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| ORIGINAL ARTICLE |
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| Year : 2018 | Volume
: 36
| Issue : 1 | Page : 37-42 |
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Molecular characterisation and phylogenetic analysis of dengue outbreak in Pasighat, Arunachal Pradesh, Northeast India
Biswajyoti Borkakoty1, Mandakini Das1, Kishore Sarma1, Aniruddha Jakharia1, Palash Kumar Das1, Chandrakanta Bhattacharya1, Basumoti Apum2, Dipankar Biswas1
1 ICMR- Regional Medical Research Centre, N. E. Region, Dibrugarh, Assam, India
2 Department of Microbiology, General Hospital, Pasighat, East Siang, Arunachal Pradesh, India
| Date of Web Publication | 2-May-2018 |
Correspondence Address:
Dr. Mandakini Das
ICMR- Regional Medical Research Centre, N.E. Region, Dibrugarh - 786 001, Assam
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/ijmm.IJMM_17_30
Background and Objectives: Dengue is one of the most prevalent arboviral diseases in the world with 390 million dengue infections per year. In this study, we report the molecular characterisation of dengue outbreak in Pasighat, Arunachal Pradesh, Northeast India during 2015. Subjects and Methods: A total of 613 dengue-suspected cases were screened for dengue virus by dengue NS1 Ag and anti-dengue IgM antibody depending on the duration of sample collection and onset of symptom. Further, molecular characterisation was done by amplifying the C-PrM region by real-time polymerase chain reaction followed by phylogenetic analysis. Results: Molecular characterisation revealed that the dengue outbreak was predominantly due to dengue virus serotype-1 (DENV-1) (90.9%) while DENV-2 was detected in 7.5% of samples. Co-infection of DENV-1 and DENV-2 was detected in one case. Phylogenetic analysis of the DENV-1 strains with the prototype revealed that the DENV-1 strains were grouped within genotype III. Similarly, DENV-2 strains were clustered within genotype IV. The study revealed a change in the predominant serotype in recent years with DENV-3 in 2012 to DENV-1, 2, 3 and 4 in 2014 to DENV-1 in 2015 in the study region. A unique L24M mutation was observed in the DENV-1 strains of Arunachal Pradesh which was absent in all the circulating strains in India except one strain from the state of Kerala in South India. Marked variation within the DENV-2 strains was observed at A102V and I163V in one strain similar to earlier circulating isolates in India. Conclusions: The present study reveals a shift in the serotype dominance in the study region. As serotype shifts and secondary infection with a heterologous DENV serotype are frequently associated with disease severity, there is an urgent need for sustained monitoring of the circulating serotypes and enhanced surveillance operations, especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in this region.
Keywords: Arunachal Pradesh, dengue virus serotype-1, dengue virus serotype-2, dengue, genotype, serotype shifts
How to cite this article:
Borkakoty B, Das M, Sarma K, Jakharia A, Das PK, Bhattacharya C, Apum B, Biswas D. Molecular characterisation and phylogenetic analysis of dengue outbreak in Pasighat, Arunachal Pradesh, Northeast India. Indian J Med Microbiol 2018;36:37-42 |
How to cite this URL:
Borkakoty B, Das M, Sarma K, Jakharia A, Das PK, Bhattacharya C, Apum B, Biswas D. Molecular characterisation and phylogenetic analysis of dengue outbreak in Pasighat, Arunachal Pradesh, Northeast India. Indian J Med Microbiol [serial online] 2018 [cited 2018 Sep 22];36:37-42. Available from: http://www.ijmm.org/text.asp?2018/36/1/37/231666 |
| ~ Introduction | |  |
Dengue is one of the most prevalent vector-borne, acute arboviral disease caused by one of the five antigenically distinct serotypes of dengue virus (DENV-1, DENV-2, DENV-3, DENV-4 and the most recently identified DENV-5).[1],[2] DENV is a member of the Flavivirus genus with a positive sense single-stranded RNA genome of about 10.7 kb in size.[3] DENV serotypes differ from one another by 25%–40% at the amino acid level and further each serotype is subdivided into distinct phylogenetic clusters or genotypes with variation up to ~3% at amino acid level.[3] DENV-1 and DENV-2 are the most prevalent serotypes worldwide while DENV-3 and DENV-4 are mostly limited to Asian and Southeast Asian countries, and the newly identified DENV-5 has been reported from Malaysia in 2013.[1],[4],[5],[6] Primarily, dengue virus is transmitted by Aedes aegypti and Aedes albopictus.[7] The geographical distribution of both the vectors and viruses along with the co-circulation of multiple virus serotypes has led to the global resurgence of epidemic dengue in the recent years.[8],[9] The World Health Organisation estimated that 3.6 billion people live in dengue-endemic areas and about 390 million dengue infections occur annually of which about 96 million manifest clinically with over 2 million causing dengue haemorrhagic fever (DHF) and 21,000 resulting in death.[10],[11]
