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 ~  Abstract
 ~ Introduction
 ~ Subjects and Methods
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
 ~  Article Figures
 ~  Article Tables

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  Table of Contents  
Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 393-400

Emergence of dengue virus 4 as the predominant serotype during the outbreak of 2017 in South India

1 Department of Microbiology, Regional Viral Research and Diagnostic Laboratory, JIPMER, Puducherry, India
2 Department of Microbiology, MGMCRI, Sri Balaji Vidyapeeth, Puducherry, India
3 Department of Microbiology, JIPMER, Puducherry, India

Date of Submission12-Sep-2019
Date of Decision06-Nov-2019
Date of Acceptance11-Dec-2019
Date of Web Publication29-Jan-2020

Correspondence Address:
Dr. Rahul Dhodapkar
Department of Microbiology, JIPMER, Puducherry - 605 006
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmm.IJMM_19_338

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 ~ Abstract 

Context: Dengue virus (DENV) causes acute febrile illness in tropical and subtropical countries. In India there is a steady increase in incidence since 1950s which could be attributed to emergence of new serotype or lineage\clade shifts in circulating DENV. Aims: We aimed to perform molecular characterisation and phylogenetic analysis on samples from the recent outbreak (August–October 2017). Settings and Design: Retrospective epidemiological analysis of dengue outbreak. Subjects and Methods: Samples positive for non-steroidal 1 antigen by enzyme-linked immunosorbent assay (n = 147) were included. The study was approved by our institute ethics committee (JIP/IEC/2018/496). Five hundred and eleven base pair of capsid and pre-membrane encoding genes (CprM) region was amplified using Lanciotti primers, followed by second round of polymerase chain reaction using serotype specific primers. Samples which were positive by second round (n = 68) were sequenced and genotyped using Basic Local Alignment Search Tool analysis and phylogenetic tree was constructed by MEGA7 software. Results: Phylogenetic analysis of CprM sequences identified all 4 serotypes in circulation during this outbreak. We observed both single (n = 50) and concurrent infections (n = 18), with DENV4 as the major contributor (64%). Within Genotype I of DENV4 we observed a distinct new clade (Clade E) which was 2.6% ± 0.9%–5.5% ± 1.1% divergent from the other clades. Among the concurrent infection, DENV 4 and DENV 2 combination was observed to form the majority (77.8%). Conclusions: Overall this study documents the emergence of DENV4 as the major serotype in circulation, replacing DENV1, 2 and 3 which had been previously reported from Tamil Nadu and Puducherry. This substantiates the need for continuous monitoring in endemic countries like India, where such data may impact the formulation of vaccine policy for dengue.

Keywords: Concurrent infections, dengue serotyping, dengue virus 4, South India

How to cite this article:
Sharmila P F, Vanathy K, Rajamani B, Kaliaperumal V, Dhodapkar R. Emergence of dengue virus 4 as the predominant serotype during the outbreak of 2017 in South India. Indian J Med Microbiol 2019;37:393-400

How to cite this URL:
Sharmila P F, Vanathy K, Rajamani B, Kaliaperumal V, Dhodapkar R. Emergence of dengue virus 4 as the predominant serotype during the outbreak of 2017 in South India. Indian J Med Microbiol [serial online] 2019 [cited 2021 Mar 7];37:393-400. Available from:

 ~ Introduction Top

Dengue is one of the most rapidly spreading mosquito–borne viral infections in tropical and subtropical countries, with an overall increase in global incidence by 30 fold over the past 50 years.[1] This disease is endemic in more than 125 countries, with rapid urbanisation and increased global transportation being the major risk factors. In 1997 WHO categorised dengue into dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) which was revised in 2009 based on levels of severity into (i) dengue without warning signs; (ii) dengue with warning signs (abdominal pain, persistent vomiting, fluid accumulation, mucosal bleeding, lethargy, liver enlargement, increasing haematocrit with decreasing platelets); and (iii) severe dengue (dengue with severe plasma leakage, severe bleeding or organ failure). This reclassification is based on dengue control study which showed that with the previous classification several overlaps existed between the categories and could not be extended to all age groups.[2]

