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

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 401-408
 

Molecular diversity of dengue virus serotypes 1–4 during an outbreak of acute dengue virus infection in Theni, India


1 Department of Microbiology, Government Theni Medical College, Theni; Department of Microbiology, Dr ALM PG IBMS, University of Madras, Chennai; Department of Microbiology, Virus Research and Diagnostic Laboratory, Government Theni Medical College, Theni, Tamil Nadu, India
2 Department of Microbiology; Department of Microbiology, Virus Research and Diagnostic Laboratory, Government Theni Medical College, Theni, Tamil Nadu, India
3 Infection Biology, Department of Life Sciences, School of Life Sciences, Central University of Tamil Nadu, Thiruvarur, Tamil Nadu, India
4 Department of Microbiology, Dr ALM PG IBMS, University of Madras, Chennai, Tamil Nadu, India

Date of Submission04-Mar-2020
Date of Decision05-Jun-2020
Date of Acceptance07-Sep-2020
Date of Web Publication4-Nov-2020

Correspondence Address:
Dr. Amudhan Murugesan
Department of Microbiology, Government Theni Medical College, Theni, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_20_89

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 ~ Abstract 


Background: Dengue fever (DF) is caused by an arthropod-borne dengue virus (DENV), has four serotypes and several genotypes. Although having clinical and epidemiological significance, the information on the circulating serotypes/genotypes is scarce in India.
Materials and Methods: Blood specimens were collected from the patients suspected of DF and they are tested for DENV NS1 antigen and DENV IgM by ELISA. Antigen-positive samples were further serotyped by reverse transcriptase polymerase chain reaction. Representative samples from each serotype were sequenced to identify the genotypes.
Results: All the four DENV serotypes were detected with the pre-dominance of DENV-1 (n = 49; 41.9%). Cases with multiple DENV serotype infections were also identified. Genotyping showed that DENV-1 belonging to genotype I, DENV-2 cosmopolitan (IV), DENV-3 genotype III and DENV-4 genotype I were active in the circulation during the outbreak in 2017.
Conclusion: Our study documents the molecular characteristics of DENV circulating in our geographical locality. The detection of heterologous DENV serotypes highlights the importance of regular molecular monitoring for the early recognition of any switch in pre-dominant serotype.


Keywords: Dengue, genotype, molecular epidemiology, phylogenetic analysis, serotype, Tamil Nadu, Theni


How to cite this article:
Murugesan A, Aridoss D, Senthilkumar S, Sivathanu L, Sekar R, Shankar EM, Manickan E. Molecular diversity of dengue virus serotypes 1–4 during an outbreak of acute dengue virus infection in Theni, India. Indian J Med Microbiol 2020;38:401-8

How to cite this URL:
Murugesan A, Aridoss D, Senthilkumar S, Sivathanu L, Sekar R, Shankar EM, Manickan E. Molecular diversity of dengue virus serotypes 1–4 during an outbreak of acute dengue virus infection in Theni, India. Indian J Med Microbiol [serial online] 2020 [cited 2020 Nov 24];38:401-8. Available from: https://www.ijmm.org/text.asp?2020/38/3/401/299849





 ~ Introduction Top


Dengue is becoming the most common vector-borne disease in the world surpassing malaria. Dengue fever (DF) is caused by dengue virus (DENV) which belongs to the genus Flavivirus, family Flaviviridae. The infection is manifested with wide spectrum of clinical illness ranging from asymptomatic to fatal dengue haemorrhagic fever (DHF) or Dengue Shock Syndrome (DSS)[1]. The WHO estimates that DENV causes >390 million new infections every year with 100 million cases of DF and 0.2–0.5 million cases of DHF. The disease is endemic in ~100 countries and about half of the global population is at risk of infection with this arbovirus.[2] DENV is a small single-stranded positive-sense RNA virus comprising of four serologically related, but genetically distinct virus serotypes named as DENV-1, DENV-2, DENV-3 and DENV-4. Studies on the evolution and molecular epidemiology of DENV have demonstrated that strain variation within a serotype allows these viruses to be classified into genetically distinct groups called genotypes.[3] Infection with one serotype confers life-long immunity to subsequent infection by the same serotype, but it does not confer strong immunity against infection with other serotypes. Hence, it is important to understand the prevailing serotypes and genotypes circulating in our region. The epidemiology of DV infection in India is very complex and has substantially changed over time in terms of prevalent strains, geographical distribution and disease severity.[4] In Tamil Nadu, Dengue infections and their complications have been well documented since 1966.[5] The incidence of dengue cases in India was the highest in 2017, with Tamil Nadu reporting the second highest number of cases in the country only after West Bengal.[6] We have previously reported that our region (Theni district) is hyper endemic to dengue infection.[7]

The present study was aimed to investigate the molecular diversity of DENV serotypes I–IV during acute DENV infection in Theni district, India.


