Continued Circulation of DENV-2 (Genotype IV) in Delhi, India

Background: Dengue is one of the rapidly emerging arboviral infection in many parts of the world including India. The metropolitan city, Delhi is one of the worst affected areas by dengue. In the last two decades, it has serotype(s)/genotype(s) NS1 reverse transcription polymerase chain reaction (RT-PCR) partial nucleotide sequencing for capsid-premembrane (CprM) gene Results: Serotypic and genotypic analysis revealed cases of DENV-2 (genotype IV) 58.33%, DENV-3 (genotype III) 33.33% and DENV-1 (genotype III) 8.33%. Presence of DENV-2 (genotype IV) in majority of the cases (58.33%) indicated its pre-dominance. Multiple sequence alignment and phylogenetic analysis revealed the presence of a new strain of DENV-2 (genotype IV); Short Communication differentiated from pre-existing strain by the substitutions; Ala102Val in capsid and Ile49Val in premembrane protein. Conclusion: This study reports, continued circulation of DENV-2 (genotype IV) in 2014. The results also indicated circulation of a new strain of DENV-2, similar to Hyderabad isolate “1392” (JX475906), along with the pre-existing strain. It is proposed that, this strain has been introduced recently and circulating at low key in the capital.


INTRODUCTION
Dengue fever (DF), a mosquito borne arboviral infection is emerging as a major public health threat on a global scale. A recent study estimated one third contribution of India in global dengue infection [1,2]. The infection from dengue virus (DENV) can cause mild fever (DF) to severe disease conditions; dengue hemorrhagic fever (DHF) /dengue shock syndrome (DSS) [3]. DENV (genus Flavivirus and family Flaviviriadae) is enveloped, single stranded positive sense RNA of nearly 11 kb length. The genomic organization consists of three structural (capsid, C; membrane, M; and envelope, E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) genes flanked by non-coding region at 5' and 3' ends [4]. DENV circulate as four antigenically distinct serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). Serotypes are further classified into genetically related forms, termed as genotypes which may differ in their virulence and pathogenesis.
Delhi, the capital city is majorly affected area by dengue. The city has witnessed a continuous changing trend of circulating serotypes(s)/ genotype(s) during the last two decades [5]. The present study was undertaken to detect the circulating serotype(s)/genotype(s) in Delhi during the post-monsoon period of 2014. To achieve this partial molecular characterization of DENV was performed on the basis of capsidpremembrane (CprM) gene region.

Study Samples
Acute phase serum samples of suspected dengue patients with fever of less than 5 days and prominent clinical symptoms such as body aches, headache, myalgia and rash were included in the study. Samples were referred to the National Centre for Disease Control from different locations in Delhi during the postmonsoon period of 2014.

NS1 Antigen Detection Assay
For detection of DENV, NS1 antigen detection assay was carried out by DENV Detect NS1 ELISA kit (InBios, USA) according to the manufacturer's instruction. Optical density was read at 450 nm. Immune status ratio (ISR) was calculated from the ratio of optical density obtained from test sample and the mean optical density of the cut-off control. ISR ≥ 1 considered positive for NS1 antigen.

RNA Extraction
Viral RNA was isolated from 140 µl serum, by spin column technique according to the manufacturer's instructions. For this purpose QIAamp Viral RNA Mini kit (Qiagen, Germany) was used. RNA was eluted in 50 µl nuclease free water and stored at -80ºC.

Sequencing and Phylogenetic Analysis
Amplified PCR products were purified, using the QIAquick PCR purification kit (Qiagen, Germany). For sequencing BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems, USA) was applied. Purification of sequencing PCR product was done, using Centri-Sep™ Spin Column (Princeton Separations, USA). Purified products were lyophilized, subsequently reconstituted in HiDi Formamide (Applied Biosystems, USA) and loaded in 3130xl Genetic Analyzer, (Applied Biosystems, USA) for capillary electrophoresis. The sequences thus obtained were submitted to GenBank and accession numbers were acquired (

