SOJ Microbiology & Infectious Diseases

The metropolitan city of Delhi is gradually heading towards the endemicity for dengue "exhibiting sudden ups and downs" of different serotypes/genotypes of Dengue Virus (DENV). In previous outbreaks, shift in dominance of circulating serotype towards the emergence of a new serotype as the dominant strain has been observed. A major shift in dominance of DENV-2 towards DENV-3 was witnessed during 2003 DF outbreak. Surprisingly yet another shift was recorded with emergence of DENV-1 in 2006 which subsequently remained in circulation at low key until the 2010 DF outbreak. This study was conducted to detect circulating serotype and genotype of DENV in Delhi during the post-monsoon period of 2012. Reverse Transcription Polymerase Chain Reaction (RT-PCR) and sequencing analysis of Capsid/Premembrane CprM gene region confirmed 16.12 % samples of DENV-1 (genotype III), 38.70 % samples of DENV-2 (genotype IV) and 45.16% samples of DENV-3 (genotype III).The result indicated dominance shift of DENV-1, which was the causative agent of 2010 dengue outbreak, toward re-emergence and co-dominant circulation of erstwhile DENV-2 (genotype IV) and DENV-3 (genotype III). This dominance shift can be imperative for dengue epidemiology as both DENV-2 and DENV-3 have been associated with major Dengue Haemorrhagic Fever/Dengue Fever (DHF/DF) outbreaks previously, and any of these (DENV-2 and DENV-3) serotype can be the cause of dengue outbreak in future.

Keywords: Dengue fever; Dengue hemorrhagic fever; Molecular epidemiology; Outbreaks

Dengue is one of the most rapidly spreading arboviral disease, transmitted by the mosquito vector Aedes aegypti. During the last five decades, up to a 30-fold rise in dengue incidence has been reported with increased geographic expansion of the disease [1]. An estimated 50-100 million dengue infections are reported annually and approximately 2.5 billion people worldwide live in dengue endemic areas [2]. Nearly 1.8 billion people at the risk of dengue infection live in member states of the WHO South East Asia and Western Pacific Regions, which bear approximately 75% of the dengue global disease burden [1]. India, one of the member states of the WHO South East Asia region has reported many dengue outbreaks since the first major one, in 1963 in Kolkata [3-5]. Dengue Virus (DENV) infections are caused by four antigenically distinct serotypes (DENV-1, 2, 3 and 4). These serotypes are further classified into distinct genotypes. Specific genotypes have been associated with higher outbreak potential and disease severity [6,7]. Beneath the level of genotype, lineages of the virus also exist, which are known to play an important role in the evolutionary dynamics and epidemiology of DENV [8].

Delhi, the capital city, situated in northern India is endemic for co-circulation of different dengue serotypes and genotypes [9]. Since the largest outbreak of Dengue Hemorrhagic Fever (DHF) in 1996, Delhi has reported periodic Dengue Fever (DF) outbreaks in 2003, 2006 and 2010 [10-12]. In Delhi, the trends of the last decade indicate unusual ups and downs of different serotypes/genotypes of DENV. The present study was conducted to detect the dominant serotype(s) and genotype(s) of DENV circulating in Delhi during the post- monsoon season in the year 2012.

Acute phase blood samples of dengue suspected patients with fever of less than 5 days, and prominent clinical symptoms such as headache, myalgia and rash were included in the study. Samples were referred to the National Centre for Disease Control (NCDC) from different locations in Delhi during the post-monsoon season of the year 2012. Approval of ethical committee of the institute was obtained to carry out the present study. "Waiver of consent” was granted by the committee on the basis of "Use of left over specimen after clinical investigation" under the Indian Council of Medical Research Guidelines 2006.