 ~ Subjects and Methods Top


Specimens

Samples were collected from patients with suspected DF attending the fever clinic of the Government Medical College, Theni (age range, 1 month–85 years) during the study period – July 2017–February 2018. All the patients included in this study exhibited fever along with any three or more of the following symptoms: myalgia, arthralgia, nausea, vomiting, rash, abdominal pain, arthralgia, retro-orbital pain, epistaxis, melena, hematemesis and pleural effusion. The choice of antigen or antibody testing for DENV by ELISA was based on the duration of the fever; patients with ≤5 days fever were tested for NS1 and patients with >5 days of fever were tested for immunoglobulin M (IgM).

With the informed consent of the patient and from parents in case of children, 5 mL of blood was collected; serum was separated and stored at −80°C until further analysis. The study was approved by the Institutional Ethical Committee of Government Medical College, Theni (Ref. No. 2443/PandD/2012).

Confirmation of DENV infection by the detection of anti-dengue-immunoglobulin M or dengue NS1

Serum samples were tested for anti-DENV-IgM antibody by NIV Dengue MAC ELISA Kits (National Institute of Virology, India) or Dengue NS1 antigen by PanBio Dengue Early ELISA (Alere, USA). Testing conditions and interpretations were done according to the manufacturer's instructions.

Determination of DENV serotypes

Samples with high OD >1.2 for DENV NS1 were subjected for viral RNA extraction by QIAamp viral RNA extraction kit (Qiagen, USA). Immediately, complementary DNA (cDNA) synthesis was done by SuperScript IIIFirst-Strand Synthesis kit (Invitrogen, USA) and DENV serotyping was carried out with CDC DENV-1-4 real-time polymerase chain reaction (RT-PCR) Assay (CDC, USA) and Superscript III platinum one-step RT-PCR kit (Thermo Fisher, USA) as per the CDC protocol. Reactions were performed in Applied Biosystems 7500 instrument using serotype-specific DENV controls provided with the kit.

Confirmation of DENV serotypes by conventional polymerase chain reaction for capsid-pre-membrance gene

Samples with Ct value ≤25 in real-time PCR were subjected for conventional PCR according to Fatima et al.[8] A 511 bp capsid-pre-membrance (C-prM) gene junction was amplified. Briefly, for a 25 μl reaction 10X Taq Buffer 2.5 μl, 10 mM dNTP mix 0.5 μl, 10 μM each forward and reverse primers 1.0 μl 5 U/μL KAPA Taq DNA Polymerase, 2.5 μL of template cDNA were added and finally made up to 25 μL with nuclease free water. The following cycling conditions were used initial denaturation at 93°C for 3 min, denaturation 93°C for 45 s, annealing 55°C for 1 min, extension 72°C for 45 s, final extension 72°C for 5 min and hold at 4°C infinitely. The amplified PCR product was analysed by gel electrophoreses using 2% agarose. The product size was identified by comparing with 100 bp DNA ladder (Thermo Scientific).

DNA sequencing and sequence analysis

Amplified C-prM gene product was sequenced by Sanger method at Scigenomics, Cochin, India. The obtained sequences were searched against the NCBI database to confirm their correct identity by BLAST and submitted to NCBI GenBank. Multiple sequence alignments were done by ClustalW tool in BioEdit.

Phylogenetic analysis

Phylogenetic tree was constructed by MEGA 7. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. The reliability of tree was estimated using 1000 bootstrap replications under Nearest-Neighbour interchange procedure with input distance determined by the Maximum-Likelihood method.[9] Fifty-four nucleotide sequences of DENV-1, 45 of DENV-2, 40 of DENV-3 and 31 sequence of DENV-4 were retrieved from the NCBI Database and used for tree construction with study strains.


