BackgroundInfluenza A viruses are a major cause of viral respiratory infections in humans, leading to a spectrum of diseases ranging from mild to severe, particularly in high-risk groups. Monitoring the frequency and evolution of A (H1N1)pdm09 viruses is essential for guiding vaccine strain selection and maintaining vaccine effectiveness.ObjectivesTo characterize the evolution of circulating influenza A (H1N1)pdm09 strains in Mar del Plata(MDQ), Argentina, and estimate vaccine efficacy from 2015 to 2020, before the SARS-CoV-2 pandemic.MethodsWe analyzed 46 influenza A (H1N1)pdm09 strains detected by RT-PCR from patients with acute respiratory infections in MDQ between 2015 and 2020. Phylogenetic analysis was performed using maximum likelihood, and vaccine efficacy was estimated with the Pepitope model.ResultsSeven genetic clades were identified: 6B in 2015, 6B.1 in 2016-2017, 6B.1A, 6B.1A.1, and 6B.1A.3 in 2018, 6B.1A.5A in 2019-2020, and 6B.1A.5a.2 in 2020. Genetic diversity and regional clustering suggested multiple strain introductions from other Argentinian regions or countries. The predicted vaccine efficacy was highest when the frequency of influenza A (H1N1)pmd09 was below 2%, decreasing as viral frequency increased, due to mutations occurring particularly in haemagglutinin epitopes B, C, and E.ConclusionOur findings highlight the dynamic evolution of influenza A (H1N1)pdm09 in MDQ and emphasize the importance of continuous molecular surveillance and regular vaccine updates. Additionally, the Pepitope model proved useful in estimating vaccine efficacy. The impact of vaccination in reducing viral frequency when circulating and vaccine strains are well-matched further supports annual influenza vaccination recommendations to minimize viral burden in the community.

Medicinal plants are used to cure diseases, and their replacement is frequent and affects public health. The genus Baccharis has representatives within the medicinal flora of Argentina, although the replacement of the species of this genus known under the vulgar name of "carqueja" by Baccharis spicata has been detected in herbalists or markets of herbal products. The genotoxic safety of this species has been established in previous work of our group. The aim of this study was to evaluate the antiviral activity of an infusion made from B. spicata leaves against hepatitis B virus with the HepG2.2.15 cellular system and to determine cytotoxicity in HepG2.2,15, A549 and Vero cell lines. Infusion of B. spicata was active to inhibit HBV replication with an EC50of 22.54 μg/mL and a CC50of 190 μg/mL.

Introduction and Objectives: Two clusters of the subgenotype F1b (basal and cosmopolitan) have recently been described. The basal cluster has been associated with the early occurrence of hepatocellular carcinoma in chronically infected patients from Alaska and Peru. In Argentina, where the cosmopolitan cluster is the most prevalent, this relationship has not been observed. In the course of chronic hepatitis B infection, mutations occur in different regions. In particular, mutations in the basal core promoter (BCP) and the preC/C regions are associated with HBe-antigen (HBeAg) seroconversion, an event related to the severity of chronic HBV infection. This study aimed to determine the HBeAg status and to characterize the molecular mutation patterns associated with HBeAg seroconversion in both subgenotype F1b clusters. Materials and Methods: Serum samples from 68 patients with subgenotype F1b chronic hepatitis B infection were analyzed. The BCP and pC/C regions were amplified and sequenced. Results: Twenty-one samples belonged to the basal cluster and 47 to the cosmopolitan cluster. No differences in age or gender were observed between the cases of both clusters. The basal cluster samples showed a lower frequency of positivity for HBeAg (38.1 vs. 57.4 %). In HBeAg negative samples, the basal cluster showed significantly higher rates of A1762T/G1764A (92.3 vs. 50.0, p:0.013) and G1896A (92.3 vs. 20.0, <0.001) mutations in relation to the cosmopolitan cluster. Conclusions: The disparity observed in HBeAg positivity frequency suggests that the basal cluster would be associated with earlier HBeAg seroconversion than the cosmopolitan cluster. The frequency of mutations associated with a worse clinical outcome was significantly higher in the basal cluster samples.Overall, this study provides new insights into the role of viral variants in the pathogenesis of chronic HBV infection and contributes to identifying molecular determinants associated with the pathogenesis of chronic HBV infection.

