Seroprevalence of dengue epidemic in Southern India during June-August 2013 Nageswar Reddy , Ranjeet Dungdung, Rajendra Pilankatta Central University of Kerala, School of Biological Sciences, Department of Biochemistry and Molecular Biology, Reverside transit campus, Padnakkad, Nileshwar -671328, Kerala, India. Abstract Dengue virus belongs to the family Flaviviridae, having four serotypes and transmitted by mosquito bites. Dengue infection causes severe mortality and morbidity, affecting mostly tropical and subtropical regions causing 500,000 death cases annually. The co-circulation of all four dengue serotypes were reported in Delhi during 2003 epidemic, which may have enhanced the DHF/DSS cases. Also, the co-circulation of multi serotypes of dengue virus has resulted in the concurrent infection of dengue virus in some patients during 2006 epidemic 1 in Delhi followed by 2008 epidemic 2 in Ernakulam. The present study was aimed to detect and identifying dengue virus isolates and concurrent dengue infection enhancement in southern part of India especially Kerala state during June - August in the year 2013. Total of 20 dengue clinical serum samples were analysed for serotyping and to find out concurrent infections among individuals. The serum samples were collected from the suspected dengue patients through various diagnostic centres and confirmed by presence of dengue virus NS1 and anti dengue IgG and IgM antibody detection by ELISA. Viral RNA was extracted from the positive serum samples and cDNA was prepared by subjecting to Reverse transcriptase PCR (RT-PCR). PCR product of 511-bp was amplified using D1 and D2 primers 3 . Unique target sequence was amplified by sero type-specific primers in a 2 nd step nested PCR. Out of 20 samples, 9 were positive for dengue viral genome and all of them showed concurrent infections with more than one serotypes.The data shows an enhancement in the concurrent infections among individuals in comparison with previous epidemic in India. Three samples shows novel combinations of concurrent infections with all four serotype of dengue viruses and one of the sample gives additional band of 300bp along with 511 bps in a 1 st step of PCR amplification. Further studies carried out by sequencing PCR products for genetic analysis of the virus fitness which are found to be co- circulating. The data is highly useful to evolve strategies to combat against DHF/DSS caused by dengue infection and well as viral evolutionary studies. Methods Results Conclusion Acknowledgement References 1. Bharaj P, Chahar HS, Pandey A, Diddi K, Dar L, Guleria R, Kabra SK, Broor S. Concurrent infections by all four dengue virus serotypes during an outbreak of dengue in 2006 in Delhi, India. Virol J. 2008 Jan 9;5:1. 2. Anoop.M, Issac A, Mathew T, Philip S, Kareem NA, Unnikrishnan R, etal. Genetic characterization of dengue virus serotypes causing concurrent infection in an outbreak in Ernakulam, Kerala, South india. Indian J Exp Biol 2010; 48:849-57. 3. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol. 1992 Mar;30(3):545-51. 4. Gupta N, Srivastava S, Jain A, Chaturvedi UC. Dengue in India. Indian J Med Res. 2012 Sep;136(3):373-90. Review Fig.1: Agarose gel image of the PCR products amplified by RT-PCR using viral RNA as a template isolated from dengue infected clinical samples of 1-10. Enhancement in dengue concurrent infections with multiple serotypes were observed in Kerala during June-August 2013 in comparison to the year 2006 epidemic in Delhi. Out of 9 positive samples, three samples shows concurrent infection of all DENV1, DENV2, DENV3 and DENV4 and found to be a novel combination. Sequence analysis shows that the gene fragment of 511bp is having identity of 99% with the DENV-3 serotype by NCBI blast analysis. Sample 3 shows one additional band of 300bps along with 511bps in a duplex PCR amplification. Concurrent infection of unreported novel combination of DENV2, DENV3 and DENV4 was observed in Kerala region. UGC-BSR start up grant 2012 Detection of NS1 antigen in clinical samples by the DENV NS1 antigen capture ELISA Detection of dengue virus-specific IgM and IgG antibodies Isolation of dengue viral RNA Serum collection Synthesis of cDNA by RT Sequence and NCBI blast analysis Amplification of 511bp using D1 and D2 primers by 1 st step of duplex PCR Amplification of type specific gene products using nested PCR in the 2 nd step. Fig. 2 : Agarose gel image of the nested PCR products amplified by serotype specific primers with D1&TS1, D1&TS2, D1&TS3, D1&TS4 primer combinations using duplex PCR product as a template. 2A,3A,4A,5A,7A,8A: Sero typing profile of sample 2,3,4,5,7 and 8 respectively. M 11 12 13 14 15 M 16 17 18 19 20 Fig. 3: Agarose gel image of the PCR products amplified by RT-PCR using viral RNA as a template isolated from dengue infected clinical samples of 11-20. M 1 2 3 4 M 5 6 7 8 9 10 2A 3 A 4A 5A 7A 8A 11A 14A 1 6A Fig.6 Graph representing enhanced concurrent infections during 2006,2010,2013 in Delhi, Ernakulam and Kasargod respectively. Table 1: Concurrent infection of Dengue serotypes in an outbreak in and around Kasargod, Kerala in 2013 epidemic . Tot No. NS1 + RNA + % Single Concurrent DV 1 and DV 2 DV2 and DV3 DV1 DV2 DV3 DV2 DV3 DV4 DV1 DV2 DV3 DV4 20 14 9 64 NIL ALL NIL 1 4 1 3 Fig. 4 : Agarose gel image of the nested PCR products amplified by serotype specific primers with D1&TS1, D1&TS2, D1&TS3, D1&TS4 primer combinations using duplex PCR product as a template. 11A,14A,16A: Sero typing profile of sample 11,14 and 16 respectively. 0 20 40 60 80 100 2006 2010 2013 % Concurrent infection Year Enhancement of Dengue concurrent infections during 2006-2013 500bp Fig.5: Sequence chromatogram of 511bp fragment of sample 2. View publication stats View publication stats