RESULTS AND DISCUSSION
Out of 112 samples, 12 were positive for NS1 antigen. The mean age of the samples (6 male and 6 female) was 22.05 years (SD ±13.51 years). The platelet count equal or less than 150,000 cells per mm 3 was observed in positive samples. All the 12 samples were amplified for the CprM gene region by RT-PCR. Amplified PCR products were sequenced and serotype was confirmed through BLAST analysis. Of the 12 samples 7 were DENV-2 (58.33%), four were DENV-3 (33.33%) and 1 was DENV-1 (8.33%).
In the phylogenetic tree (Fig. 1)   All the 4 serotypes of DENV circulate in Delhi. During outbreaks, many cases of mixed infection, with more than one serotype have also been documented [7][8][9]. But so far, only three serotypes have been reported as a main etiological agent in different outbreaks; DENV-1, DENV-2 and DENV-3. The last three outbreaks in 2013, 2010 and 2006 were predominated by DENV-2, DENV-1 and DENV-3 respectively [7][8][9][10][11][12]. Since the three serotypes (DENV-1, DENV-2 and DENV-3) had also been associated with DF outbreaks throughout the country, it is of prime importance to understand molecular epidemiology of these serotype(s). This study was focused on identifying the circulating serotype(s)/genotype(s) during the postmonsoon period in 2014. A low positivity rate, 10.71% (12/112) was observed in virus detection as compared to the previous years [5,9]. However several factors, one of them could be the presence of DENV NS1 antibodies (secondary infection) in the samples as well as to the fact that the RT-PCR was performed only in the samples NS1 antigen positive. Sequencing and BLAST analysis confirmed the majority of DENV-2 (58.33%) cases as compared to DENV-3 (33.33%) and DENV-1 (8.33%). The single DENV-1 sequence clustered within the same clade with previous DENV-1 isolates, being circulated from 2010 to 2013 (Fig. 1). High sequence identity to these isolates (99.51-99.71%) with no amino acid change confirmed the circulation of the same strain of DENV-1 since 2010. Similar analysis revealed clustering of all the four DENV-3 sequences with previous Delhi isolates (from 2012 and 2013) with no changes in amino acid sequence and hence; circulation of the same strain of DENV-3 for the past three years was observed. Analysis of DENV-2 revealed the circulation of different strains of DENV-2. Four sequences (KT180256, KT180257, KT180261 and KT180262) clustered with pre-existing strain from 2012 and 2013 (Fig. 1) and showed similar genetic make-up at the amino acid level (Fig. 2). The newly introduced strain of DENV-2 (KT180258, KT180259 and KT180260) was characterized by the presence of Ala102Val and Ile49Val substitutions in capsid and premembrane protein.

CONCLUSION
Among all four serotypes DENV-2 is genetically most diverse [13]. Studies have shown existence of 6 different genotypes of DENV-2. Genotype IV of DENV-2 is most widely distributed genotype and has been isolated in different countries of Asia, Africa, American continents and Australia. Due to its global occurrence, it is also known as cosmopolitan genotype [2,14]. The predominance of DENV-2 (genotype IV) during an outbreak has been documented from different areas in the country including Delhi [9,[15][16][17]. This genotype has already been associated with DHF outbreaks in 1996 in Delhi [15]. After being in circulation silently for past few years, its reemergence in 2012, followed by the predominance in 2013 was recorded [5,9].
This study reports continued circulation of DENV-2 (genotype IV) in 2014. The sequence based studies are helpful to monitor the genomic changes occurring in virus as well as its spread. The persistence of DENV-2 (genotype IV) in the capital is a major concern for public health. Its continuous circulation may give rise to another DF/DHF outbreak. The results also indicated circulation of a new strain of DENV-2, similar to Hyderabad isolate "1392" (JX475906), along with the pre-existing strain. It is proposed that, this strain has been introduced recently and circulating at low key in the capital. The assessment of its impact on the population still needs to be determined. Further study of different gene regions of this strain may be required to detect important mutations and their role in pathogenesis, virulence and outbreak potential, but till then close monitoring of circulating strains along with clinical co-relation will be essential.

ETHICAL APPROVAL
Ethical approval was obtained from the institute to carry out the present study.