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.

Hepatitis B virus (HBV) is the main etiological agent of hepatocellular carcinoma (HCC) worldwide. It has been classified into nine genotypes and several subgenotypes, with uneven global distribution. There is growing evidence that the viral genotype influences the course and outcome of chronic hepatitis B infection. Two evolutionarily different clusters of the subgenotype F1b, called basal and cosmopolitan, have been described. The two clusters have constrained geographical distribution, with the particular feature that the basal cluster is present in regions of high HCC incidence, while the Cosmopolitan cluster is found in regions of low HCC incidence. The BCP/pC region was sequenced in 68 cases chronically infected with the F1b subgenotype to determine if there was a differential pattern of pathogenic-associated mutations between both clusters. Twenty-two of the 68 cases belonged to the subgenotype F1b basal cluster and 46 to the cosmopolitan cluster. Among the HBeAg-negative patients the A1762T/G1764A and G1896A mutations were more frequently found in the basal samples (85.7 and 92.9%) compared to the cosmopolitan ones (50 and 18.2%). Interestingly, no HBeAg loss-associated mutations were observed in 7.1 and 36.4% of the basal and cosmopolitan cases, respectively. The different rate of mutations associated with a more severe course of chronic hepatitis in the basal cluster would support the difference in the HCC incidence rate in the geographical regions where the basal cluster is restricted.

BACKGROUND:JC polyomavirus (JCV) has an ethno-geographical distribution across human populations. OBJECTIVE:Study the origins of the population of Misiones (Argentina) by using JCV as genetic marker. METHODS:Viral detection and characterization was conducted by PCR amplification and evolutionary analysis of the intergenic region sequences. RESULTS:22 out of 121 samples were positive for JCV, including 5 viral lineages: MY (n = 8), Eu-a (n = 7), B1-c (n = 4), B1-b (n = 2) and Af2 (n = 1). MY sequences clustered within a branch of Native American origin that diverged from its Asian counterpart about 21,914 years ago (HPD 95% interval 15,383-30,177), followed by a sustained demographic expansion around 5000 years ago. CONCLUSIONS:JCV in Misiones reflects the multiethnic origin of the current population, with an important Amerindian contribution. Analysis of the MY viral lineage shows a pattern consistent with the arrival of early human migrations to the Americas and a population expansion by the pre-Columbian native societies.

BACKGROUND:Few studies about the evolutionary history of the hepatitis E virus (HEV) have been conducted. The aim of our work was to investigate and make inferences about the origin and routes of dispersion of HEV-3 in Argentina.METHODS:Phylogenetic, coalescent and phylogeographic analyses were performed using a 322-bp ORF2 genomic fragment of all HEV-3 sequences with known date and place of isolation published at GenBank until May 2018 (n=926), including 16 Argentinian sequences (isolated from pigs, water and humans).RESULTS:Phylogenetic analysis revealed two clades within HEV-3: abchij and efg. All Argentinian samples were grouped intermingled within clade 3abchij. The coalescent analysis showed that the most recent common ancestor for the clade 3abchij would have existed around the year 1967 (95% highest posterior density (HPD): 1963-1970). The estimated substitution rate was 1.01×10-2 (95%HPD: 9.3×10-3-1.09×10-2) substitutions/site/y, comparable with the rate previously described. The phylogeographic approach revealed a correspondence between phylogeny and place of origin for Argentinian samples, suggesting many HEV introductions in the country, probably from Europe and Japan.CONCLUSIONS:This is the first evolutionary inference of HEV-3 that includes Argentinian strains, showing the circulation of many HEV-3 subtypes, obtained from different sources and places, with recent diversification processes.ACCESSION NUMBERS:[KX812460], [KX812461], [KX812462], [KX812465], [KX812466], [KX812467], [KX812468], [KX812469].

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.