Dengue virus (DENV) belongs to the family Flaviviridae, a group of positive-sense single-stranded RNA virus and has four distinct serotypes (DENV1-4) identified so far which are primarily transmitted between humans by the vectors Aedes aegypti and Aedes albopictus. All the 4 serotypes are known to cause DF, a self-limiting febrile illness, while a variable proportion of patients progress towards life threatening DHF, characterised by thrombocytopenia and haemorrhage. On account of nucleotide sequence variability, dengue serotypes are further classified into distinct genotypes that differ >6% within a single serotype. The variation in severity of the disease during outbreaks has been attributed to the emergence of such new genotypes which acts as enhancing risk factors.[3]

The global disease burden of dengue has been reported to be 96 million cases in 2010, whereas in India a recent study showed an overall seroprevalence of 48.7%.[4] The burden of dengue infection in India was heterogeneous, with evidence of high transmission in northern, western and southern regions.[5] Phylogenetic analysis of E gene from Indian strains archived over a period of 50 years revealed that these strains formed temporally distinct clusters.[6] Genotype III of DENV3, which originated in India, is more virulent and has caused haemorrhagic outbreaks in many countries and was attributed as the cause of increased incidence of DHF/DSS in northern India. Recent study from Central India revealed both single and concurrent infections with DENV 3 as the major contributor.[7] Spatio-temporal analysis revealed Asian genotype of DENV 1 to be the predominant circulating strain in Southern states which includes Tamil Nadu and Kerala while American/African genotype in the western states. Also this Asian genotype of DENV1 was reported as the major cause for the large outbreak in 2012 with 12,000 cases.[8] Analysis of 2016 outbreak samples from Pune showed emergence of novel Clade (D) within Genotype I of DENV4.[9]

Severity of dengue is known to be associated with certain serotypes and genotype or presence of heterotypic immunity. Sequential circulation of all serotypes and/or occurrence of concurrent infections are thus considered to be potential population-level risk factors for severe dengue. In this regard up to date information on prevailing strains is essential. Hence this study aimed to characterise the DENVs circulating in the southern states of Tamil Nadu and Pondicherry which are known endemic states[6] during the outbreak of August–October 2017.

 ~ Subjects and Methods Top

Sample collection

This retrospective study was designed to investigate the dengue genotypes which circulated in the southern states of India, Tamil Nadu and Puducherry, during the August–October 2017 outbreak. The study was approved by our institute ethics committee (JIP/IEC/2018/496). Serum of patients presenting with dengue like symptoms (headache, retro-orbital pain, myalgia, arthralgia, rash, haemorrhagic manifestations and leucopenia) in Medicine outpatient department/clinic of our tertiary care hospital in Puducherry were tested for non-steroidal (NS) 1 enzyme-linked immunosorbent assay (ELISA) (Dengue NS1 Ag Microlisa-J. Mitra and Co Pvt). Those samples which were positive for NS1 antigen were stored in −80°C and included in the study.

Viral RNA extraction and cDNA conversion

Total RNA was extracted from 200 μl of human serum using Roche life science High Pure Viral Nucleic acid extraction kit (Roche Molecular Systems USA, Inc.), as per manufacture's instruction and the nucleic acid was eluted in 60 μl. Single stranded cDNA was synthesised from total RNA using TaKaRa PrimeScript RT reagent kit (Takara Bio USA, Inc) as per the manufacturer's instruction and was then stored immediately at −20°C until use.

Polymerase chain reaction and sequencing

Nested polymerase chain reaction (PCR) was used for serotyping the samples. Amplification of the junction of dengue capsid and pre-membrane encoding genes (CprM) was done using Lanciotti primers.[10] 511 bp product of C-PrM gene was amplified with D1 and D2 primers using Ampliqon Taq DNA polymerase master mix. The thermal profile for first round was: initial denaturation at 94°C for 2 min followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 52°C for 45 s and extension at 72°C for 2 min. A final extension was given at 72°C for 10 min. The second round of PCR using the type-specific primers[11] was same to the thermal profile of first-round, only the annealing was carried out at 54°C for 30 s in 35 cycles. The size of the second round PCR products of partial CprM gene of DENV1, DENV2, DENV3 and DENV4 are 486 bp, 119 bp, 290 bp and 392 bp, respectively. The PCR amplified products were analysed by electrophoresis on 2% agarose gel. The PCR products from the second round were purified using ExoSap-IT PCR clean-up reagent and sequenced by ABI 3500 sequencer. The resulting sequence reads were assembled using SeqScape® software v2.5 (Life Technologies, Foster City, CA, USA) SeqScape, version 2.5 (Applied Biosystems), with additional manual adjustment performed when manual inspection of the assembly showed some discrepancies.

