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 ~  Materials and Me...
 ~ Results
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 37  |  Issue : 1  |  Page : 12-18
 

Molecular studies on dengue viruses detected in patients from Central India


Division of Virology and Zoonoses, ICMR-National Institute of Research in Tribal Health, Jabalpur, Madhya Pradesh, India

Date of Web Publication16-Aug-2019

Correspondence Address:
Dr. Pradip V Barde
ICMR-National Institute of Research in Tribal Health, Nagpur Road, Garha, Jabalpur, Madhya Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_18_377

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 ~ Abstract 


Purpose: Dengue viruses (DENVs), the causative agents of dengue (DEN), are classified into four serotypes and several genotypes. Identifying circulating serotypes and genotypes has clinical and epidemiological importance; however, limited information in this regard is available from Central India. This laboratory-based study was done to fill this lacuna. Materials and Methods: The samples collected in the acute phase of illness were subjected to DEN NS1 ELISA, and NS1-positive samples (n = 80) were subjected to serotyping; representative samples from each serotype were sequenced to identify genotypes. Results: Seventy-one (88.75%) samples could be serotyped. All the four DENV serotypes with dominance of DENV-3 (n = 33; 47%) were detected. DENV-4 was detected after a gap of 3 years. Cases with multiple DENV serotype infection were identified. Genotyping showed that DENV-1 belonging to genotype III, DENV-2 cosmopolitan (IV), DENV-3 genotype III lineage C and DENV-4 genotype I were in circulation in the year 2016. Conclusion: Our study documents the molecular characteristics of DENV circulating in the area. Detection of heterologous DENV serotype with dominance of DENV-3 emphasises the need for regular molecular monitoring.


Keywords: Central India, dengue, genotype, serotype/s


How to cite this article:
Barde PV, Shukla MK, Joshi P, Sahare L, Ukey MJ. Molecular studies on dengue viruses detected in patients from Central India. Indian J Med Microbiol 2019;37:12-8

How to cite this URL:
Barde PV, Shukla MK, Joshi P, Sahare L, Ukey MJ. Molecular studies on dengue viruses detected in patients from Central India. Indian J Med Microbiol [serial online] 2019 [cited 2019 Sep 23];37:12-8. Available from: http://www.ijmm.org/text.asp?2019/37/1/12/264481





 ~ Introduction Top


Dengue (DEN) is the most important arboviral disease.[1] Every year, about 100 million infections are detected across the world,[1] of which 500,000 manifest in haemorrhagic cases and 22,000 deaths are attributed to DEN.[2] The disease is reported from more than 100 countries, and is prevalent in tropical and subtropical regions. Among World Health Organization (WHO) regions, South-East Asia, the Americas and Western Pacific regions are the most seriously affected,[3] and 75% of the population is at the risk of exposure, thus the situation in the South-East Asian region is alarming.[4]

DEN Virus (DENV) belongs to the genus Flavivirus of Flaviviridae family. These are single-stranded positive-sense RNA viruses. The 10.7 kb long RNA codes for a single polyprotein that is cleaved to produce seven nonstructural and three structural functional proteins.[5] DENV has four serotypes (DENV-1–4) which are antigenically distinct. Based on the nucleotide sequence difference of >6%, these four serotypes are further divided into different genotypes with several clades/lineages.[6] A recent study from Pune, India, classified DENV-1 and DENV-2 into six genotypes and classified DEN-3 and DENV-4 into five different genotypes.[6] However, a study has classified DENV-1, DENV-2 and DENV-3 into five distinct genotypes and classified DENV-4 into four genotypes,[7] whereas another study by Chen and Vasilakis classified DENV-1 and DENV-3 into five different genotype, DENV-2 into six genotypes and DENV-4 into four different genotypes.[8]

The infecting DENV serotype and genotype are shown to have an impact on the clinical picture and outcome.[2] DENV serotypes and genotypes are also related to their outbreak, causing potentials of varying severity.[9] Further, the introduction of new serotype or genotype may have both clinical and epidemiological implications.[2] Moreover, infection by multiple serotypes is shown to have a severe outcome.[10] It is also established that the transmission dynamics of arbovirus can change with even a single amino acid change. Thus, to understand epidemiological and clinical outcome/s, it is important to monitor circulating serotypes and their genotypes by conducting molecular studies;[1],[11] this has now become more relevant in the context of recent licensing of vaccine Dengvaxia (CYD-TDV) by the WHO.[12]

Reports from Central India confirm that all the four serotypes are in circulation causing sporadic cases, and DENV-1 and DENV-2 are shown to cause outbreaks with high positivity.[13],[14],[15],[16],[17] Hence, we present this molecular analysis identifying serotypes and their genotypes of DENV circulating in Central India in the year 2016.