Serum was separated from blood specimens by centrifugation and RNA was extracted from the serum by using QIAamp Viral RNA Mini kit (Qiagen, Germany) as per the manufacturer's protocol. For detection of DENV, reverse transcription polymerase chain reaction (RT-PCR) was performed using Capsid/Premembrane (CprM) gene junction specific primers D1 (5'-TCAATATGCTGAAACGCGCGAGAAACCG-3') and D2 (5'-TTGCACCAACATCAATGTCTTCAGTTC-3') [13]. RT-PCR was carried out, using the Access Quick one-step RT - PCR kit (Promega, USA) in ABI 9700 Thermal cycler (Applied Biosystems, USA). Reverse transcription was performed at 42°C for 45 min followed by initial denaturation at 95°C for 2 min and 35 cycles of denaturation (95°C for 1 min), annealing (55°C for 1 min) and extension (72°C for 2 min). Final extension was carried out at 72°C for 10 min. The PCR product of 511 bp was visualized on ethidium bromide stained 1.2 % agarose gel.

PCR product purification of positive samples was done using QIAquick PCR purification kit (Qiagen, Germany). For sequencing Big Dye Terminator cycle sequencing ready reaction kit v. 3.1 (Applied Biosystems, USA) was used on automatic DNA sequence (ABI 3130xl Genetic Analyzer, Applied Biosystems). After obtaining sequences, BLAST search (www.ncbi.nlm.nih. gov/Education/BLASTinfo/information3.html) was carried out to confirm type of DENV. Thus obtained sequences were submitted to GenBank (www.ncbi.nlm.nih.gov) to acquire accession numbers (Table 1).

After sequence comparison from different geographical locations (Table 1) CLUSTAL W based multiple sequences alignment was done using BioEdit v 7.0.5.3. For phylogenetic analysis and tree construction MEGA v 6.0 was used by employing "Neighbor-Joining" method with bootstrap analysis of 1000 replicates.

Out of 107 samples, 31 were found positive for CprM gene region by RT-PCR. Sequencing and further BLAST analysis confirmed 5, 12 and 14 samples of DENV-1, DENV-2 and DENV- 3 serotypes respectively. In the phylogenetic tree construction (Figure 1) sequences were found to be segregated in three separate groups, DENV-1 (genotype III), DENV-2 (genotype IV) and DENV-3 (genotype III). All 5 DENV-1 sequences (KJ438859, KJ438860, KJ438861, KJ438862 and KJ438863) grouped with Delhi isolates of 2010 and 2011 and formed a single lineage of DENV-1 (lineage I) which was found to be closely related with Comoros isolate (DQ285562). All 12 isolates of DENV-2 serotype were split into two lineages (lineage I and lineage II). Both the lineages were found to be closely related with GWL228 isolate (DQ448237) of India and FJ-10/11 isolates (AF276619 and AF359579) of China. Sequence comparison of all sequences of DENV-2 (lineage I and II) with GWL 228 isolate revealed average sequence similarity of 98.70%. While average sequence similarity with Delhi isolates (AF047394 and AF047396) of 1996 was found to be 98.57%. The estimate of mean evolutionary diversity (D) between the two lineages and within the lineages was found 1.45 and 0.75 respectively. Phylogeny of all DENV-3 sequences also revealed the presence of two lineages (lineage I and lineage II). Five of DENV-3 sequences (KJ451722, KJ451724, KJ451729 KJ451731 and KJ451734) were clustered with DENV isolates of Delhi from 2006 and 2007 (EU181211, EU846235 & EU846236), within lineage II while 9 sequences formed distinct clade (lineage I) in close proximity with Guangzhou and Yiwu isolates (GU363549, JN662391 and JF504679) of China. The estimate of mean evolutionary diversity (D) between the two lineages of DENV-3 (lineage I and lineage II) was 3.18 while within the lineages was 1.12. Sequence comparison of 5 DENV- 3 isolates (KJ451722, KJ451724, KJ451729, KJ451731 and KJ451734) of (lineage II) with Delhi isolates of 2007 (EU846235 and EU846236) revealed average sequence similarity of 99.28%, while the remaining isolates falling under lineage I were found to be 97.88% to 98.11% (average 98.08%) similar to same isolates (EU846235 and EU846236).