Background: Hepatitis B virus (HBV) has been classified into 10 genotypes (A-I) and numerous subgenotypes (SGTs). There is growing evidence that HBV genotypes (GTs) influence clinical outcomes, HBeAg seroconversion rates, severity of liver disease, and response to interferon therapy. However, there is a paucity of data regarding their distinctive biological characteristics, in particular for GTF, the most prevalent in Latin America. Aim: To investigate the impact of HBV genotypes on HBV-DNA levels and viral antigen expression. Materials and Methods: Full-length HBV genomes representing GTs A-D and SGTs F1b and F4 were transfected in Huh7 cell line. Secreted HBeAg and intra and extracellular HBsAg were quantified by EIA. HBV-DNA was analyzed by real-time PCR. Results: Marked differences were observed in HBV replicative capacity as well as HBeAg and HBsAg antigen expression across genotypes (Table 1). GTC secreted significantly higher levels of HBeAg in relation to the other GTs. GTD showed lower HBsAg extracellular levels than all other GTs, while GTA showed the highest HBsAg intracellular levels. Finally, SGTs F1b and F4 showed significantly lower HBV-DNA levels. Regarding the ratio of extra and intracellular HBsAg, GTs A and D showed the lower ratios compared to SGTs F1b and F4, while SGTs F1b and F4 showed the highest HBsAg/HBV-DNA ratio. Discussion: This study provides new insights into the impact of HBV genotypes on HBV antigen expression and HBV-DNA levels. The uneven expression of antigens, as well as their intracellular accumulation, could be associated with the role of genotypes in pathogenesis. Likewise, the extracellular levels of HBsAg and the replication rate might have implications in immunopathogenesis as well as in the exhaustion of the host's immune system. The virus-cell interaction in different genotypes deserves further study to understand its role in the pathogenesis of HBV infection.

Objective: In this study, we investigated the occurrence of papillomavirus (PV) infection in non-human primates (NHP, Platyrrhine) of northeastern Argentina by using broad-spectrum PCR primers at the L1 gene. In addition, we conducted a phylogenetic and coalescence analysis of viral sequences to explore their evolutionary history and evaluate the co-speciation hypothesis in the context of primate evolution. Methods: We obtained samples of 57 individuals from wild and captive populations of Alouatta caraya , Sapajus nigritus and Sapajus cay . We assessed PV infection by PCR amplification with the CUT primer system and sequencing of 337 bp (112 amino acids) of the L1 protein. The viral sequences were analyzed by phylogenetic and Bayesian coalescence methods to estimate the age of the most common recent ancestor (tMCRA) with BEAST, v1.4.8 software. We evaluated viral/host tree congruence with TreeMap v3.0. Results: We identified two novel putative PV sequences of the genus Gamma- PV in Sapajus sp and Alouatta caraya (SPV1 and AcPV1, respectively). The tMRCA of SPV1 was estimated at 11,941,682 years before present (ybp) and that of AcPV1 at 46,638,071 ybp, both predating the coalescence times of their hosts: 6.4 million years (MYA) and 6.8 MYA, respectively. Based on the comparison of primate and viral phylogenies, we could not reject the null hypothesis that the PV tree is no more congruent with the host tree than a random tree would be (P>0.05). Thus, a model of virus-host coevolution was rejected. Conclusion: This study presents the first report of PV infection in Platyrrhine species from Argentina, expands the range of described hosts for these viruses, and proposes new scenarios for their origin and dispersal.

Human adenoviruses (HAdV) are one of the most frequent causes of respiratory infections around the world, causing mild to severe disease. In Argentina, many studies focused on the association of HAdV respiratory infection with severe disease and fatal outcomes leading to the discovery in 1984 of a genomic variant 7h associated with high fatality. Although several molecular studies reported the presence of at least 4 HAdV species (B, C, D and E) in Argentina, few sequences were available in the databases. In this study, sequences from the hexon gene region were obtained from 141 patients as a first approach to assess the genetic diversity of HAdVs circulating in Buenos Aires, Argentina. Phylogenetic analysis of these sequences and others recovered from public databases confirmed the circulation of the four above-mentioned species represented by 11 genotypes, with predominance in species B and C and shifts in their proportion in the studied period (2000 to 2018). The variants detected in Argentina, for most of the genotypes, were similar to those already described in other countries. However, uncommon lineages belonging to genotypes C2, C5 and E4 were detected, which might indicate the circulation of local variants and will deserve further studies of whole-genome sequences.

