RESUMO

Hepatitis B e antigen (HBeAg) loss represents a late stage of chronic hepatitis B virus (HBV) infection associated with a drastic decrease in HBV-DNA, a lower risk of disease progression, and the occurrence of several mutations in the preCore/core region. However, the underlying mechanisms supporting the downregulation of viral replication have yet to be elucidated. In the present study, the analysis of the frequency of subgenotype D1 core protein (HBc) mutations associated with HBeAg status revealed a higher mutation rate in HBeAg-negative sequences compared to HBeAg-positive ones. Particularly, 22 amino acids exhibited a higher frequency of mutation in HBeAg-negative sequences, while the remaining residues showed a high degree of conservation. Subsequently, the assessment of HBc mutants derived from HBeAg-negative patients in viral structure and replicative capacity revealed that HBc mutations have the ability to modulate the subcellular localization of the protein (either when the protein was expressed alone or in the context of viral replication), capsid assembly, and, depending on specific mutation patterns, alter covalently closed circular DNA (cccDNA) recycling and up- or downregulate viral replication. In conclusion, HBc mutations associated with HBeAg-negative status impact on various stages of the HBV life cycle modulating viral replication during the HBeAg-negative stage of infection.

Assuntos

RESUMO

After more than four decades of hepatitis B virus (HBV) vaccine implementation, its safety and efficacy in preventing HBV infection have been proven and several milestones have been achieved. Most countries have included HBV immunization schedules in their health policies and progress has been made regarding universalization of the first HBV vaccine dose at birth. All of these actions have significantly contributed to reducing both the incidence of HBV infection and its related complications. However, there are still many drawbacks to overcome. The main concerns are the deficient coverage rate of the dose at birth and the large adult population that has not been reached timely by universal immunization. Additionally, the current most widely used second-generation vaccines do not induce protective immunity in 5% to 10% of the population, particularly in people over 40-years-old, obese (body mass index > 25 kg/m2), heavy smokers, and patients undergoing dialysis or infection with human immunodeficiency virus. Recently developed and approved novel vaccine formulations using more potent adjuvants or multiple antigens have shown better performance, particularly in difficult settings. These advances re-launch the expectations of achieving the World Health Organization's objective of completing hepatitis control by 2030.

Assuntos

RESUMO

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.

RESUMO

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.

RESUMO

Hepatitis B virus (HBV) is classified into ten genotypes and numerous subgenotypes (sgt). In particular, sgt F1b and sgt F4, native of Latin America, have been associated with differences in clinical and virological characteristics. Hepatitis B virus X protein (HBx) is a multifunctional regulatory protein associated with the modulation of viral transcription and replication. In this work, we analyzed the role of the X gene and the encoded X protein in sgtF1b and sgtF4 replication. Transfection with HBx deficient genomes revealed remarkable differences in the replicative capacity of sgtF1b and sgtF4 mutants. The silencing of HBx increased sgtF1b X(-) transcription and replication by more than 2.5 fold compared to the wild type variant, while it decreased sgtF4 X(-) transcription and replication by more than 3 fold. Trans-complementation of HBx restore sgtF1b and sgtF4 wild type transcription and replication levels. In addition, transfection with chimeric variants, carrying wild type (F1b/XF4 and F4/XF1b) or mutated (F1b/X(-)F4 and F4/X(-)F1b) X gene of one sgt in the backbone of the other sgt, showed that the nucleotide sequence of the X gene, that includes regulatory elements that modulate pgRNA transcription, was responsible for the disparity observed between sgtF1b X(-) and sgtF4 X(-). These results showed that sgtF1b and sgtF4 X gene play a central role in regulating HBV transcription and replication, which eventually lead to a common purpose, to reach wild type replication levels of sgtF1b and sgtF4 viruses.