Dengue is emerging as one of the most important arboviral infections in India. Several parts of the country are endemic for dengue infection. During the last 2 decades Delhi has experienced several dengue outbreaks. High rates of Aedes infestation and climatic conditions (temperature and humidity) after the postmonsoon season are important contributing factors for the occurrence of dengue in Delhi. All four serotypes of DENV have been reported in Delhi at different times; however, the dominance of one serotype during a given outbreak has been observed. In the DHF outbreak of 1996, DENV-2 (genotype IV) emerged as the dominant serotype, which remained in circulation till 2002 [10- 14]. Emergence of DENV-3 (genotype III) was observed during a 2003 DF outbreak, which continued and dominated till 2006 [11,15]. However, in 2006 the emergence of DENV-1 (genotype III) was also observed and reported in nearly 30% of the cases [11]. In the following years, a continued presence and predominance of DENV-1 was reported, which resulted in the 2010 DF outbreak [12,16]. In our study, we confirmed the presence of DENV-1 in 16.12%, DENV-2 in 38.70% and DENV-3 in 45.16% of the samples inform the year 2012. These results suggest a shift from DENV-1 circulation towards the co-dominant circulation of both DENV- 2 and DENV-3. A recent study by another group of researchers has reported the dominance of DENV-2 in 2013 [17]. Possibly, this could be a further shift from co-dominance of DENV-2 and DENV-3 towards predominance of DENV-2. However, this might be further authenticated by additional studies involving larger geographical areas and a larger number of samples. The codominant re-emergence of DENV-2 and DENV-3 during 2012 is an important event for dengue epidemiology. In the coming years, any of these re-emerging serotypes can be the major circulating serotype in the population and may cause a new dengue outbreak. The phylogenetic analysis of DENV-2 and DENV-3 also revealed the presence of multiple lineages of DENV circulating and evolving in a parallel manner. Comparatively, the low sequence similarity (98.08%) of lineage I (DENV-3) with DENV-3 isolates from 2007 (lineage II) depicts the genetic variability accumulated over this period of time by the viruses. In a similar way, an average sequence similarity of 98.57% between DENV-2 (lineage I and II) and 1996 DENV-2 isolates (from Delhi) also indicates accumulation of genetic changes. However, the low divergence (d = 1.45) between the two lineages of DENV-2

suggests the need of further investigation for the genetic changes and sites under positive selective pressure in other gene regions of the virus.
Both, DENV-2 and DENV-3 have been the causative agents of major dengue outbreaks in the country, including Delhi [10,11,14,18]. The re-emergence and co-dominant circulation of DENV-2 and DENV-3 not only pose the risk of dengue outbreak but also the threat of increased disease severity as a large portion of population of Delhi may have been infected with DENV-1 during previous outbreak and the secondary dengue infection with different serotype may result in increased severity of the disease due to antibody dependent enhancement [19]. The evolvement and circulation of multiple lineages further complicates the problem as clinical manifestation such as encephalitis may be associated with new lineages with increased pathogenic potential. To manage dengue outbreaks and institute effective preventive and control measures, continuous surveillance, vector control and monitoring of dengue cases will be required, as this co-dominant circulation of DENV-2 and DENV-3 serotypes can emerge as a major public health problem.

The authors gratefully acknowledge the partial financial support from Indian Council of Medical Research during the course of this study.

