HCV NS2 induces nuclear foci formation of phosphorylated Chk2.

<p>(A) Confocal microscopy was performed for Chk2 localization in HepG2 cells stably expressing vector control or HCV NS2. HepG2 cells were treated with 15 µM cisplatin for 6 hrs and used as a positive control. Chk2 was stained with specific monoclonal antibody (green color) and nuclei were stained with DAPI (blue color). Merged photographs were shown in right panel. (B) Confocal microscopy was performed for phospho-Chk2 (Thr 68) localization in vector control and cisplatin treated HepG2 cells or HepG2 cells transfected with HCV NS2. Immunofluorescence images displayed the expression of HCV NS2 (green color) and phospho-Chk2 (red color) in control, cisplatin treated and HCV NS2 transfected HepG2 cells. The nuclei (blue color) were stained with DAPI. Merged photographs (right panel) were superimposed digitally for fine comparison.</p

Generation of stable cell lines expressing HCV NS2 protein.

<p>(A) HepG2 cells are stably transfected with HCV-NS2 or HCV-FL. Immunofluorescence images displayed the expression of HCV NS2 (green color) in stably transfected HepG2 cells with HCV NS2 and HCV-FL. The nuclei (blue color) were stained with DAPI (4′, 6′-diamidino-2-phenylindole). Merged photographs are shown in right panel. (B) Cell lysates were prepared from HepG2 cells stably expressing HCV NS2 and HCV-FL. Protein expression of NS2 were analyzed by Western blot using specific antibodies. The blot was reprobed with actin for a comparison of protein load.</p

HCV NS2 promotes cellular proliferation.

<p>(A) Cell proliferation was measured in control, HCV NS2 and HCV-FL stably transfected HepG2 cells by trypan blue exclusion method. The number of viable cells was counted at day 2 and day 4. The results are presented as means of three different experiments with standard errors. (B) Cell lysates prepared from HCV NS2 or HCV-FL stably transfected HepG2 cells were analyzed by western blot for the expression of cyclin E and p27 with respect to control vector transfected cells using specific antibody. The blot was reprobed with actin for comparison of equal protein load.</p

HCV NS2 does not activate p53 or its downstream molecule.

<p>(A) p53 expression was examined in HepG2 cells stably expressing HCV NS2 or HCV-FL and compared with control vector transfected cells using monoclonal antibody to p53. The blot was reprobed with an antibody to actin for comparison of equal protein load. (B) Immunofluorescence staining using a polyclonal antibody to p53 exhibits nuclear localization of p53 (red color) in mock-transfected cells (top row) or perinuclear localization of p53 in HCV NS2 (middle row) or HCV-FL (bottom row) transfected cells. HCV NS2 was stained with a monoclonal antibody (green color). The nuclei were stained with DAPI (blue color). Fluorescence images of left and middle panels were superimposed digitally for fine comparison (right panel). (C) p21 expression was examined in HepG2 cells stably expressing HCV NS2 or HCV-FL and compared with control vector transfected cells using monoclonal antibody to p21. The blot was reprobed with actin for comparison of equal protein load.</p

HCV NS2 activates Chk2 in hepatocytes.

<p>(A) Cell lysates were prepared from HepG2 cells stably expressing HCV NS2 and HCV NS5A. Protein expression of phospho-Chk2 (Thr-68), total Chk2, HCV proteins were analyzed by Western blot using specific antibodies. The blot was reprobed with actin for a comparison of protein load. (B) Cell lysates were prepared from HepG2 cells stably transfected with vector control or HCV-FL. Protein expression of phospho-Chk2, and total Chk2 were analyzed by Western blot using specific antibodies. The blot was reprobed with actin for a comparison of protein load. (C) Cell lysates were analyzed from mock or 15 µM cisplatin treated HepG2 cells, and protein expression of phospho-Chk2 and total Chk2 were analyzed by Western blot using specific antibodies. The blot was reprobed with actin for a comparison of protein load. (D) Cell lysates were prepared from HepG2 cells expressing HCV NS2 and HCV-FL. Protein expression of phospho-H2AX (Ser-139) was analyzed by Western blot using specific antibody. UV treated cell lysates were used as positive control. The blot was reprobed with actin for a comparison of protein load.</p

Genotyping analyses of CprM gene sequences of DENV-2 serotype isolates (n = 26) from Pune.

<p>Each strain is indicated by Genbank accession number followed by country and year of isolation. Numbers at the nodes are support values for the major branches (bootstrap; 1000 replicates). The sequences obtained in this study are marked in filled colored circles. Scale bar indicates number of base substitutions per site.</p

Genotyping analyses of complete gene sequences of DENV-4 serotype isolates (n = 3) from Pune.

<p>Each strain is indicated by Genbank accession number followed by country and year of isolation. 141 Genotype II sequences obtained from Genbank are shown here as compressed tree (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192672#pone.0192672.s001" target="_blank">S1 Text</a> provides the accession numbers). Numbers at the nodes are support values for the major branches (bootstrap; 1000 replicates). The sequences obtained in this study are marked in filled colored circles. Scale bar indicates number of base substitutions per site.</p

Nucleotide diversity in CprM region between (A) clades within genotype I and (B) across genotypes of DENV-4 viruses.

<p>Nucleotide diversity in CprM region between (A) clades within genotype I and (B) across genotypes of DENV-4 viruses.</p

Phylogenetic analyses of CprM gene sequences from 64 DENV positive cases for serotype determination.

<p>Each strain is identified by Genbank accession number followed by country and year of isolation. Numbers at the nodes are support values for the major branches (bootstrap; 1000 replicates). The sequences obtained in this study are marked in filled colored circles. Scale bar indicates number of base substitutions per site.</p

Co-circulation of all the four dengue virus serotypes and detection of a novel clade of DENV-4 (genotype I) virus in Pune, India during 2016 season

<div><p>Dengue is the most common mosquito-borne viral infection in tropical and sub-tropical countries. In recent years, India has reported increased incidences of concurrent infection with multiple serotypes of dengue viruses (DENV). In the present study, we have characterized DENV circulating during a single season of 2016 in Pune, India. A total of 64 serum samples from NS1 ELISA positive dengue patients were used for PCR amplification of CprM region of the viral genome and sequencing. Phylogenetic analysis documented circulation of all the four DENV serotypes with predominance of DENV-2 (40.6%). DENV genotyping classified DENV-1 to Genotype V, DENV-2 to Genotype IV, DENV-3 to Genotype III and DENV-4 to Genotype I. Further analysis revealed emergence of a novel clade (D) of genotype I of DENV-4. Subsequent isolation of three DENV-4 viruses in cell culture followed by complete genome sequence analysis confirmed this observation. Additionally, a new genotype within serotype-4 with >6.7% sequence variation from other genotypes was identified. This first report of significant co-circulation of all the four serotypes in a single outbreak in Pune reconfirms need for molecular monitoring of DENV.</p></div