avates to haemorrhage, plasma leakage and organ impairment9. A vaccine for dengue is not available yet as the virus exists as 4 serotypes which do not cross-protect and enhance the risk of severe disease by antibody dependent enhancement of infection10. There was an increase in the number of cases of dengue fever reported from Delhi and all over India in the year 201311. The present study was undertaken to characterize dengue viruses detected during the outbreak in Delhi in 2013.
Materials and Methods
The study was granted approval by Institutional Ethics Committee, Jamia Millia Islamia and was done in accordance with the World Medical Association Declaration of Helsinki. Acute phase blood samples were collected from suspected dengue patients from Dr. M. A. Ansari Health Centre, Jamia Millia Islamia, New Delhi. Dr. M. A. Ansari Health Centre is a small health centre situated in the University campus which provides basic medical facilities to approximately 40,000 university students, employees and their dependents. Patients showing symptoms of fever accompanied by any two of the following; nausea, vomiting, rash, aches and pains, positive tourniquet test, leukopenia, were enrolled in the study. Written informed consent in English and Hindi was obtained from all adult subjects or a parent or guardian in case of minors. The age/sex, clinical symptoms and duration of fever of the patients were recorded in proformas. Serum was separated from the blood samples and stored at-80°C in aliquots. Dengue viruses were detected in the serum samples by semi nested RT-PCR for C-prM region as described previously12. RNA was extracted from the serum samples using QIAamp Viral RNA Mini kit (Qiagen, Germany) according to the manufacturer’s instructions. Extracted RNA was aliquoted and stored at -80°C. RNA was reverse transcribed into complimentary DNA (cDNA) copy in a 25µL reverse transcription (RT) reaction mixture using 20ng of random primers (Promega, USA), 1mM dNTPs (Promega, USA), 8U of rRNAsin (Promega, USA) and 10U of Avian Myeloblastosis Virus reverse transcriptase (Bangalore Genei, India) and 13.75µL RNA. The RT reaction mixture was incubated at 37°C for 90 minutes followed by enzyme inactivation at 65°C for 15 minutes. First round (external) PCR was carried out in a 25µL reaction using 1X Taq buffer, 200µM dNTPs (Promega, USA), 0.4µM primers (D1 and D2)12, 1.5U of Taq DNA polymerase (Bangalore Genei, India) and 3 µL cDNA. The PCR was carried out as follows: 94°C for 2 minutes followed by 35 cycles of 94°C for 30s, 52°C for 30s and 72°C for 60s and a final extension at 72°C for 10 minutes. A 511bp segment specific to all 4 dengue serotypes was amplified in this external PCR.
External PCR was followed by semi nested PCR using D1 primer and 4 serotype-specific primers; TS1, TS2, TS3 and TS412. Nested PCR was carried out in a 25µL reaction using, 1X Taq buffer, 200µM dNTPs (Promega, USA), 0.4µM primers (D1, TS1, TS2, TS3 and TS4), 1.5U of Taq DNA polymerase (Bangalore Genei, India) and 1 µL of the diluted (1:5) external PCR product. The nested PCR was run as follows: 94°C for 1 minute followed by 25 cycles of 94°C for 30s, 54°C for 30s and 72°C for 60s and a final extension at 72°C for 10 minutes. The sizes of the nested PCR products were specific for each dengue serotype i.e. 482-bp for DENV-1, 119-bp for DENV-2, 290-bp, for DENV-3 and 392-bp for DENV-4. PCR products were electrophoresed through 2% agarose gel, stained with ethidium bromide and examined under ultraviolet light using a gel documentation system (Wealtec, USA). Singleplex nested PCR reactions with D1 and one of the type specific primers (as applicable) were carried out for confirmation of the co-infection of serotypes using same conditions as mentioned above.
Partial segments (448bp, 574bp and 582 bp) of envelope protein gene were amplified using published primers for DENV-113, DENV-214 and DENV-315 strains respectively. The respective amplicons were visualized on 2% agarose gel. The specific bands were cut from the gel and DNA was extracted from the gel using QIAquick 