Dengue was first reported in India during 1945, and then there was a latent period after which the initial dengue epidemic was reported in 1956 from Vellore in Southern India.[12],[13] Rapid urbanisation and population growth and significant climate change contributed to the increase in dengue fever (DF) cases in India.[14] Gradually, dengue took an epidemic form in India with DF being reported from both urban and rural areas of India.[13],[15]
The DF slowly spread to the hilly regions of Northeast India, and during 1963, there was reported dengue activity in the Lohit district of Arunachal Pradesh.[12] The first laboratory-confirmed dengue outbreak in Arunachal Pradesh was reported from Pasighat in 2012 where 164 suspected cases of DF were subjected for DENV serology at ICMR-, Dibrugarh, of which 107 (65.2%) cases were positive for DENV by Elisa test. The predominant serotype of that outbreak was DENV-3, though DENV-1 and DENV-2 were also detected in few cases.[12] The second outbreak of DENV occurred in 2014, in which 154 suspected cases were subjected for DENV serology by Elisa and 27 were found positive for DENV. In 2014 dengue outbreak, all the four DENV serotypes were detected almost equally, though search for the presence of the novel fifth serotype which has been reported from Malaysia has not been undertaken. There was a latent period in 2013 with no suspected cases reported from the hospital outpatient departments in the region. In 2015, a major outbreak of DENV which took an epidemic form occurred in the town of Pasighat, the district headquarters of East Siang district of Arunachal Pradesh during June to November 2015. Due to climate change and probably with effect of high EL Nino activity in 2015, unreasonably warm weather may have benefitted the Aedes vectors to a higher density. It is known that the extrinsic incubation period for DENV in the mosquitoes shortens from 15 days to 6.5 days at 30°C compared to 25°C.[16] Therefore, unreasonably warm climatic condition along with the presence of the vectors and the agent may have facilitated a massive outbreak of DF in Pasighat, in 2015. To identify which DENV serotypes caused the outbreak in Pasighat region, serum samples from dengue-suspected cases were tested for dengue virus infection and molecular characterisation.
| ~ Subjects and Methods | | |
Investigating teams from the ICMR- Regional Medical Research Centre, N.E. Region, Dibrugarh investigated the dengue outbreak in Pasighat of Arunachal Pradesh during August–September 2015. A subset of 613 cases from the 6195 suspected cases of DF from the region was examined and a 2 ml venous blood sample was collected from consenting patients in K3 EDTA vial for testing of DENV NS1 Ag EIA kits (DRG, USA) for cases within 5 days of onset of illness and by NIV IgM Mac ELISA kit for cases with > 5 days of onset of fever. All the DENV NS1 Ag-positive cases were further processed for RNA extraction using QIAamp Viral Mini kit (Qiagen, USA) according to the manufactures' instructions. DENV serotyping was performed on 5 μl of extracted RNA by a one-step real-time polymerase chain reaction (RT-PCR) using QIAGEN OneStep RT-PCR kit (Qiagen, USA) using DENV type-specific multiplex oligonucleotide primers (covering the C-preM region) for DENV-1, 2, 3 and 4 based on the method described by Lanciotti et al. 1992.[17]
Molecular characterisation of DENV strains was carried out by sequencing the C-prM gene of DENV in the present study. Phylogenetic analysis of the DENV strains was carried out based on the sequenced region. The sequences were analysed using BioEditV 7.0.5.3.[18] Nucleotide sequences corresponding to core pre-membrane (CprM) region or complete genome sequences were downloaded from NCBI-GenBank and aligned with partial nucleotide sequences of DENV from Northeast India using CLUSTAL W in MEGA 6.0. The phylogenetic tree was generated using the maximum likelihood method implemented in the MEGA 6.0 software with Tamura-Nei Model. Nucleotide substitution model was selected based on model test module of MEGA 6.0. Reliability of the individual nodes in the phylogenetic tree was assessed with 1000 bootstrap resampling. Branches corresponding to partitions reproduced in >50% bootstrap replicates are collapsed. Serotype identity was determined on the basis of the phylogenetic tree.
| ~ Results | | |
Amongst 613 dengue-suspected cases, 91 (14.8%) were positive for DENV NS1 Ag, while another 24 (3.9%) were positive for DENV IgM antibody. Thus, a total of 115 cases (18.7%) tested positive for DENV infection amongst the subset. Amongst the 91 NS1 Ag-positive cases subjected to DENV typing by RT-PCR, 72.5% (66/91) could be typed by RT-PCR. The predominant serotype of DENV detected in the 2015 outbreak in Pasighat was DENV-1 (60/66). DENV-2 infection was detected in five cases and co-infection with DENV-1 and 2 was detected in one case. Based on molecular typing, 15 DENV-1, one co-infected and five DENV-2 strains were sequenced for further phylogenetic analysis.