1. World Health Organization. Dengue: guidelines for diagnosis, treatment, prevention and control. New edition. Geneva: WHO. 2009; pp. 3-4.
2. World Health Organization. Dengue and severe dengue. Factsheet N°117. [Accessed 2014 March 23]; Available from: http://www.who. int/mediacentre/factsheets/fs117/en/
3. Sarkar JK, Chatterjee SN, Chakravarty SK. Haemorrhagic fever in Calcutta: some epidemiological observations. Indian J Med Res. 1964; 52: 651-59.
4. Chatterjee SN, Chakravarti SK, Mitra AC, Sarkar JK. Virological investigation of cases with neurological complications during the outbreak of haemorrhagic fever in Calcutta. J Indian Med Assoc. 1965; 45(6):314-16.
5. Gore MM. Need for constant monitoring of dengue infections. Indian J Med Res. 2005; 121(1): 9-12.
6. Rico-Hesse R, Harrison LM, Salas RA, Tovar D, Nisalak A, Ramos C, et al. Origins of dengue type 2 viruses associated with increased pathogenicity in the Americas. Virology. 1997; 230(2): 244-51.
7. Leitmeyer KC, Vaughn DW, Watts DM, Salas R, Villalobos I, de C, et al. Dengue virus structural differences that correlate with pathogenesis. J Virol. 1999; 73(6):4738-47.
8. Patil JA, Cherian S, Walimbe AM, Patil BR, Sathe PS, Shah PS, et al. Evolutionary dynamics of the American African genotype of dengue type 1 virus in India (1962-2005). Infect Genet Evol. 2011; 11(6):1443-8. doi: 10.1016/j.meegid.2011.05.011.
9. Dar L, Gupta E, Narang P, Broor S. Cocirculation of dengue serotypes, Delhi, India, 2003. Emerg Infect Dis. 2006; 12(2): 352-53. doi:10.3201/ eid1202.050767.
10. Dash PK, Parida MM, Saxena P, Kumar M, Rai A, Pasha ST, et al. Emergence and continued circulation of dengue-2 (genotype IV) virus strains in northern India. J Med Virol. 2004; 74(2): 314-322. doi:10.1002/ jmv.20166.
11. Kukreti H, Chaudhary A, Rautela RS, Anand R, Mittal V, Chhabra M, et al. Emergence of an independent lineage of dengue virus type 1 (DENV-1) and its co-circulation with predominant DENV-3 during the 2006 dengue fever outbreak in Delhi. Int J Infect Dis. 2008; 12(5): 542-49. doi:10.1016/j. ijid.2008.02.009.
12. Singh P, Mittal V, Rizvi MM, Chhabra M, Sharma P, Rawat DS, et al. The first dominant co-circulation of both dengue and chikungunya viruses during the post-monsoon period of 2010 in Delhi, India. Epidemiol Infect. 2012; 140(7): 1337-42. doi:10.1017/S0950268811001671.
13. 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 ClinMicrobiol. 1992; 30(3): 545-51.
14. Singh UB, Maitra A, Broor S, Rai A, Pasha ST, Seth P. Partial nucleotide sequencing and molecular evolution of epidemic causing Dengue 2 strains. J Infect Dis. 1999; 180(4): 959-65.
15. Kumar M, Pasha ST, Mittal V, Rawat DS, Arya SC, Agarwal N, et al. Unusual emergence of guate98-like molecular genotype of DEN-3 during 2003 dengue outbreak in Delhi. Dengue Bulletin. 2004; 28: 457-461.
16. Chakravarti A, Chauhan MS, Kumar S, Ashraf A. Genotypic characterization of dengue virus strains circulating during 2007-2009 in New Delhi. Arch Virol. 2013; 158(3):571-81. doi:10.1007/s00705-012- 1522-5.
17. Afreen N, Deeba F, Naqvi I, Shareef M, Ahmed A, Broor S, et al. Molecular investigation of 2013 dengue fever outbreak from Delhi, India. PLOS Currents Outbreaks 2014; Edition 1. doi:10.1371/currents.outbreaks.041 1252a8b82aa933f6540abb54a855f.
18. Dash PK, Parida MM, Saxena P, Abhyankar A, Singh CP, Tewari KN, et al. Reemergence of dengue virus type-3 (subtype-III) in India: implications for increased incidence of DHF & DSS. Virol J. 2006; 3:55-64. doi:10.1186/1743-422X-3-55.
19. Halstead SB. Pathogenesis of dengue: challenges to molecular biology. Science. 1988; 239(4839): 476-81.