 ~ Materials and Methods Top


Study area, study period and source of samples

Madhya Pradesh (MP) is geographically situated in the central part of India. The virology laboratory of the institute is designated as an apex referral laboratory for the diagnosis of DEN in Central India. Blood/serum samples from different government health facilities from all over MP are referred for diagnosis and serotyping of DENV. The samples collected/referred from 1 January to 31 December 2016 from DEN-suspected cases were included in the study.

Testing of samples

NS1 ELISA

Serum was separated from whole blood sample by centrifugation at 3000 revolutions per minute at 4°C. Samples collected in the acute phase of illness (0–5 days of illness, n = 363) were tested for the presence of DENV NS1 antigen by ELISA (PAN BIO, Australia catalogue no. 01P40) as described by manufactures.

Dengue virus serotyping

The acute-phase samples found positive by NS1 ELISA (n = 80) were subjected to serotyping using the Centers for Disease Control and Prevention (CDC)-developed quantitative reverse transcription-polymerase chain reaction (qRT-PCR) kit by following manufacturer's protocol.[18]

Molecular analysis

To establish the genotypes of circulating DENVs, the representative qRT-PCR-positive samples were subjected to one-step RT-PCR. Briefly, RNA was extracted using viral RNA extraction kit (Qiagen, Hilden, Germany catalogue no. 52906), and then the extracted RNA was amplified using one-step RT-PCR kit with high-fidelity platinum Taq polymerase (Invitrogen catalogue no. 11732-088) and different primer sets. For DENV-1, 2 and 3, the primer sets described by Kanakratne et al.[19] were used, whereas the primers suggested by Lanciotti et al. were used for DENV-4.[20] The resulting PCR products were sequenced as described earlier.[15] After manual curation, 11 sequences were submitted to GenBank database of the National Centre for Biotechnology Information (NCBI).

The sequences obtained in the study were subjected to Basic Local Alignment Search Tool at the NCBI database to identity and confirm percent identity and homology. Sequences from the study were further compared with sequences from this laboratory and other global sequences downloaded from the NCBI database, using BioEdit v7.2.5 (Tom Hall, Ibis Therapeutics, CA, USA). Multiple alignment and phylogenetic analysis were performed using CLUSTAL W available with MEGA 5.[21] A neighbour-joining tree was separately constructed with p-distance model for each DENV; the reliability of each node was assessed by bootstrap resembling with 1000 replicates. The DENV-1 tree was constructed using 39 sequences of three major genotypes; similarly, DENV-2 tree was constructed using 39 sequences of six genotypes; for DENV-3 tree, 29 sequences of four genotypes were used and for DENV-4 tree, 16 sequences of four genotypes were constructed. For each serotype, tree sequence of some other serotype was used as out-group [Figure 1], [Figure 2], [Figure 3], [Figure 4].
Figure 1: The phylogenatic tree inferred using the Neighbor-Joining method showing DENV-1 circulating in 2016 belonged to III (American/African) genotype. The sequences in this study are marked with black diamond symbol (◾). DENV-3 sequence was used as out-group

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Figure 2: The phylogenatic tree inferred using the neighbor-Joining method showing DENV-2 circulating in 2016 belonged to genotype IV (Cosmopolitan). The sequences in this study are marked with black square symbol (). DENV-3 sequence was used as out-group

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Figure 3: The phylogenatic tree inferred using the Neighbor-Joining method showing DENV-3 circulating in 2016 belonged to genotype III (linage C). The sequences in this study are marked with black circle symbol (•). DENV-1 sequence was used as out-group

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Figure 4: The phylogenatic tree inferred using the Neighbor-Joining method showing DENV-4 circulating in 2016 belonged to genotype I. The sequences in this study are marked with black circle symbol (•). DENV-1 sequence was used as out-group

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All the diagnosis and experiments were done by following good laboratory practices, ensuring proper positive, negative and no template controls in each test. The laboratory also participated in external quality assurance programme by the national apex laboratory.

Ethical statement

The study was done as a part of the project titled 'Establishment of Grade II Virology laboratory', and this has clearance of the institutional ethical committee (approval no. RMRCT/Ethics committee/2715/2011), and written informed consent was taken from the patients.[22]


 ~ Results Top


Serotyping

Out of eighty NS1-positive samples, 71 (88.75%) could be serotyped using qRT-PCR. Seventeen (23.9%) were positive for DENV-1, 18 (25.4%) were positive for DENV-2, 33 (46.5%) were positive for DENV-3 and only one (1.4%) was detected as DENV-4. Two samples had dual infection of DENV-2 and DENV-3. DENV-3 was the dominant serotype circulating during the study period, DENV-1 and DENV-2 were detected in almost equal proportion, whereas DENV-4 was the least detected serotype that too after a gap of 3 years.