Assuntos

RESUMO

Hepatitis B virus (HBV) circulates as a collection of genetically related variants that evolve throughout the chronic infection. Those viral variants that have the greatest fitness are fixed. We recently showed different fitness for HBV variants involved in two epidemiological situations. To understand these fitness differences better, we determined the levels of extracellular HBV DNA, the synthesis of HBV DNA intermediates, and the expression of HBeAg and HBsAg in transfection and cotransfection assays. Our results show that for the subgenotype (sgt) D1, which has an 8-nucleotide deletion (sgtD1del) and exhibits lower fitness, the levels of extracellular DNA and intracellular replicative intermediates were much lower than with sgtD1wt or sgtD1mut (G1896A), which had higher fitness. In addition, in the cotransfection assay, sgtD1del inhibited sgtD1mut but not sgtD1wt replication. We also found that sgtF1b, which exhibits higher fitness, produces significantly higher levels of both extracellular DNA and intracellular replicative intermediates than does the lower-fitness sgtF4. These results demonstrate a relationship between fitness and the replicative ability of the HBV genome in the transfection assay. In addition, the data obtained by cotransfecting cells with sgtD1del and sgtD1mut provide new information about the impact of simultaneous replication of two viral variants in the same cell system on HBV replication.

Assuntos

RESUMO

Hepatitis B virus (HBV) genotypes and mutants have been associated with differences in clinical and virological characteristics. Autophagy is a cellular process that degrades long-lived proteins and damaged organelles. Viruses have evolved mechanisms to alter this process to survive in host cells. In this work, we studied the modulation of autophagy by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. HBV subgenotypes F1b and F4 replication induced accumulation of autophagosomes in hepatoma cells. However, no autophagic protein degradation was observed, indicating a blockage of autophagic flux at later stages. This inhibition of autophagy flux might be due to an impairment of lysosomal acidification in hepatoma cells. Moreover, HBV-mediated autophagy modulation was independent of the viral subgenotypes and enhanced in viruses with BCP and preCore naturally occurring mutations. These results contribute to understand the mechanisms by which different HBV variants contribute to the pathogenesis of HBV infections. In addition, this study is the first to describe the role that two highly prevalent naturally occurring mutations exert on the modulation of HBV-induced autophagy.

Assuntos

RESUMO

Hepatitis B virus (HBV) has a high mutation rate and exists as a mixture of genetically different but closely related variants. We present a HBV DNA co-transfection fitness assay and use it to evaluate the relative fitness of different HBV variants in two scenarios: seroconversion process and occupation of an ecological niche. In the seroconversion experiment, subgenotype D1 (sgtD1) deletion (1763-1770) had significantly lower fitness comparing with both sgtD1 wild type and sgtD1mut G1896A, while, in the case of occupation of ecological niche experiment, the results showed the same relative fitness between all of the genotype combinations, except F1b-F4. In this case sgtF1b clearly overgrow sgtF4, which is in accordance with the observation that F1b is the most prevalent in the new infections in Argentina. In summary, we present a method aimed to evaluate HBV viral fitness which improve the analysis of the relative frequency of viral variants during the HBV infection process.

Assuntos

Assuntos

RESUMO

Hepatitis B virus (HBV) is a globally distributed human pathogen that leads to both self-limited and chronic infections. At least eight genotypes (A-H) with distinct geographical allocations and phylodynamic behaviors have been described. They differ substantially in many virological and probably some clinical parameters. The aim of this study was to analyze full-length HBV genome sequences from individuals with symptomatic acute HBV infections using phylogenetic and coalescent methods. The phylogenetic analysis resulted in the following subgenotype distribution: F1b (52.7%), A2 (18.2%), F4 (18.2%) and A1, B2, D3 and F2a 1.8% each. These results contrast with those previously reported from chronic infections, where subgenotypes F1b, F4, A2 and genotype D were evenly distributed. This differential distribution might be related to recent internal migrations and/or intrinsic biological features of each viral genotype that could impact on the probability of transmission. The coalescence analysis showed that after a diversification process started in the 80s, the current sequences of subgenotype F1b were grouped in at least four highly supported lineages, whereas subgenotype F4 revealed a more limited diversification pattern with most lineages without offspring in the present. In addition, the genetic characterization of the studied sequences showed that only two of them presented mutations of clinical relevance at S codifyng region and none at the polymerase catalytic domains. Finally, since the acute infections could be an expression of the genotypes currently being transmitted to new hosts, the predominance of subgenotype F1b might have epidemiological, as well as, clinical relevance due to its potential adverse disease outcome among the chronic cases.

Assuntos