Nucleotide and amino acid sequence analysis

The obtained sequences were edited and Basic Local Alignment Search Tool search was conducted to confirm the identity of the sequences. The amino acid sequences were translated using BioEdit software (v7.0.5) (Carlsbad, CA, USA) and it was also used to align amino acid and protein sequences. The phylogenetic and molecular evolutionary analyses were conducted using MEGA 7.0 (Pennsylvania, USA).[12] The phylogenetic tree was drawn by using the Neighbor-Joining method[13] with bootstrap analysis of 1000 replicates.

 ~ Results Top

Characteristics of patients

A total of n = 147 samples were positive for NS1 antigen by ELISA, of which we were able to amplify and sequence 68 samples (46%). The demographics of the patient included in the current study are given in [Table 1]. The average age of the patients was 28 years. Males (n = 33) to females (n = 35) ratio was 1:0.9. Based on recent WHO guidelines (2009) all the patients were categorised as dengue ± warning signs. The average platelet value was 154 × 10^3/uL, with a minimum value of 15 × 10^3/uL.
Table 1: Demographics of dengue patients

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Phylogenetic analysis

Serotyping using type-specific primers revealed DENV1 (n = 4), DENV2 (n = 6), DENV3 (n = 2) and DENV4 (n = 38) serotypes among single serotype infections and n = 18 concurrent infections. The concurrent infections included: DENV1-DENV4 (3/68), DENV2-DENV4 (14/68) and DENV2-DENV3-DENV4 (1/68). Considering both single and concurrent infections DENV1 = 7, DENV2 = 21, DENV3 = 3, DENV4 = 56 [Supplementary Table 1]. Clearly this shows circulation of all 4 serotypes during this outbreak, with DENV4 forming the majority (65%) among single serotype infection and DENV4-DENV2 combination among concurrent infection [Table 1]. The genotypic distribution of DENV within each serotype was determined by sequencing the partial CprM gene and constructing phylogenetic tree along with reference sequences obtained from National Centre for Biotechnology Information (NCBI) [Figure 1], [Figure 2], [Figure 3], [Figure 4].

Figure 1: Molecular phylogenetic analysis of capsid and pre-membrane encoding genes sequence of dengue virus 1 samples by neighbour-joining method. Phylogenetic analyses of capsid and pre-membrane encoding genes sequence for genotype determination. The evolutionary distances were computed using the p-distance method. The analysis involved a total of 41 nucleotide sequence, of which 34 are reference sequences. Current study samples are indicated by ♦ followed by Genbank accession numbers. The numbers in the nodes are support values for the major branches (bootstrap; 1000 replicates)

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Figure 2: Molecular phylogenetic analysis of capsid and pre-membrane encoding genes sequence of dengue virus 2 samples by neighbour-joining method. Phylogenetic analyses of capsid and pre-membrane encoding genes sequence for serotype determination. The evolutionary distances were computed using the p-distance method. The analysis involved a total of 67 nucleotide sequence, of which 21 are reference sequences. Current study samples are indicated by ♦ followed by Genbank accession numbers. The numbers in the nodes are support values for the major branches (bootstrap; 1000 replicates)

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Figure 3: Molecular phylogenetic analysis of capsid and pre-membrane encoding genes sequence of dengue virus 3 samples by neighbor-joining method. Phylogenetic analyses of capsid and pre-membrane encoding genes sequence for serotype determination. The evolutionary distances were computed using the p-distance method. The analysis involved a total of 27 nucleotide sequence, of which 24 are reference sequences. Current study samples are indicated by ♦ followed by Genbank accession numbers. The numbers in the nodes are support values for the major branches (bootstrap; 1000 replicates)

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Figure 4: Molecular phylogenetic analysis of capsid and pre-membrane encoding genes sequence of dengue virus 4 samples by neighbour-joining method. Phylogenetic analyses of capsid and pre-membrane encoding genes sequence for serotype determination. The evolutionary distances were computed using the p-distance method. The analysis involved a total of 89 nucleotide sequence, of which 33 are reference sequences. Current study samples are indicated by ♦ followed by Genbank accession numbers. The numbers in the nodes are support values for the major branches (bootstrap; 1000 replicates)

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Dengue virus 1

Phylogenetic tree was constructed using 41 sequences including 15 sequences from India (7 sequences from the current study and 8 published). The study sequences showed a nucleotide distance of 4%–7% and an amino acid distance of 2%–4% with respect to the prototype strain (NC_001477.1) whereas the nucleotide and amino acid distance between themselves was 2% each. Strains from the present study belonged to Genotype V (American/African genotype) and was found close to KJ420624 (Delhi, India 2011), MF065948 (Assam, India 2016), AY732476 (Thailand 1980), and KM242113 (Tamil Nadu, India 2013), respectively [Figure 1]. Protein sequence analysis revealed known variations G70S, N90S in all seven samples and two unknown variations S129T and L158M in MK427043 [Table 2].
Table 2: List of amino acid variations in capsid and pre-membrane region of all dengue virus serotypes on comparison with the reference strain NC_001477.1 (dengue virus 1), NC_001474.2 (dengue virus 2), NC_001475.2 (dengue virus 3), NC_002640.1 (dengue virus 4)

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Dengue virus 2

Phylogenetic analysis revealed the distribution of current strains into 3 genotypes. Of the 21 samples, majority of the samples (n = 19) grouped along with Genotype IV (Cosmopolitan), while MK408550 was close to KX702403 (Haiti 2016) belonging to Genotype III and MK408544 was near HM582101 (Fiji 1971) belonging to Genotype V. Among the Genotype IV samples, they exhibited 2 clusters: one close to KJ438865 (India 2012), and KX380828 (Singapore 2013), while the other was near KM274895 (India 2013) [Figure 2]. The study sequences showed a nucleotide distance of 8%–9% and an amino acid distance of 3%–5% with respect to the prototype strain (NC_001474.2), while between themselves the nucleotide distances was 4% and amino acid distances of 2%. Total of 6 known amino acid variations were observed within DENV2 sequences K76R, S101T, A102V, M104V, I106V and V112A [Table 2].

Dengue virus 3

Phylogenetic analysis revealed all 3 samples to cluster in Genotype III along with MH606210 (Assam, India 2017). While this cluster exhibited wide geographic distribution from Asia, Africa, America and Europe, our samples were closer to DENV3 strains from Asian continent including KF954946 (China 2013), JN940919 (India 2010), KF041254 (Pakistan 2008), JQ717295 (India 2011) and a single isolate from Africa (Senegal-KU509282 2009) [Figure 3]. The study sequences showed a nucleotide distance of 1%–5% and an amino acid distance of 1%–2% with respect to the prototype strain (NC_001475.2), while between themselves the nucleotide distances and amino acid distances were 1% each. A single known amino acid change was observed Q97K when compared with the prototype strain [Table 2].

Dengue virus 4

This was the major strain in circulation during this outbreak and they aligned at 235–536 bp corresponding to the prototype strain NC_002640.1, covering 46th to 145th amino acid of the CprM region. Molecular phylogenetic analysis revealed clustering of DENV4 sequences into 4 genotypes, with the current sequences falling under Genotype I. Within this genotype, we observed 5 different clusters/clades with the current samples distributed among Clades C, D, E. N = 26 of current study samples grouped into Clade D along with isolates from Andhra Pradesh (EU652498), Kerala (JN882277) and Pune (MG272273-MG272274) and n = 4 samples were grouped along with a strain from Pakistan (KF041260) [Figure 4]. We also observed about 46% of the strains formed a distinct new clade (Clade E) within Genotype I which was 0.7%±0.4 to 6±1.2 divergent from the other clades [Table 3] which could suggest emergence of a new clade within genotype I. (However the difference between clades D and E were minimal.). The study sequences showed a nucleotide distance of 2% and an amino acid distance of 2%–3% with respect to the prototype strain, while between themselves the nucleotide distances was 1% and amino acid distances was 0.5%. A total of 7 amino acid variations were observed of which 5 are known I81T, I102M, V111A, S115H and A128T and 2 are unknown E122G and L136F [Table 2].
Table 3: Nucleotide diversity in capsid and pre-membrane region between clades within genotype I of dengue virus 4 serotype

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 ~ Discussion Top

Here we report the circulation of all four dengue serotypes during the August–October 2017 outbreak in Puducherry and Tamil Nadu, with DENV4 emerging as the major causative serotype. DF has gained attention as an important emerging disease affecting tropical and subtropical regions. The incidence of dengue has increased steadily in India since its recorded outbreak in 1950s.[6] Current statistics (2012–2016) indicate a decrease in morbidity of dengue cases but marginal rise in mortality, nationally.[14] This is substantiated by the increased incidence of co-circulation and concurrent infections as observed in the present study which pose a serious threat for occurrence of severe forms of the disease in future.