 ~ Results Top


During the study period, a total of 3925 patients with suspected DF were enrolled that included 466 patients with ≤5 days fever. Among the 466 serum samples, tested for antigen, 181 (38.8%) were positive for DENV NS1; and among the 3459 samples tested for IgM 1907 (55.1%) were positive for DENV specific IgM [Table 1]. Seropositivity rate was higher among the paediatric population, especially the age group ≤9 (63.7%) followed by the age group of 10 years–19 years (57.4%).
Table 1: DENV serology tests performed age-group wise, total tested, positive and negative

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Prevalence of DENV serotypes

DENV Serotyping was performed on all 181 dengue NS1 positive samples, out of which 117 (73.75%) were positive by RT-PCR; also revealed the presence of all four serotypes with DENV-1 in 49 (41.9%) being the pre-dominant serotype followed by DENV-2 in 41 (35.0%), DENV-4 10 (8.5%) and DENV-3 in 8 (6.8%) [Table 2]. Further, we found combined infection with different serotypes in 9 (7.7%) samples, among these, combined infection DENV-1 and 2 was found in 5 (55.5%), DENV-2 and 4 was in 3 (33.3%) and DENV-1 and 3 was in 1 (11.1%).
Table 2: Frequency of samples tested for DENV by reverse transcriptase polymerase chain reaction and its outcome

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Phylogeny of DENV-1 strains

Phylogenetic analysis of 9 DENV-1 strains from this study showed that they were grouped in genotype I. Interestingly, strains MG516715, MG544876, MN123806 and MN108138 clustered with Vietnam, Thailand, Sri Lanka and Chinese strains, whereas MN108142, MN123804, MN123805, MN123807 and MG544874 clustered with Indian strain of 2013 (KJ755855.1) [Figure 1].
Figure 1: Phylogenetic tree based on capsid-premembrance gene of dengue virus-1. The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model. Evolutionary analyses were conducted in MEGA7

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Phylogeny of DENV-2 strains

A phylogenetic tree constructed on the basis of 6 DENV-2 sequences from this study and reference sequences from the different parts of the world segregated into different genotypes namely Genotype IV (cosmopolitan), Asian-II, Asian-1, American-Asian and American [Figure 2]. DENV-2 strains of the present study clustered within Genotype IV (cosmopolitan) of DENV-2 along with previously reported Indian DENV-2 strain isolated from Gwalior in 2001.
Figure 2: Phylogenetic tree based on capsid-premembrance gene of dengue virus-2. The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model. Evolutionary analyses were conducted in MEGA7

Click here to view


Phylogeny of DENV-3 strains

Nucleotide sequences of DENV-3 strains of this study and 40 global reference strains were utilised in the construction of phylogenetic tree. DENV-3 strains of this study belonged to genotype III [Figure 3]. The MG516716 was found to be more closely related to Indian strains reported from Gwalior III and German traveller returning from India 2009, other two strains (MN108410 and MN108139) were related to the strains of Singapore and China.
Figure 3: Phylogenetic tree based on capsid-premembrance gene of dengue virus-3. The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model. Evolutionary analyses were conducted in MEGA7

Click here to view


Phylogeny of DENV-4 strains

Nucleotide sequences of DENV-4 strains and 31 global reference strains were utilised in the construction of phylogenetic tree. DENV-4 strains belonged to genotype I [Figure 4]. They were found to be more closely related to Indian strains reported from Pune and Pakistan in 2009.
Figure 4: Phylogenetic tree based on capsid-premembrance gene of dengue virus-4. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model. Evolutionary analyses were conducted in MEGA7

Click here to view



 ~ Discussion Top


Since 1950's, the co-circulation of all four serotypes of DENV is common in South-east Asia and now it has expanded widely to tropics and subtropics of the world wherever the Aedes sp. mosquitoes are distributed.[10] The co-circulation of various DENV serotypes in an endemic area plays a major role in the severity of illness and disease outcome.[11] Hence, understanding the information on circulating serotypes and genotypes is crucial for the development of an effective vaccine and epidemic management.[12],[13] Circulation of multiple serotypes with concurrent infections are being reported from most parts of the country and major outbreaks of DF and DHF are caused by DENV-2 and DENV-3.[14] Multiple serotypes were reported in southern states of Kerala,[15] Karnataka[16] and Telangana.[17] To the best of our knowledge, this is the first study from our region where we observed the circulation of all four serotypes of DENV. Further, we observed that 7.7% of samples had concurrent infection with multiple serotypes while another study from Pondicherry showed 26.4% of concurrent infections. However, we found the similar rate of concurrent infection with DENV-1 and DENV-2 (4.4%) with respect to their study but the above said study showed a higher prevalence of DENV-2 and DENV-4 co-infection[18] whereas we found DENV-1 and DENV-2 and 2 co-infection to be pre-dominant.