Molecular analysis

We could successfully sequence and submit 11 qRT-PCR-positive samples (DENV-1 [n = 5] [GenBank accession no. KY315509-KY315513], DENV-2 [n = 2] [GenBank accession no. KY315514 and KY315515], DENV-3 [n = 3] [GenBank accession no. KY315516 to KY315518] and DENV-4 [n = 1] [GenBank accession no. KY315519]).

DENV-1 sequences (KY315509–KY315513) from the study showed maximum homology (99%), with the sequences deposited from Gwalior (GenBank accession no. KF289072, JQ692085). The DENV-2 sequences (KY315514 and KY315515) from the study showed 99% homology from Jabalpur, MP (GenBank accession no. KF887485-KF887489); Gwalior, MP (GenBank accession no. DQ448237, DQ448234); and Thiruvananthapuram, Kerala (GenBank accession no. KY427085). DENV-3 (KY315519), which was dominant during the study period, showed a maximum (99.7%) homology with the sequence (GenBank accession no. KY315516) reported from Singapore in 2013. It showed 98.37% and 98.61% nucleotide-level similarity with sequences of DENV-3 that were circulating in the year 2013 (GenBank accession no. KM983551) and 2014 (GenBank accession no. KM983556), respectively, from the study area. DENV-4 detected in 2016 (GenBank accession no. KY315519) showed 98.33% identity with DENV-4 detected in 2010 from Central India (GenBank accession no. JF929180).

The details of serotype (n = 71) and genotype (n = 11) detected in these samples along with other details are given in [Table 1] and [Table 2], respectively. The phylogenetic trees of each serotype showing genotypes are depicted in [Figure 1], [Figure 2], [Figure 3], [Figure 4].
Table 1: Dengue virus serotypes detected from different districts of Madhya Pradesh

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Table 2: Details of genotype of dengue viruses detected in Madhya Pradesh

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 ~ Discussion Top


We could successfully serotype 88.75% of the samples found positive by NS1 ELISA; loss of RNA during transportation, reporting incorrect days of onset of illness by patients, sensitivity of NS1 ELISA, specificity of qRT-PCR and very low amount of viral RNA in the sample could be the reasons for failing of serotyping nine samples.

Detection of all the four serotypes of DENV from cities and villages of MP indicates that DENV has become a perpetual problem. Co-circulation of all the four serotypes is a matter of concern, as this may lead to co-infection by multiple serotypes and/or secondary infections which are known for severe disease outcomes.[23]

The genotyping studies showed a single genotype of each serotype which was circulating during the year 2016. The DENV-1 detected in 2016 showed close relationships with the DENV-1 detected earlier from outbreaks of Central India[13] and belonged to genotype III (American/African [AM/AF] genotype) [Figure 1]. The molecular studies suggest that the Indian DENV-1 belong to different lineages of the AM/AF genotype (genotype III) till 2012; however, in a large outbreak of 2012 in Tamil Nadu, south India infection by Asian genotype (AG) of DENV 1 resulted in higher than average case fatality rates.[9] Although AG was not detected during 2016, it is essential to monitor the circulating genotype of DENV-1 at local level as the emerging AG is more virulent and dominant.[9]

DENV-2 belonging to the Cosmopolitan genotype (IV) was detected during the study period from the area [Figure 2]; viruses which belong to this genotype have caused an outbreak in the tribal area of Central India in 2013.[12],[13] DENV-2 is regarded as the most virulent serotype of DENV,[3] and was the predominant serotype associated with the outbreaks of DEN fever and DEN haemorrhagic fever cases during 1970–2000.[7] The molecular evolutionary analysis of DENV-2 detected in India has revealed that the American genotype was in circulation from 1956 to 1971, which was replaced by Cosmopolitan genotype of different lineages. However, the detection of American genotype in 1980 from Kolkata (West Bengal) and viruses clustering in different lineages warn for molecular surveillance.[24]

