Genotyping analyses of CprM gene sequences of DENV-1 serotype isolates (n = 9) 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 CprM gene sequences of DENV-3 serotype isolates (n = 17) 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

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

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

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

Demographics and clinical parameters of patients infected with DENV with different serotypes.

<p>Demographics and clinical parameters of patients infected with DENV with different serotypes.</p

Nucleotide and amino acid diversity in complete genome between (A) clades within genotype I and (B) across genotypes of DENV-4 viruses.

<p>Pairwise distances and standard errors of nucleotide and amino acid diversity are displayed in lower-left and upper-right matrix respectively.</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