Detection of acidic vacuoles in cells transfected with full-length HBV genomes.

<p>Huh-7 cells were either starved or transfected with pUC19 empty vector (control), F1b wt, F1b BCPdm, F1b preCore, F4 wt, F4 BCPdm and F4 preCore variants. Forty-eight hours post-transfection, cells were fixed and stained with acridine orange (AO). AO was imaged through a 515/530-nm band-pass filter (green) or a 580-nm long-pass filter (red). (A) Representative images are shown. (B) Quantification of AO-red dots per cell. Shown values represent the mean ± standard deviation of three independent experiments. Statistically significant changes compared to control cells are indicated with asterisks above bars. *** <i>p</i> < 0.0001.</p

Detection of LC3 positive puncta in cells transfected with full-length HBV genomes.

<p>Huh-7 cells were either starved or transfected with pUC19 empty vector (control), F1b wt, F1b BCPdm, F1b preCore, F4 wt, F4 BCPdm and F4 preCore variants. Forty-eight hours post-transfection, cells were fixed and stained with anti-LC3 antibody. Nuclei were stained with DAPI, and distribution of LC3 protein was visualized with a fluorescence microscope. (A) Representative images are shown. (B) Quantification of LC3 positive puncta per cell. Shown values represent the mean ± standard deviation of three independent experiments. Statistically significant changes compared to control cells are indicated with asterisks above bars and statistically significant changes between the viral variants are indicated with asterisks above brackets. * <i>p</i> < 0.05; ** <i>p</i> < 0.005 and *** <i>p</i> < 0.0001.</p

Detection of autophagic vacuoles by transmission electron microscopy in cells transfected with full-length HBV genomes.

<p>(A) Representative transmission electron micrographs of Huh-7 cells transfected with pUC19 empty vector (control), F1b wt, F1b BCPdm, F1b preCore, F4 wt, F4 BCPdm and F4 preCore variants. Arrows indicate representative autophagic vacuoles. Inset: enlargement of autophagic vacuoles in the electron micrograph. (B) Quantification of autophagic vacuoles (autophagosomes and autolysosomes). Shown values represent the mean ± standard deviation of three independent experiments. Statistically significant changes compared to control cells are indicated with asterisks above bars and statistically significant changes between the viral variants are indicated with asterisks above brackets. *** <i>p</i> < 0.0001.</p

Detection of acidic vacuoles in cells transfected with full-length HBV genomes.

<p>Huh-7 cells were either starved or transfected with pUC19 empty vector (control), F1b wt, F1b BCPdm, F1b preCore, F4 wt, F4 BCPdm and F4 preCore variants. Forty-eight hours post-transfection, cells were fixed and stained with acridine orange (AO). AO was imaged through a 515/530-nm band-pass filter (green) or a 580-nm long-pass filter (red). (A) Representative images are shown. (B) Quantification of AO-red dots per cell. Shown values represent the mean ± standard deviation of three independent experiments. Statistically significant changes compared to control cells are indicated with asterisks above bars. *** <i>p</i> < 0.0001.</p

Inhibition of autophagic flux in cells transfected with full-length HBV genomes.

<p>(A) Huh-7 cells were either starved or transfected with pUC19 empty vector (control), F1b wt, F1b BCPdm, F1b preCore, F4 wt, F4 BCPdm and F4 preCore variants. Forty-eight hours post-transfection, cells were treated with or without CQ (100 μM) for 2 hours, total cell proteins were extracted and cellular levels of p62 were assessed by Western Blot. (B) Relative intensity of the bands was quantified by normalization to β-actin, using ImageJ software. Shown values represent the mean ± standard deviation of three independent experiments. Statistically significant changes compared to control cells are indicated with asterisks above bars and statistically significant changes between the viral variants and starved cells are indicated with asterisks above brackets. * <i>p</i> < 0.05; ** <i>p</i> < 0.005 and *** <i>p</i> < 0.0001.</p

Expression of the tandem protein mRFP-GFP-LC3 in cells transfected with full-length HBV genomes.

<p>Huh-7 cells transfected with pUC19 empty vector (control), F1b wt, F1b BCPdm, F1b preCore, F4 wt, F4 BCPdm and F4 preCore variants, starved or treated with CQ (100 μM) were transiently transfected with mRFP-GFP-LC3 expression plasmid. Forty-eight hours post-transfection, cells were fixed and imaged through a 515/530-nm band-pass filter (green) or a 580-nm long-pass filter (red). (A) Representative images are shown. (B) Quantification of yellow puncta (mRFP-GFP-LC3 positive) and red puncta (mRFP-LC3 positive) per cell. Shown values represent the mean ± standard deviation of three independent experiments. Statistically significant changes compared to control cells are indicated with asterisks above bars and statistically significant changes between the viral variants are indicated with asterisks above brackets. ** <i>p</i> < 0.005 and *** <i>p</i> < 0.0001.</p

Conversion of LC3 in cells transfected with full-length HBV genomes.

<p>(A) Huh-7 cells were either starved or transfected with pUC19 empty vector (control), F1b wt, F1b BCPdm, F1b preCore, F4 wt, F4 BCPdm and F4 preCore variants. Forty-eight hours post-transfection, total cell proteins were extracted and expression levels of LC3-II were determined by Western Blot. (B) Relative intensity of the bands was quantified by normalization to β-actin, using ImageJ software. Shown values represent the mean ± standard deviation of three independent experiments. Statistically significant changes compared to control cells are indicated with asterisks above bars and statistically significant changes between the viral variants are indicated with asterisks above brackets. * <i>p</i> < 0.05; ** <i>p</i> < 0.005 and *** <i>p</i> < 0.0001.</p

Analysis of the Molecular Evolution of Hepatitis B Virus Genotypes in Symptomatic Acute Infections in Argentina

<div><p>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.</p></div

MCCT for subgenotype F1b.

<p>Maximum clade credibility tree (MCCT) performed by calibration with the terminal nodes for complete genome of acute subgenotype F1b samples. The MCCT presents for each node plots of its correspondent posterior probability. Labels I_F1b-IV_F1b represent the clusters found in the circulating strains. The scale at the bottom of the tree represents time (years).</p

Subgenotype distribution of the acute isolates.

<p>Maximum-likelihood phylogenetic tree performed with the PhyML (v3.0) program constructed on the complete genome sequences including fifty-five sequences of symptomatic acute HBV isolates from the city of Buenos Aires (marked with ♦) and reference sequences retrieved from GenBank, indicated by their accession numbers. The numbers at each node correspond to bootstrap values obtained with 1000 replicates. The scale bar indicates the genetic distances.</p