DENV-3 was isolated for the first time from India at Vellore in 1966 during the epidemics, and then from Kolkata in 1983 and 1990. It was also detected from Jalore city, Rajasthan in 1985, Dhule, Maharashtra in 1994, Gwalior, MP in 2003-04; and Tiruppur, Tamil Nadu in 2010.[5] DENV-3 is becoming the main serotype responsible for large outbreaks and sporadic cases of DEN since 2003 in North Indian cities such as Delhi, Gwalior and Lucknow.[2],[25],[26] Dash et al.[27] reported DENV-3 as an aetiological factor for an outbreak in 2003 from Gwalior, an urban area of North MP.[17] We have also detected DENV-3 earlier in the years 2012 and 2013; however, during these 2 years, DENV-1 and DENV-2 were the most frequently detected serotypes. In the year 2016, DENV-3 was detected in every second sample serotyped, indicating its dominance. DENV-3 detected in Central India belonged to genotype III [Table 1] and [Figure 3]. Similar findings were reported from the northern part of the country.[28] The analysis revealed that the DENV-3 belongs to lineage C, which is reported as circulating in the Indian subcontinent. The viruses belonging to this lineage are allegedly causing more severe diseases.[29]

The DENV-4 was the least detected serotype and was detected after a gap of 3 years.[6],[15],[30] Similar to DENV-2 and 3, DENV-4 was also detected in the early 1960s from Vellore, Tamil Nadu;[5] however, except for few outbreaks, Kanpur (1968–1969)[31] and Andhra Pradesh (2007),[32],[33] it is rarely associated with major outbreaks.[7] DENV-4 virus detected belonged to genotype I [Table 1] and [Figure 4], in recent past DENV-4 belonging genotype I was detected from Pune, Maharashtra and Jabalpur, MP from sporadic cases and outbreaks.[15],[29],[33],[34] This is known to be circulating in India and other Asian countries including Thailand, Cambodia, Malaysia, China and Sri Lanka.[33] This virus has shown to be responsible for causing outbreaks in South India.[15] It is interesting to note that DENV-4 is the least detected serotype and is rarely responsible for outbreaks; however, it is also reported that the secondary infection by DENV-4 has severe outcomes such as haemorrhagic manifestations[35] and has higher epidemic potential.[36] Further detection of a novel clade DENV-4 with almost 6% nucleotide differences from Pune, India,[6] warns for isolation and in-depth full-length genome analysis of DENV-4 from Central India.

Several known and undiscovered intrinsic and extrinsic factors influence the transmission dynamics and disseminating potential of arboviruses. Adaptability and maintenance of DENV strain in the vertebrate and arthropod population, ability of DENV to achieve high titres during human infections enabling their easy pickup by vectors and vector competence of mosquitoes for particular strain of virus are few to name. Studies have shown that DENV-3 could persist in mosquitoes via transovarial transmission for several generations,[37] and few strains of DENV-3 have lower extrinsic incubation period and achieve rapid transmission.[38] Studies elucidating factors such as molecular analysis of dominating viruses, herd immunity of vertebrate hosts and the entomological factors responsible for the dominance of circulating strain of DENV-3 and scanty presence of DENV-4 are needed.

Further, regular molecular monitoring is essential to keep track of the ever-changing epidemiology of DEN as it is emerging in rural MP with high attack rates.[13],[14],[15] This study has few limitations such as not sequencing all serotyped virus and focusing on partial genome sequences instead of full genome owing to limited resources; nonetheless, this study presents sufficient information on circulating serotypes and genotypes of DENV in Central India.

Studies have shown that change in circulating serotype/genotype increases the severity of DENV infection and outbreak-causing potential.[23],[29],[39] Moreover, due to shortening of travel time, the virus can move along with both humans and vectors, which may result in the introduction of new serotype/genotype of virus to the area. All these factors call for close, comprehensive vigilant monitoring of DENV at molecular level.


 ~ Conclusion Top


Detection of all four DENV serotypes and infection to patients with multiple serotypes are alarming. DENV-3 belonging to genotype III lineage C was dominant during the study period in MP, whereas DENV-4 seems to reappear in the region.

Acknowledgement

The authors are grateful to the Secretary of the Government of India, Department of Health Research, Ministry of Health and Family Welfare, and the Director General, Indian Council of Medical Research (ICMR) (Grant No. VIR/43/2011-ECD-I) and 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 Health Services, Government of MP, and doctors and patients of medical colleges and district hospitals for referring samples. The authors acknowledge help by Dr L Shivlata for her help in molecular analysis. The manuscript has been approved by the Publication Screening Committee of ICMR-NIRTH Jabalpur and assigned with the number ICMR-ICMR-NIRTH/PSC/14/2018.

Financial support and sponsorship

This study was financially supported by the Indian Council of Medical Research (Grant No. VIR/43/2011-ECD-I).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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

  [Table 1], [Table 2]



 

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