Gel Extraction Kit (Qiagen, Germany). The amplicons were sequenced in both forward and reverse directions by commercial sequencing services (Xceleris Labs, India). The identity of the sequences were checked and confirmed by BLAST. The forward and reverse sequences were aligned and manually edited in Genedoc (v2.7.000) software to obtain the consensus sequence. The sequences obtained in the present study and other sequences retrieved from the GenBank, were aligned in ClustalW (2.1). Phylogenetic trees were constructed using Maximum Likelihood method in MEGA 6.06 software. Genetic distances were calculated using Tamura- Nei model of nucleotide substitution. The robustness of the tree was assessed with 1000 bootstrap replicates. Nauru strain (GenBank Accession Number: U88535) was used as prototype strain for DENV-1, NGC 44 strain (GenBank Accession Number: AF038403) for DENV-2 and H87 strain (GenBank Accession Number: M93130) for DENV-3.
A total of 378 acute phase blood samples were collected from suspected dengue patients attending the Out Patient Department (OPD) of Dr. M. A. Ansari Health Centre, Jamia Millia Islamia from 29 August, 2013 to 19 November, 2013. Patients included in the study showed symptoms of fever, headache, body-ache, nausea, vomiting and weakness. Information regarding age was available for 326 patients, regarding sex for 334 patients and regarding duration of illness for 315 patients. The duration of acute phase of illness ranged from 1-15 days (mean ± SD: 3.4± 2.4). The mean age of patients was 20.68 years (SD ± 12.3years) with male to female ratio of 1.8: 1. Dengue virus specific RT-PCR was conducted on all samples. DENV was detected in 269 (71.16%) samples out of 378 samples tested. The male to female ratio among the dengue virus positive samples was 1.59:1. DENV-2 was detected in highest number of patients (232, 86.24%) followed by DENV-1 (52, 19.4%) and DENV-3 (23, 8.58%) among the DENV positive samples. Co-infection with more than one DENV serotype was detected in 37 (13.75%) samples. Majority of concurrent infection samples were co-infected by DENV-1 and DENV-2 (n=26). Ten samples were co-infected by DENV-2 and DENV-3 and one sample was co-infected by DENV-1 and DENV-3. Singleplex nested PCR reactions with D1 and one of the type specific primers (as applicable) were carried out for all the concurrent infection samples and single specific bands were visualized on agarose gel.
Molecular characterization of DENV strains detected in the 2013 outbreak was also carried out by sequencing domain III of the envelope protein gene. E gene fragments of 10 DENV-1, 12 DENV-2 and 7 DENV-3 strains were sequenced in the present study. The sequences were deposited in the GenBank database with following accession numbers; GenBank: KJ729163-KJ729172 (DENV-1 strains) GenBank: KJ729151-KJ729162 (DENV-2 strains) and GenBank: KJ729173-KJ729179 (DENV-3 strains). Phylogenetic analysis of the DENV strains was carried out based upon the sequenced region. A 353 bp (117 amino acid) region of envelope genes of 46 DENV -1 strains including 10 DENV-1 strains sequenced in the present study were aligned. The aligned region spanned 1921-2273 bp of DENV-1 genome, 993-1345 bp of the E protein gene and 332-448 amino acid of the E protein (numbering based on the prototype). These strains grouped within the American African Genotype (Fig.1). Six mutations i.e. PheE339Ile, SerE341Thr, AlaE371Thr, Val E382Ile, Ile E438Val, Ile E441Val were detected in the study strains in comparison to the prototype strain. All these mutations have been previously reported. These study sequences showed nucleotide distance of up to 0.3% amongst themselves while amino acid sequences were identical. The study sequences showed nucleotide distance of 11.2-11.6% and amino acid distance of 5.3 % when compared to the prototype.
For DENV-2 strains, a 449bp (149aa) region of E gene of 45 strains including 12 strains sequenced in the present study was aligned. The region corresponded to 1801-2249 bp of the DENV-2 genome, 865-1313 bp of the E protein gene and 289-4