37 amino acid of the E protein (numbering based on the prototype). The DENV- 2 strains of the present study clustered within the Cosmopolitan genotype (Fig. 2). The study strains showed nucleotide distance of up to 3% whereas amino acid sequences showed distances of up to 0.7%. Nucleotide distance of 5.8 to 6.3% and amino acid distance of 1.4-2% were identified when compared to the prototype strain. Two mutations, AsnE390Ser and IleE402Phe were detected in the study strains as compared to the prototype strain which are all previously reported.
Phylogenetic analysis of DENV-3 strains was carried out by aligning 485 bp (161 amino acid) region of E gene of 27 strains including 7 DENV-3 strains sequenced in the present study. The aligned region was from 1600 to 2084 bp of the DENV-3 genome, 666-1150 bp of the E protein gene and 223-383 amino acid of the E protein (numbering based on the prototype). The DENV- 3 strains of the present study grouped within the genotype III (Fig. 3). The DENV-3 strains showed nucleotide distances of up to 1.3% and amino acid distances of up to 0.6% among themselves. Nucleotide distance of 5.6 to 6.1% and amino acid distance of 3.2-3.8% were detected when compared to the prototype strain. Five mutations, all previously reported, were detected in the sequenced strains, LysE225Glu, ThrE270Asn, LysE291Glu, LeuE301Thr and LysE383Asn with reference to the prototype.
Both serotypes detected in co-infected samples could not be sequenced for the E gene. Therefore, three co-infected samples were sequenced for C-prM gene junction. Two of such samples were co-infected by DENV-1 and 2 and one sample was co-infected by DENV-2 and 3. The sequences were confirmed by BLAST. These sequences were submitted to the GenBank with accession numbers KM114683- KM114685. The DENV-2 sequences were <200 bp and hence were not accepted by the GenBank (available upon request). As described by Chinnawirotpisan et al.16, the sequences were subjected to a preliminary phylogenetic analysis including prototype strain of all four dengue virus serotypes. Phylogenetic trees showed their similarity to their prototypic strains and not to the prototypic strain of other three serotypes confirming their identity (Fig.4).
An outbreak of dengue fever occurred in Delhi after monsoon season in 2013. We have investigated the outbreak by conducting dengue virus specific RT-PCR on 378 suspected acute phase blood samples. Dengue viruses were detected in 71% of the samples confirming that the fever outbreak was caused by dengue viruses. The outbreak was mainly caused by DENV-2 serotype viruses. Dengue fever outbreaks are generally dominated by a particular serotype as reported from different parts of the world. DENV-2 dominated fever outbreaks were reported from Africa in 2007 and again in 201017. DENV-3 dominated outbreaks have been reported in 2013 in Solomon Islands18, in 2009-2010 from Nicaragua19 and in 2004 from Indonesia20. Dengue type 1 dominated outbreak was reported in 2001 from Myanmar21. A dengue 4 dominated outbreak was reported in 2009 in French Polynesia22.
Three dengue virus serotypes were detected in the present study. DENV-2 dominated the outbreak with DENV-1 and DENV-3 being detected in small number of samples. DENV-4 was not detected in our study. This is the first report of molecular detection of dengue viruses during 2013 dengue fever outbreak from Delhi, India. Chakravarti and colleagues23 had reported detection of all 4 serotypes of dengue during 2007-2009 in Delhi. Another recent study has reported a DENV-2 dominated outbreak in Orissa state of India in 2010-2011 in which 72% samples were detected positive by RT-PCR and IgM ELISA24. A DENV-2 dominated outbreak was earlier reported in the year 199625 in Delhi. All four serotypes were detected without any dominance by a particular serotype in 2003 outbreak in Delhi26 whereas DENV-3 was found to be the dominant serotype in the 2006 dengue outbreak27.The last dengue epidemic occurred in Delhi in 2010 in which 12.5% samples were detected positive for dengue viruses by RT-PCR and