Maximum Likelihood Tree for the NS5B region obtained using GTR+Γ+I as a model of nucleotide substitutions. Gray circles

<p>: Samples from Mar del Plata (Buenos Aires, Argentina). The reference samples were named after their Genotype and Accession Number. The number above/below the branches represents the bootstrap value (over 1000 pseudo-replica). The relevant groups were highlighted with parentheses depicting the genotype and the bootstrap value (<b>BV</b>) of the genotype's clade.</p

Molecular Survey of Hepatitis C Virus in the Touristic City of Mar Del Plata, Argentina

<div><p>The global epidemiology of Hepatitis C Virus (HCV) may be roughly described by two groups of genotypes: the worldwide distributed ones (subtypes 1a, 1b, 2a and 3a, among others) and the endemic ones (subtypes 4a, 5a, 6a, among others). Epidemiological and population dynamic studies of the worldwide distributed genotypes have shown that subtypes 1a and 3a are common among intravenous drug users (IDUs) and that they are also in expansion in some countries. The molecular survey of HCV provides some clues about the epidemiological status of the infections in a local scale and the phylogenetic and demographic reconstruction analyses complement this study by inferring whether the infections of certain subtypes are in a steady state or expanding. Here, a molecular survey of the HCV variants that circulate in the touristic city of Mar del Plata (Buenos Aires, Argentina) was performed in samples obtained from 42 patients. The subtypes detected were 1a (32 patients), 3a (8 patients) and 1b (2 patients). The demographic history of subtype 1a inferred using the sequence data showed an exponential growth in the 1990′s. The period of viral expansion was delayed compared with that observed for the same genotype in other countries where the transmission was associated with IDUs. Also, the phylogeographic analysis of HCV-1a showed a statistically significant association between the location of the samples and the phylogeny, which may be the result of the local transmission of HCV in the city. The molecular analysis helped in the description of the complex epidemiological context of a touristic city, and pointed out that some sanitary measures should be taken in order to reduce the transmission of HCV (and maybe of HIV) among IDUs.</p> </div

Maximum Likelihood Tree for the NS5B region in the context of the most similar sequences deposited at Genbank. A.

<p>Tree obtained for HCV-1a sequences (GTR+Γ+I). <b>B.</b> Tree obtained for HCV-3a (TrN+Γ+I). <b>C.</b> Tree obtained for HCV-1b (HKY+Γ+I). <b>Gray circles</b>: samples from Mar del Plata. The rest of the samples were the most similar sequences (obtained by BLAST of each sequence from Mar del Plata) named after their GenBank accession number, following the three-letter ISO code of the country: <b>ARG</b>: Argentina; <b>AUS</b>: Australia; <b>BRA</b>: Brazil; <b>CAN</b>: Canada; <b>CHE</b>: Switzerland; <b>CYP</b>: Cyprus; <b>DEU</b>: Germany; <b>ESP</b>: Spain; <b>FRA</b>: France; <b>GBR</b>: the United Kingdom; <b>GRC</b>: Greece; <b>IND</b>: Indonesia; <b>IRL</b>: Ireland; <b>MEX</b>: Mexico; <b>MTQ</b>: Martinique; <b>PHL</b>: the Philippines; <b>PRT</b>: Portugal; <b>TUN</b>: Tunisia; <b>USA</b>: the United States of America. The numbers either above or below the branches are the bootstrap value (over 100 pseudo-replica for HCV-1a, and 1000 pseudo-replica for HCV-3a and HCV-1b).</p

Results of Bayesian Tip-association Significance testing (BaTS).

<p>Association Index (<b>AI</b>) and Parsimony Score (<b>PS</b>) test the global association between a trait and tree topology, taking into account the level of uncertainty in the phylogenetic reconstruction. The Monophyletic Clade (<b>MC</b>) index tests which traits are associated with phylogeny. The observed mean and its associated 95% confidence intervals (<b>Upper and Lower CI</b>) were obtained by analyzing trees sampled during the Bayesian phylogenetic reconstruction. The null mean and its associated confidence intervals were obtained after randomly distributing the traits in the phylogeny (100 replica). The significance level is the <b>p</b> value for the statistical hypothesis test for equality between the index observed and that expected under no association. Each state was defined following the three-letter ISO code of the country where the sample was collected (see <i>Phylogeography</i>).</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

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

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

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