the usefulness of the proviral load would be a prognostic marker of HTLV-1 disease progression. Moreover, host, viral or socio-environmental factors cannot be excluded as determinant of high proviral load

Characterization of the basal core promoter and precore regions in anti-HBe-positive inactive carriers of hepatitis B virus

Background: The study of hepatitis B virus (HBV) genomic heterogeneity has become a major issue in investigations aimed at understanding the relationship between HBV mutants and the wide spectrum of clinical and pathological conditions associated with HBV infection. Although most chronically infected HBV patients are inactive carriers, several virological aspects of this state remain unclear. Methods: In order to determine the prevalence and clinical significance of mutations in the basal core promoter (BCP) and precore (pC) regions among inactive carriers, the nucleotide sequences from 41 inactive carriers were analyzed and compared with those from 29 individuals with chronic active hepatitis. Results: Genotypes A (24.3%), D (37.1%), F1b (12.9%), and F4 (18.6%) were the most prevalent. Mutations in the BCP/pC regions were observed in most of the inactive carriers (92.7%) and in most of the patients with chronic active hepatitis (93.1%). The prevalence of mutation 1764A was significantly higher in patients with chronic active hepatitis (65.5%) than in inactive carriers (36.6%) (p=0.038), whereas the prevalences of mutations at the other positions analyzed were not significantly different. Older patients (>50 years) showed BCP/pC patterns with a higher number of substitutions. Mutations were found to be biased by genotype: the 1896A mutation was highly prevalent in genotypes D and F4, while alternative substitutions in the pC region were more prevalent in genotypes A and F1b. Conclusions: Mutations in the BCP/pC regions are the hallmark of chronic anti-HBe-positive individuals; nevertheless, the even distribution of mutations in active and inactive carriers suggests that BCP/pC mutations may occur during HBV infection not strictly related to the HBV infection activity.Fil: González López Ledesma, María Mora. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Galdame, Omar. Hospital Italiano; ArgentinaFil: Bouzas, Belén. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Tadey, Luciana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Livellara, Beatriz I.. Hospital Italiano; ArgentinaFil: Giuliano, Silvina. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Viaut, Marcela. Hospital Italiano; ArgentinaFil: Paz, Silvia. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Fainboim, Hugo. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas "Dr. Francisco Javier Muñiz"; ArgentinaFil: Gadano, Adrián Carlos. Hospital Italiano; ArgentinaFil: Campos, Rodolfo Hector. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Flichman, Diego Martin. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin