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Table of Contents
RESEARCH ARTICLE
Year : 2019  |  Volume : 56  |  Issue : 3  |  Page : 231-236

Circulation of Asian-I and Cosmopolitan genotypes of Dengue-2 virus in northeast India, 2016–2017


Manipal Institute of Virology, Manipal Academy of Higher Education, Manipal, Karnataka, India

Date of Submission25-Apr-2018
Date of Acceptance28-Aug-2018
Date of Web Publication09-Jul-2020

Correspondence Address:
Dr G Arunkumar
Professor and Head, Manipal Institute of Virology, Manipal Academy of Higher Education, Manipal–576 104, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.289402

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  Abstract 

Background & objectives: Dengue is a major public health problem in northeast India where the majority of the cases go unreported and undiagnosed. Even though all four dengue serotypes are reported, there is a dearth of information on genetic diversity. The present cross-sectional study was undertaken during 2016–17 to determine the genetic variance of dengue virus serotype 2 (DENV-2) based on the envelope (E) glycoprotein gene.
Methods: The serum samples collected from the northeast parts of India, as a part of hospital-based acute febrile illness surveillance, were serotyped. Viral RNA was extracted from DENV-2 serum samples using QIAquick® RNA Extraction Kit. The E gene was amplified by conventional reverse-transcriptase polymerase chain reaction (RT-PCR) and the PCR products were sequenced.
Results: The E glycoprotein gene of nine serum samples with high viral RNA concentration (Ct <25) was sequenced. The E gene sequences of eight DENV-2 strains from Assam and Meghalaya aligned with genotype IV (Cosmopolitan) and one strain from Tripura segregated with Asian-I genotype.
Interpretation & conclusion: Ongoing laboratory-based surveillance is mandatory to understand the transmission dynamics of dengue in endemic countries. This study concluded that in northeast India, presently two distinct genotypes of DENV-2, namely genotype IVb (Cosmopolitan) and Asian-I genotype are in circulation.

Keywords: Dengue; DENV-2; envelope gene; genotypes; northeast India


How to cite this article:
Ujwal S, Sabeena S, Bhaskar R, D'Souza G, Santhosha D, Auti A, Kumar R, Ramachandran S, Hindol M, Aithal A, Bhandari Y, Jazeel A, Arunkumar G. Circulation of Asian-I and Cosmopolitan genotypes of Dengue-2 virus in northeast India, 2016–2017. J Vector Borne Dis 2019;56:231-6

How to cite this URL:
Ujwal S, Sabeena S, Bhaskar R, D'Souza G, Santhosha D, Auti A, Kumar R, Ramachandran S, Hindol M, Aithal A, Bhandari Y, Jazeel A, Arunkumar G. Circulation of Asian-I and Cosmopolitan genotypes of Dengue-2 virus in northeast India, 2016–2017. J Vector Borne Dis [serial online] 2019 [cited 2020 Oct 20];56:231-6. Available from: https://www.jvbd.org/text.asp?2019/56/3/231/289402




  Introduction Top


Dengue has re-emerged in tropical and subtropical countries as one of the most prevalent arboviral diseases. The dengue virus (DENV) belonging to the genus Flavivirus and family Flaviviridae has four serotypes[1] called DENV-1, DENV-2, DENV-3, and DENV-4. All the four serotypes can cause a spectrum of disease manifestations ranging from asymptomatic or mild self-limiting febrile illness (dengue fever) to severe and fatal manifestations such as dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The documentation of the genetic variants of the dengue virus is essential in understanding the origin of outbreaks and the liable virulent strains among the human population.

Globally 3.9 billion people in 128 countries reside in dengue-endemic areas[2]. After the global decline in dengue cases during the Zika epidemic, the year 2019 witnessed an increase in several Asian countries, central America and south America[3]. India is home to two-thirds of the global population at risk of dengue and northeast states account for 5.6% of the dengue burden in the country. The state of Assam alone contributes to 68.4% population of northeast India[4] accounting for 1.2% dengue burden in the country[5]. Assam is home to 85% dengue cases reported from the northeast region with Kamrup Metro district reporting the highest number of cases[5],[6]. Meanwhile, the neighbouring states of Tripura and Meghalaya reported 100 and 61 dengue cases, respectively with no fatalities[5]. Northeastern India has a humid tropical climate with heavy rains which is highly conducive to the breeding of Aedes aegypti mosquitoes. Even though there are reports of all the four dengue virus serotypes from the northeast, there is a lack of molecular data which is addressed in this study.


  Material & Methods Top


The present cross-sectional study was carried out to understand the genetic nature of the DENV-2 serotypes based on envelope glycoprotein gene (E gene) in northeast India, using serum samples archived as a part of ongoing “Hospital-based surveillance of Acute Febrile Illness (AFI) in India”. Informed written consents were obtained from all the study participants.

The serum samples serotyped positive for DENV-2 by real-time reverse-transcriptase polymerase chain reaction (RT-PCR)[7] representing the period 2016–17 were included by purposive sampling. The viral RNA was extracted from 150 μl serum using QIAquick® RNA Extraction Kit (Qiagen, Germany) as per the manufacturer’s instructions. The E gene amplification was performed to obtain the product size of 1616 bp spanning the complete E gene, by conventional RT- PCR using the forward primer (D2Seq3F) and reverse primer[8] (D2Seq7R) [Table 1]. The extracted nucleic acid (5 μl RNA) was added to the 20 μl reaction mixture containing 1μmol of each primer, 15 μl buffer mix (Ambion® Life Technologies, USA), 1 μl enzyme mix (Ambion Life Technologies®, USA) and 2 μl of nuclease-free water (NFW). The amplification was carried out in the ProFlex™ PCR system (Life Technologies, USA). The reverse transcription was carried out at 50 °C for 30 min and initial denaturation at 94 °C for 10 min. The cDNA was amplified by 40 cycles of denaturation (94 °C, 30 sec), primer annealing (58 °C, 30 sec), primer extension (60 °C, 2 min) and a final extension at 65 °C for 5 min. The PCR products were electrophoresed on 1 % (w/v) agarose gel and were purified using the GenElute™ gel purification kit (Sigma Aldrich, Merck). The samples with higher RNA concentration were included for sequencing. The purified PCR products were directly sequenced using specific primers[8] and the Big Dye terminator kit (Applied Biosystems, USA) as per the manufacturer’s instructions in a 3500 XL Genetic analyzer (Applied Biosystems).
Table 1: Primers used for amplification and sequencing of the E gene of DENV-2

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Table 2: Variation in DENV-2 strains in comparison with global and regional strains at nucleotide and amino acid level

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A total of nine DENV-2 sequences were analysed using Sequencher 5.4.6 software. All the nine sequences were subjected to multiple sequence alignment (MSA) using Clustal W in MEGA 7.0 (http://www. megasoftware. net/). The nucleotide and deduced amino acid sequences of the partial E gene (1248 nucleotides) of the strains were compared with previously published E gene sequences in the NCBI nucleotide database. The phylogenetic tree was constructed using the maximum-likelihood method with reliability testing by 1000 bootstrap replications. Tamu-ra-Nei model, namely TN93+G+I, TN93+G and TN93+I were found to be the best evolutionary model for codon positions 1, 2 and 3, respectively using Model selection in MEGA 7.0 where ‘I’ represents invariant and ‘G’ represents gamma, a shape parameter for the model. To assess the amino acid diversity of the Cosmopolitan strains, we used earlier Indian strains since 1974 and NCBI global reference genome for DENV-2 (NC_001474) [Supplementary Table 1*].[Additional file 1] Since the present cross-sectional study was the first study reporting Asian-I genotype in India, Tripura strain was compared with NCBI global reference genome for DENV-2 (NC_001474) and with a strain of nearest phylogeny from Myanmar (KX357995)[9] [Supplementary Table 2*].[Additional file 2] Socio-demographic, clinical and laboratory details of the cases were obtained from the AFI database.

The statistical analysis was carried out using SPSS 15.0 for Windows (SPSSTM Inc, USA). Number and percentages summarised the demographic data and baseline characteristics. Meanwhile, continuous data were represented by the mean and standard deviation.

Ethical statement

The study was reviewed and approved by the Institutional Ethical Committee of Manipal Academy of Higher Education (IEC No. UEC/32/2013–14, MUEC/Renew- al-08/2017). All the procedures performed in the studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.


  Results Top


During the study period, 63 serum samples were serotyped as DENV-2 from northeast India, out of which 58 were from Assam, four were from Meghalaya and one was from Tripura. The mean age of the DENV-2 positive cases included in the study was 31.7 yr (SD =13.6). About 55 % of enrolled cases belonged to the age group of 19–39 yrs. Among these, 34 (53%) samples were collected from males and 29 (47%) were retrieved from females. In total, nine serum samples with high viral RNA concentration (Ct value <25) were included for sequencing. Out of these nine samples, six were collected from Amerigog and Saruti Gaonpanchayats, located in Kamrup Metro district of Assam; two were procured from Baridua panchayat, Meghalaya and one sample belonged to Dhalai district of Tripura state.

The phylogenetic analysis showed that the (E) gene sequences of eight DENV-2 strains collected from patients in 2016 grouped along with the genotype IV (Cosmopolitan) forming a diversified subclade within the clade and one strain from Tripura representative of the year 2017 clustered with Asian-I genotype as in [Figure 1]. Tripura strain grouped with DENV-2 Asian-I genotype strains reported from Cambodia 2009 (JF967989, Myanmar (KX357995) and Australia (JN568246). Tripura strain showed 100% query coverage and 99% identity with DENV-2 Asian-I genotype Myanmar strain (KX357995). The genotype IV (Cosmopolitan) has been previously reported from Delhi (KX061419), Gwalior (DQ448231) and Hyderabad (JX 475906) as shown in [Figure 2]. The nine E gene sequences of the DENV-2 strains, identified in the present study were submitted to the GenBank under the accession numbers MG271942–MG1949 and MG885749.
Figure 1: Phylogenetic tree of DENV-2 genotypes, northeast India 2016–17. The tree was based on 1248 nucleotides encoding the partial envelope gene. Classification and naming of DENV-2 genotypes were established as per the reports of Wang et al[28], and Weaver and Vasilakis[29]. Only bootstrap values >75% were shown on the major nodes. The genotype IV is represented as black circle • and Asian-I genotype is shown as a black triangle ▴.

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Figure 2: DENV-2 genotypes circulating in southeast Asia (India and neighbouring countries), 2016–17. DENV-2 genotype IV (Cosmopolitan) and Asian-I genotype, and Asian-II are represented as circles, triangles, and squares. DISCLAIMER: This map and the data it contains are a graphical representation provided for informational purposes only. This map is not for legal land survey nor does it represent correct coordinate data. Boundary representations are not authoritative.

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When partial nucleotide sequences of the envelope region of all the eight Cosmopolitan study strains were analyzed in comparison to earlier Indian strains, there were 170 nucleotide changes resulting in 43 amino acid mutations in the translated E gene [Supplementary Table 3*] [Additional file 3]and [Supplementary Table 1*]. A total of 84 nucleotide variations and 10 amino acid (AA) variations were reported when eight Cosmopolitan study strains were compared with NCBI global reference genome for DENV-2 (NC_001474). However, there were no insertions or deletions. All the eight sequenced DENV-2 Cosmopolitan strains had a sequence homology of 99.60–99.76%. Multiple sequence alignment using Clustal O (1.2.4) for percentage identity matrix showed an average identity of 93.64% for all the eight Cosmopolitan study strains with NCBI global reference genome for DENV-2 (NC_001474) and 98.99% with the regional strain (JX475906) of closest phylogeny [Supplementary Table 4*].[Additional file 4] As shown in and [Supplementary Table 2*], unlike the closest phylogeny strain (JX475906) at position 46, Isoleucine (I) was replaced by Valine (V) in all the eight Cosmopolitan genotypes. At position 176, Threonine (T) was replaced by Isoleucine (I) by one of the Assam study strains (MG271949). At position 366, Asparagine (N) was substituted by Tyrosine (Y) in two study strains from Assam (MG271946 and MG271947).

The present paper reports the first case of Asian-I genotype of DENV-2 in India. The DENV-2 sequences were compared with reference strains and NCBI global reference genome for DENV-2 (NC_001474) [Supplementary Table 5].[Additional file 5] At position 46, Valine (V) was replaced by Isoleucine (I) in comparison to Myanmar strain[9] (KX357995). Meanwhile, the NCBI global reference genome had Isoleucine at position 46. At remaining sites such as 83,120,129,141,164, 226, 228, and 346 there were amino acid variations when compared with NCBI reference sequence. However, there were no amino acid changes when compared with the Myanmar strain (KX357995).

In the present study, all the patients with DENV-2 infection presented with mild febrile illness and had an uneventful recovery. Out of the nine patients whose serum samples were genotyped, one patient with infection by Cosmopolitan genotype had moderate thrombocytopaenia (<1 lakh/mm3) and none had leucopenia. Seven patients presented with warning signs such as vomiting and lethargy. To the best of our knowledge, this is the first study revealing the circulating genotypes of DENV-2 from northeast India.


  Discussion Top


The close clustering of the genotype IV sequences identified in the present study with Delhi and Gwalior sequences suggest that this genotype might have been introduced from the northern region of the country. The adjacent Meghalaya state shares the same geographical and climatic properties of Assam. Assam is located >2000 km from Delhi and a lot of people from northeast India travel to Delhi for job and livelihood. The genotype IV (Cosmopolitan) was reported to be circulating in India, Bhutan and Nepal[8],[10],[11]. The earlier molecular studies from Hyderabad (2009), Kerala (2008–2010) and Uttar Pradesh (2009-2012) also have reported the occurrence of genotype IV (Cosmopolitan)[10],[12],[13].

Dengue-2 is the most genetically diverse dengue serotype with six distinct genotypes[14]. The DENV-2 isolates representative of the years 1960 and 1967 from Delhi were grouped into genotype V (American) which was later replaced by genotype IV (Cosmopolitan) in 1996[13],[14],[15],[16],[17]. The replacement of genotype was characteristic of den- gue-2 virus evolution in India[10] which is well-established since 1980. In Srilanka, genotype I strains of the time period 1968–69 were replaced more than two decades later by Cosmopolitan genotype in the 1990s[18]. Dengue fever with haemorrhagic manifestations and plasma leakage were reported from India, Sri Lanka and some Latin American countries when a new genotype replaced the circulating genotype[15],[18],[19].

The phylogenetic analysis of GenBank data suggested the occurrence of two distinct Cosmopolitan sublin- eages[14]. The sublineage IVa has been reported only from southeast Asia, China, and Oceania. Meanwhile, sub- lineage IVb is widespread in the Indian subcontinent[20]. The phylogenetic analysis of CPrM region of DENV-2 samples procured during the 2009 dengue outbreak in the neighbouring state of Kerala[21] revealed the circulation of Cosmopolitan genotype (IV). The study strains belonging to genotype IV grouped under Cosmopolitan sublineage IVb. According to Mishra et aP, DENV-2 genotype IVb is rampant in India causing severe and widespread outbreaks. We also observed the circulation of Asian-I and genotype IV in the present study. The co-circulation of two distinct genotypes such as Asian-II and IVa was reported from Nepal[8] during the dengue outbreak of 2013. Asian-I strains are currently circulating in southeast Asian countries such as Thailand, Malaysia, Cambodia, Vietnam, and Myanmar. Meanwhile, the Asian-II genotype had been reported from China, Sri Lanka, Taiwan, Vietnam and Nepal[8],[22].

In the present study, we did not observe any significant mutation in nucleotide and amino acid sequences. There are three E glycoprotein functional domains such as domains I, II and III which correspond to E antigenic domains, namely A, B, and C. Even though four sites at 64, 126, 310 and 360 were under positive selection, only site 126 in the Cosmopolitan genotype was under significant positive selection[23]. The change from acidic Asparagine (N) to neutral Aspartic acid (D) in E390 was considered significant as there was an increased number of DHF cases in south America[24]. The mutation from Glu (G) to Lysine (K) at position 126 resulted in a more neurogenic dengue virus in mice[25]. These mutations were more pronounced in Cosmopolitan genotype in comparison to Asian strains.

The most important challenge in preventing dengue epidemics at the population level is to predict and terminate the progress of emerging infections. Genomic sequencing is vital in correlating predominant circulating genotypes with virulence[26]. The greatest dengue genetic diversity was observed in southeast Asia from where the spread of viral lineages results in widespread epidemics globally, and multicentric laboratory-based studies have much significance from the public health perspective[27].


  Conclusion Top


The study indicates that presently DENV-2 genotype IVb (Cosmopolitan) and Asian-I genotype are in circulation in northeast India. Constant virological surveillance at various sentinel points is required to understand the transmission dynamics of dengue in India.

Conflict of interest: The authors declare no financial or commercial conflict of interest.


  Acknowledgements Top


The study was partially supported by CDC Cooperative Agreement (5U01GH001051) and ICMR grant File No. 5/8/7/15/2010/ECD-I). The authors sincerely acknowledge the help of the clinicians in Sonapur District Hospital, Assam and Sub-District Hospital, Longtarai, Manu, Tripura state and express their gratitude to the State health officials of Assam and Tripura for supporting the study.





 
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