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.

The genotype F (HBV-F) is an autochthonous Native American strain of the hepatitis B virus. In this study, we reconstruct the HBV-F long-term evolution under a hypothesis of co-divergence with humans in Central and South America, since their entry into the region 14.5-16 thousand years ago. The Bayesian phylogeographic reconstruction supported a virus-host co-expansion; however, two evolutionary scenarios would have been present. Whereas subgenotype F1 spreads along a Pacific coastal route and would have evolved associated with Central American and Andean cultures from the west of the continent, subgenotypes F2-F6 spread along the Atlantic coastline and inner pathways associated with communities inhabiting the tropical forest lowlands. Then, we propose a model for HBV-F evolution in which the selection of differential biological characteristics in these two main groups would be related to their evolution in host populations with different genetic backgrounds and dissimilar demographic conditions.

Marcetia taxifolia is a neotropical plant present in South America and it has been evaluated in several biological models due to the presence of active metabolites. Nevertheless, there is a limited quantity of studies related to the antiviral activity of the compounds present in this genus. In our work, the antiviral effect of the compounds isolated from the aerial parts of Marcetia taxifolia was evaluated against Hepatitis B virus (HBV), Herpes Simplex Virus type 1 (HSV-1), and Poliovirus type 1 (PV-1). The cytopathic effect and viral quantification by qPCR were determined as indicative of antiviral activity. Our data show that myricetin rhamnoside (MyrG), myricetin-3-α-O-ramnosil (1→6)-α-galactoside (MyrGG), 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (PMF), 5-hydroxy-3,6,7,3',4'pentamethoxyflavone (PMF-OH) had antiviral activity without cytotoxic effects. The methoxyflavones PMF and PMF-OH were the most active compounds, showing an antiviral effect against all the evaluated viruses. Computational studies showed that these compounds could interact with the Reverse Transcriptase. Altogether, these results suggest that the flavonoids (related to myricetin and methoxyflavones) are the main antiviral compounds present in the aerial parts of Marcetia taxifolia. Furthermore, our results showed that the methoxyflavones have a broad antiviral activity, which represents an opportunity to evaluate these flavonoids as lead molecules to develop new antiviral compounds.

Equine influenza virus (EIV) causes severe acute respiratory disease in horses. Currently, the strains belonging to the H3N8 subtype are divided into two clades, Florida clade 1 (FC1) and Florida clade 2 (FC2), which emerged in 2002. Both FC1 and FC2 clades were reported in Asian and Middle East countries in the last decade. In this study, we described the evolution, epidemiology, and molecular characteristic of the EIV lineages, with focus on those detected in Asia from 2007 to 2017. The full genome phylogeny showed that FC1 and FC2 constituted separate and divergent lineages, without evidence of reassortment between the clades. While FC1 evolved as a single lineage, FC2 showed a divergent event around 2004 giving rise to two well-supported and coexisting sublineages, European and Asian. Furthermore, two different spread patterns of EIV in Asian countries were identified. The FC1 outbreaks were caused by independent introductions of EIV from the Americas, with the Asian isolates genetically similar to the contemporary American lineages. On the other hand, the FC2 strains detected in Asian mainland countries conformed to an autochthonous monophyletic group with a common ancestor dated in 2006 and showed evidence of an endemic circulation in a local host. Characteristic aminoacidic signature patterns were detected in all viral proteins in both Asian-FC1 and FC2 populations. Several changes were located at the top of the HA1 protein, inside or near antigenic sites. Further studies are needed to assess the potential impact of these antigenic changes in vaccination programs.IMPORTANCE The complex and continuous antigenic evolution of equine influenza viruses (EIVs) remains a major hurdle for vaccine development and the design of effective immunization programs. The present study provides a comprehensive analysis showing the EIV evolutionary dynamics, including the spread and circulation within the Asian continent and its relationship to global EIV populations over a 10-year period. Moreover, we provide a better understanding of EIV molecular evolution in Asian countries and its consequences on the antigenicity. The study underscores the association between the global horse movement and the circulation of EIV in this region. Understanding EIV evolution is imperative in order to mitigate the risk of outbreaks affecting the horse industry and to help with the selection of the viral strains to be included in the formulation of future vaccines.