The epidemiology of DF in India has been very complex pertaining to the circulating strains, the geographical locations affected and severity of disease. Most of the studies on serotype distribution are from the northern states like Delhi, Uttar Pradesh, Rajasthan, West Bengal, Gujarat, Punjab and Assam. DENV2 and DENV3 are the most common serotypes involved in outbreaks of these regions.[15]

Majority of studies from southern states are from Pune, Maharashtra[16] with very few studies from Tamil Nadu and Puducherry. In these states DENV3 is the most common serotype that has been involved in epidemics during 1966 and 1968 (Vellore),[17] 2001 (Chennai),[18] 2005 (Coimbatore),[19] 2003 (Kanyakumari),[20] and 2007 (Madurai).[21] Myers et al. had reported the presence of DENV3 in patients and A. aegypti at Vellore during the epidemic of 1966, while during the epidemic of 1968, all the four serotypes of DENV were isolated from patients and mosquitoes.[22] Other serotypes including DENV2,[23] and DENV1[24] have also been reported. Cocirculation of all 4 serotypes has also been reported in Southern India during the outbreaks of 2012–2015.[25] Similar to these reports we also observed circulation of all four serotypes. Further characterisation of the current sequences revealed presence of Genotype III–V of DENV2 and Genotype III of DENV3 which are similar to earlier reports from India.[26],[27],[28] Our samples (n = 7) of DENV1 (Genotype V) differed from recent reports stating Genotype I in this area.[29] The probable reason being the Asian genotype is again replaced by the American/African genotype.

This is the first report to observe DENV4 to be the predominant circulating serotype. This rare serotype in India had been reported earlier from Maharashtra,[9],[16] Andhra Pradesh[30] and Kerala.[31] This serotype has been associated with severe form the disease. Cecilia et al. 2011, reported two cases one with severe primary infection (Grade III) who survived, while another case with secondary infection died. Current study being a retrospective one we were unable to get details as to the nature of infection whether it was primary/secondary. However in the present study we did not observe any correlation with severity of the disease. The identification of a new clade in this study emphasises the high rate of genomic diversity observed within Genotype I of DENV4. A recent study on vaccine offered protection had revealed large differences on antigen neutralisation between the DENV4 genotypes.[32] All these warrant for continuous monitoring to identify and document the various dengue strains circulating in highly endemic areas including India.

Additionally this serotype also presented as concurrent infections in combination with other serotypes. It is a known fact that sequential infection with more than one serotype in a particular region leads to severe disease manifestation, owing to antibody dependent immune response.[33] The first known concurrent infection in dengue was reported in Puerto Rico in 1982,[34] following which several other countries including Somalia,[35] Mexico, Indonesia,[36] and China[37] have also reported the same. The probable cause for concurrent infection is attributed to the unique feeding behaviour of female Aedes egypti mosquitoes. Since they feed on several individuals during a single gonotrophic cycle, there is high chance of dual infection and then transmitting multiple viruses to a single individual.[38],[39] Earlier reports of concurrent infection from India includes: Tamil Nadu,[24] Delhi,[40] Kerala,[41],[42] Hyderabad,[43] and Karnataka.[44] This is the first report to observe high percentage of concurrent infection with DENV2 and 4 (14/68) serotypes.

CprM gene of DENV genome is involved in formation of basic structure of the virus. Therefore, variations in this region can lead to structural and functional changes. Also it is the most common one used for dengue serotyping as the amplification and sequencing primers are the same. Moreover genotyping this region had helped in identification of emergence of new clade earlier[9] as well as in this study. The amino acid variations observed in this study, both novel and known needs to be further analysed for their significant effect on the structure.

 ~ Conclusion Top

We observed all 4 serotypes to be circulating as well as presenting as concurrent infections during the August–October 2017 outbreak, with DENV4 being the major contributor. Evolution of new clades and concurrent infections pose a serious threat to society especially in endemic countries like India, which makes continuous surveillance using molecular methods a mandatory.

Financial support and sponsorship

DHR, India (VIR/14/2012/ECD-I).

Conflicts of interest

There are no conflicts of interest.

 ~ References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2], [Table 3]


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2004 - Indian Journal of Medical Microbiology
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