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  Table of Contents  
Year : 2017  |  Volume : 35  |  Issue : 2  |  Page : 293-295

Co-circulation of all four dengue virus serotypes: First report from Odisha

1 Department of Microbiology, All India Institute of Medical Sciences, Bhubaneswar, India
2 School of Botechnology, KIIT University, Bhubaneswar, Odisha, India
3 Department of Medicine, All India Institute of Medical Sciences, Bhubaneswar, India

Date of Web Publication5-Jul-2017

Correspondence Address:
Baijayantimala Mishra
Department of Microbiology, AIIMS, Bhubaneswar - 751 019, Odisha
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_15_536

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 ~ Abstract 

The present report describes the detection of co-circulation of all the four dengue serotypes along with rarely detected dengue viruses (DENVs)-4 for the first time in Odisha. One hundred and forty-eight blood samples were tested for dengue NS1 antigen ELISA and IgM antibody (Ab), and twenty early samples were subjected for type-specific multiplex reverse transcription-polymerase chain reaction (RT-PCR). Twenty-three samples found positive for dengue NS1 and/or IgM Ab; five were positive by RT-PCR. DENV-4 was detected in one sample, DENV-2 in two and 2 were co-infected with DENV-1 and 3. Co-circulation of all four dengue serotypes in Eastern India emphasises the need of molecular monitoring of circulating DENV serotypes.

Keywords: Co-circulation, co-infection, dengue, dengue-4, India, serotypes

How to cite this article:
Mishra B, Turuk J, Sahu SJ, Khajuria A, Kumar S, Dey A, Praharaj AK. Co-circulation of all four dengue virus serotypes: First report from Odisha. Indian J Med Microbiol 2017;35:293-5

How to cite this URL:
Mishra B, Turuk J, Sahu SJ, Khajuria A, Kumar S, Dey A, Praharaj AK. Co-circulation of all four dengue virus serotypes: First report from Odisha. Indian J Med Microbiol [serial online] 2017 [cited 2018 Feb 20];35:293-5. Available from:

 ~ Introduction Top

Dengue is one of the important mosquito-borne viral infections of public health concern. Dengue viruses (DENVs) belong to the genus Flavivirus and family Flaviviridae and have four antigenically related serotypes designated as DENV-1, DENV-2, DENV-3 and DENV-4.[1] All the four serotypes can cause clinical manifestation ranging from mild self-limiting illness to severe dengue haemorrhagic fever (DHF) and dengue shock syndrome. However, severity in dengue viral infection is known to be affected by secondary infection with heterologous antibodies (Abs) or with certain DENV serotypes and genotypes.[2],[3] The changing pattern of dengue serotypes in a geographic location necessitates the continuous molecular surveillance of the circulating serotypes.[4]

The present study for the first time reports the co-circulation of all four dengue serotypes in Odisha, during 2014 dengue season.

 ~ Materials and Methods Top

A total of 148 samples were received from clinically suspected dengue patients during the months of September–December 2014. Of these, twenty samples were received from the patients with <3 days illness.

All the 148 samples were subjected to dengue-specific MAC-ELISA (Pan Bio, Australia) and NS1 antigen detection by ELISA (Pan Bio, Australia) for detection of dengue IgM Ab and dengue NS1 antigen, respectively. Twenty samples of <3 days of fever were subjected to type-specific nested reverse transcription-polymerase chain reaction (RT-PCR) for detection of dengue viral RNA and typing.

Viral RNA was extracted from serum samples using pure link viral RNA/DNA kit (Invitrogen, USA), followed by cDNA synthesis using High capacity RNA–cDNA Reverse Transcription Kit (Invitrogen, USA). Briefly, 5 μl of cDNA was amplified using D1 and D2 primers. The dengue serotype detection was done in second round multiplex nested PCR with 5 μl of 1:100 diluted positive first PCR product using D1 and type-specific primers (TS1-TS4) as described previously.[5]

PCR products were visualised by 2% agarose gel electrophoresis using digital gel documentation and analysis system (Syngene, Cambridge, UK). The study was approved by the Institute Ethics Committee.

 ~ Results Top

Of the 148 samples, 23 were positive for NS1 antigen ELISA and/or dengue-specific IgM Ab by MACELISA. DENV RNA was positive in five of twenty early acute samples tested. The details of serology report along with RT-PCR result are given in [Table 1]. One sample was found to be infected with DENV-4 serotype, DENV-2 serotype was detected in two cases and two samples were found to be co-infected with DENV-1 and DENV-3 [Figure 1]. All the RT-PCR positive samples were also positive for dengue NS1 antigen. The age of the dengue cases was in the range of 15–70 years and male outnumbered the female patients (2.1:1). All the dengue-positive patients had dengue fever with uneventful outcome including the patients suffering from co-infection with two different dengue serotypes and patient infected with DENV-4.
Table 1: Dengue positive result by serology and reverse transcription-polymerase chain reaction

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Figure 1: Agarose gel analysis of dengue type-specific reverse transcription-polymerase chain reaction. Lane 1: Blank; Lane 2 and 3: Co-infection of type 1 (482 bp) and type 3 (290); Lane 4 and 5: Type 2 dengue viruses infection (119 bp); Lane 6: Type 4 dengue viruses infection (392 bp); Lane 7: No template control; Lane 8: 100 bp molecular marker

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 ~ Discussion Top

The present study has been carried out in a newly established tertiary care hospital by Government of India, in Odisha.