We found DENV-1 to be pre-dominant in our region followed by DENV-2, DENV-4 and DENV-3, this is corroborated with another study from Bangalore which showed similar serotypes prevalence;[19] however, another study during the same period reported DENV-4 as pre-dominant serotype in Pondicherry.[18] In the present study, DENVs were sequenced directly from the patient serum sample as performed elsewhere with one difference being the primer set used, this was done to avoid the selection and sequencing of mutant viral RNA which may occur during isolation/propagation in mouse brain/cell culture with additional benefits of reduction in cost and turn-around time.[20] Although the DENV genome has three major structural genes, known as the capsid, pre-membrane and envelope genes, along with seven non-structural genes, many studies have reported the C-prM junction as a powerful target in genotyping also this shorter fragment of C-prM is much more convenient to sequence than E gene which is 1857 bp without comprising the output of sequence results.[21] Hence, this C–prM gene junction was selected for this study, as it harbours epidemiologically important sequence information and a single set of primer pair could be used for the amplification and sequencing of any of the four serotypes.

Most Indian studies on the prevalence of DENV-1 serotypes showed the prevalence of genotype III.[22] Here, we show that all our isolates belonged to genotype I in similarity with a recent report analysing samples of 2012 epidemic from Tirunelveli, Tamil Nadu[23] and our isolates showed close homology with many South Asian strains of Vietnam, Thailand and Cambodia. This genotype is more cosmopolitan in the nature with strains from East Africa, Asia and South Pacific. Furthermore, we noticed that our isolates formed two different lineages within genotype I.

DENV-2 genotype IV has a wide distribution in tropics and subtropics. It has been associated with explosive epidemics on the other islands of South Pacific in previous years.[24] Two different genetic types (genotypes) of DENV-2 have been recognised based on phylogenetic studies: the 'South-East Asian' and 'American' genotypes. The former has been associated with DHF, and the latter, with mild clinical manifestations of dengue.[25] In India, DENV-2 genotype IV was first isolated during the 1996 dengue epidemic in New Delhi[26] later on reported from Kerala 2008–2010.[27] This indicates that the genotype IV of DEN-2 is still the pre-dominant genotype involved in the major outbreaks in India. In our study too, we found that all our DENV-2 isolates to be closely related and formed a separate lineage within genotype.

The first study from the state in 1966 had reported DENV-3 GIII.[28] Since then, different studies investigating DENV-3 across the country had reported multiple strains of GIII.[29] The present study also documented genotype-III and clustered with South Asian isolates. Most reported genotype of DENV-4 in India is genotype I, and our study also substantiate this. However, in 2014, Andhra Pradesh had reported genotype 2.[30]


 ~ Conclusion Top


The study reports the prevalence of all four DENV serotypes in Theni, Tamil Nadu during the 2017–2018 outbreak with the pre-dominance of DENV-1, genotype I in contrast to genotype III in earlier reports. While DENV-2 genotype IV, DENV-3 genotype III and DENV-4 genotype I was observed in concordance with earlier reports. Considerable proportion of patients had co-infection with multiple serotypes which poses high risk for severe dengue. Continuous surveillance and stringent control strategies are required to prevent and control dengue-related mortality and morbidity.

Ethical approval

This study was approved by Institutional Ethical Committee vide letter: 2443/PandD/2012.

Acknowledgements

The authors like to thank the Secretary, Department of Health Research, Ministry of Health and Family Welfare, (Grant No. VIR/66/2013/ECD-I), National Vector Borne Disease Control Programme, Directorate of Health Services, Government of India, for financial support and providing DEN IgM kits. The authors are thankful to the CDC, National Center for Emerging and Zoonotic Infectious Diseases, Division of Vector-Borne Diseases, DEN Branch, San Juan, USA, for providing DENV diagnostic kits for qRT-PCR. The authors also thank the State Directorate of Medical Education, Government of Tamil Nadu, and doctors.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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

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