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.

Hepatitis B virus (HBV) infection is a leading cause of severe chronic liver disease worldwide. The HBV epidemiology in Latin American countries is complex and the data is still scanty and fragmentary. The aim of this study was to investigate the distribution of HBV genotypes in Paraguay and to estimate the viral population dynamic and spread pattern of the main phylogenetic group. To this end, partial and complete genome sequences were obtained from 60 blood donor candidates and analysed by phylogenetic and Bayesian phylodynamic approaches. The phylogenetic analysis based on sequences of partial Polymerase/Pre-S1 overlapping region showed a predominance of the Native American subgenotype F4 (81.7%), the presence of the European subgenotypes A2 (1.7%) and D3 (8.3%), the African subgenotype A1 (3, 5%) and the Asian subgenotypes B2 (1.7%) and C2 (1.7%). The distribution of HBV genotypes was in accordance with the ethnic composition of the population. The phylogeographic analysis of subgenotype F4 complete genomes suggests that this lineage emerged and spread in the last 300 years. Paraguay was the most probable location of the common ancestor. The lineage diverged into two main clades and spread to neighbor regions, mainly Bolivia and Northwest Argentina, and Buenos Aires. The phylogeny showed a scanty geographical structure and a complex migratory pattern. In conclusion, the HBV genotypes circulating in Paraguay reflect the ethnic origin of the population. The distribution of genotypes and the phylogeographic reconstruction showed the impact of both global and local migrations in shaping the HBV molecular epidemiology in the region.

BACKGROUND:The use of human and viral genetic markers offers a novel way to study human migration in multiethnic populations of Latin America.OBJECTIVES:Our goal was to characterize the genetic diversity and geographical origins of JC Polyomavirus (JCPyV) and the genetic ancestry of mitochondrial DNA (mtDNA) in inhabitants from 25 de Mayo, Misiones-Argentina, a small village of largely German ancestry located close to the border with Brazil. We also evaluated the extent of agreement between viral and mtDNA markers for the different ancestry components of this population.STUDY DESIGN:68 individuals were analyzed for JCPyV and mtDNA diversity. JCPyV detection and typing was conducted in urine samples by PCR amplification, sequencing and phylogenetic analysis of the VP1 gene. mtDNA ancestry was assessed through HVS1 sequencing, with the resulting haplotypes being classified into haplogroups of Amerindian, European and African origin. The distribution of JCPyV diversity and mtDNA ancestry in the population was statistically evaluated by Fisher exact test and the level of agreement of both markers at the individual level was evaluated by Cohen's kappa coefficient.RESULTS:Our analysis showed that 57.4% of the samples were positive for JCPyV. Of these, the 47.6% were Asian-American Type 2, 33.3% European Type 1 and 19.1% African Type 3 in origin. The mtDNA ancestry of the study participants was 33.3% Amerindian and 66.7% European. There was a significant difference among the distribution of JCPyV diversity and mtDNA ancestry (p = 0.009) and at the individual level there was no correlation between the distribution of the both markers (κ = 0.154, p = 0.297).CONCLUSION:The apparent incongruence between JCPyV diversity and mtDNA ancestry may reflect the original settlement process and more recent migration to 25 de Mayo, the latter involving viral spread through migrants from Brazil. Some potential limitations to our interpretations are also discussed.

In the context of pathogenesis of HBV infection, HBV genotypes and mutants have been shown to affect the natural course of chronic infection and treatment outcomes. In this work, we studied the induction of apoptosis by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. Both subgenotypes F1b and F4 HBV genome transfections induced cell death by apoptosis in human hepatocytes. The BCPdm (A1762T/G1764A) and preCore (G1896A) mutants induced higher levels of apoptosis than the wt virus. This increase in apoptosis was not associated with the enhanced viral replication of the variants. HBV-mediated apoptosis was independent of viral subgenotypes, and associated with the modulation of members of the regulatory Bcl-2 family proteins expression in the mitochondrial apoptotic pathway. Finally, the apoptosis induction increase observed for the preCore mutants suggests that HBeAg might have an anti-apoptotic effect in human hepatocytes.

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.