Dengue serotype 4 (DENV-4) has recently been reported from North, West, South and Central India, and the present study reports this rare serotype for the first time from Odisha, a state in the eastern part of the country, indicating a gradual spread of DENV-4 across the country.[6],[7],[8],[9] The rarity of DENV-4 has been attributed to the low vectorial competency by Aedes aegypti which is the main vector for dengue virus in India. However, reports regarding the association of DENV-4 virus with severe cases [6] and its high rate of transmission are cause of concern.[10] The single case with DENV-4 serotype in the present study had dengue fever with uneventful outcome.

Co-circulation of all the four dengue serotypes in a single outbreak is a rare occurrence.[6],[7],[8],[9],[10],[11] Co-circulation of dengue serotypes 2 and 3 with predominance of DENV-2 has been reported from Odisha state.[12] To the best of our knowledge, this is the first report of co-circulation of all four dengue serotypes during a single season from this part of the country. However, due to small number of dengue RNA-positive cases, predominant serotype of the season could not be determined.

Co-infection with more than one DENV serotypes has been reported from places where multiple dengue serotypes co-circulates.[6],[13] During 2006 dengue outbreak in Delhi, high percentage (19%) of co-infection has been reported. Recently, studies from Kolkata and Odisha have reported co-infection with multiple dengue serotypes to the tune of 30.6% and 15%, respectively.[12],[14] In the present study, two of five cases had co-infection with DENV serotypes 1 and 3. The occurrence of co-infection with multiple DENV serotypes possesses the risk of emergence of recombinant strains.[15]

The limitation of the present study is that the RT-PCR-positive samples could not be further confirmed by nucleotide sequencing.

The present study for the first time reports the co-circulation of all the four DENV serotypes from the eastern part of the country. This highlights the importance of continuous molecular monitoring of DENV, as the circulating serotype usually gets replaced with a new serotype which may bear the potential of causing severe outbreak.

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Conflicts of interest

There are no conflicts of interest.

 ~ References Top

Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 1998;11:480-96.  Back to cited text no. 1
Martina BE, Koraka P, Osterhaus AD. Dengue virus pathogenesis: An integrated view. Clin Microbiol Rev 2009;22:564-81.  Back to cited text no. 2
Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S, et al. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 2000;181:2-9.  Back to cited text no. 3
Gupta E, Dar L, Kapoor G, Broor S. The changing epidemiology of dengue in Delhi, India. Virol J 2006;3:92.  Back to cited text no. 4
Mishra B, Sharma M, Pujhari SK, Ratho RK, Gopal DS, Kumar CN, et al. Utility of multiplex reverse transcriptase-polymerase chain reaction for diagnosis and serotypic characterization of dengue and chikungunya viruses in clinical samples. Diagn Microbiol Infect Dis 2011;71:118-25.  Back to cited text no. 5
Bharaj P, Chahar HS, Pandey A, Diddi K, Dar L, Guleria R, et al. Concurrent infections by all four dengue virus serotypes during an outbreak of dengue in 2006 in Delhi, India. Virol J 2008;5:1.  Back to cited text no. 6
Cecilia D, Kakade MB, Bhagat AB, Vallentyne J, Singh A, Patil JA, et al. Detection of dengue-4 virus in pune, western india after an absence of 30 years – Its association with two severe cases. Virol J 2011;8:46.  Back to cited text no. 7
Dash PK, Sharma S, Srivastava A, Santhosh SR, Parida MM, Neeraja M, et al. Emergence of dengue virus type 4 (genotype I) in India. Epidemiol Infect 2011;139:857-61.  Back to cited text no. 8
Barde PV, Godbole S, Bharti PK, Chand G, Agarwal M, Singh N. Detection of dengue virus 4 from central India. Indian J Med Res 2012;136:491-4.  Back to cited text no. 9
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Carrington CV, Foster JE, Pybus OG, Bennett SN, Holmes EC. Invasion and maintenance of dengue virus type 2 and type 4 in the Americas. J Virol 2005;79:14680-7.  Back to cited text no. 10
Kumar NP, Jayakumar PR, George K, Kamaraj T, Krishnamoorthy K, Sabesan S, et al. Genetic characterization of dengue viruses prevalent in Kerala State, India. J Med Microbiol 2013;62(Pt 4):545-52.  Back to cited text no. 11
Das B, Das M, Dwibedi B, Kar SK, Hazra RK. Molecular investigations of dengue virus during outbreaks in Orissa State, Eastern India from 2010 to 2011. Infect Genet Evol 2013;16:401-10.  Back to cited text no. 12
Mishra B, Sharma M, Pujhari SK, Appannanavar SB, Ratho RK. Clinical applicability of single-tube multiplex reverse-transcriptase PCR in dengue virus diagnosis and serotyping. J Clin Lab Anal 2011;25:76-8.  Back to cited text no. 13
Sarkar A, Taraphdar D, Chatterjee S. Molecular typing of dengue virus circulating in kolkata, India in 2010. J Trop Med 2012;2012:960329.  Back to cited text no. 14
Worobey M, Rambaut A, Holmes EC. Widespread intra-serotype recombination in natural populations of dengue virus. Proc Natl Acad Sci U S A 1999;96:7352-7.  Back to cited text no. 15


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