Molecular characterisation of DENV strains detected in the 2015 outbreak was carried out by sequencing the C-prM gene as it harbours epidemiologically important sequence information along with an economic alternative since a single set of primer pair could be used for amplification and sequencing of all the DENV serotypes. Since sequencing was done for serotype confirmation and mutation analysis, therefore only representative samples were selected randomly. Phylogenetic analysis of the DENV strains was carried out based on the sequenced region [Figure 1]. The sequences were deposited in the GenBank database with the following accession numbers KU948525-KU948538. CprM region of dengue virus was aligned with the Indian DENV-1 prototype isolated from Vellore in 1956[13] (GenBank Accession Number: EU626489; numbering based on the prototype Vellore strain in 1956). The DENV-1 strains in the present study were found to cluster within genotype III on phylogenetic analysis by maximum likelihood method [Figure 2]. F84 L mutation present in all the circulating strains of DENV-1 in India was also found in the DENV-1 strains of Arunachal Pradesh. However, L24M mutation which was found to be conserved in all the study strains were absent in the reported circulating strains in India except one strain isolated from Kerala (AFK94064). Strikingly, this particular L24M mutation was found to be present in few of the isolated strains from Guangzhou province, China (AKF11887, AKF11883, AKF11881, AKF11880 and AKF11877). Comparison of 2014 DENV-1 strains with the DENV-1 strains in 2015 reveals three mutations such as G61S, L116S and K118N which were unique in DENV1 strains from Arunachal Pradesh in 2014 but subsequently absent in DENV1 strains from Arunachal Pradesh in 2015. Moreover, these mutations were not found in any of the circulating dengue virus strains as per BLASTp search against the nr (non-redundant) database with the default settings in the NCBI database. | Figure 1: Phylogenetic tree based on the nucleotide variations in the core pre-membrane region of dengue virus
Click here to view |
 | Figure 2: Phylogenetic tree based on the nucleotide variations in the core pre-membrane region of dengue virus serotype 1 serotypes
Click here to view |
The Arunachal Pradesh DENV-1 strains showed 0.1% divergence at nucleotide level and a close homology amongst themselves at amino acid level. However, when compared to prototype reference strain from Vellore, the nucleotide distance was found to be 3.8% and amino acid distance was 1.5%.
Phylogenetic analysis of DENV-2 strains was carried out by aligning the CprM region of the Arunachal Pradesh strains with the reference strains retrieved from NCBI GenBank. All the DENV-2 strains of Arunachal Pradesh clustered within Genotype IV [Figure 3]. The New Guinea C DENV-2 strain in 1944 (GenBank Accession number AF038403) was used as the prototype and numbering was based on this prototype. Compared to DENV-2 prototype strain from New Guinea, four conserved mutations at positions E19A, V112A, K166N and F169 L were identified within the study strains. Marked variations within the DENV2 strains from Arunachal Pradesh were observed at A102V and I163V in one strain (GenBank Accession number KU948527) compared to DENV2 circulating in India. | Figure 3: Phylogenetic tree based on the nucleotide variations in the core pre-membrane region of dengue virus serotype 2 serotypes
Click here to view |
Genetic diversity within the Arunachal Pradesh DENV-2 strains was observed with intrastrain nucleotide diversity of 0%–4.5% (mean 2.1%) and intrastrain amino acid diversity of 0%–2.5% (Mean 1.1). Comparison of the study strains with prototype revealed interspecies nucleotide diversity of 0%–7.7% (mean 3.9) and interspecies amino acid diversity 3.1%–3.7% (mean 3.4). Moreover, the Arunachal Pradesh DENV-2 outbreak strains shared close identity with the DENV-2 Gwalior strains.
Co-infection with DENV-1 and DENV-2 was detected in one study strain. Unique A-T mutation at position 148 in the DENV-1 strain of this mixed strain was observed which was absent in the other DENV strains circulating in Arunachal Pradesh.
| ~ Discussion | | |
An outbreak of DF occurred in Pasighat of Arunachal Pradesh after the monsoon season in 2015. The outbreak was investigated by conducting dengue virus-specific RT-PCR on 613 suspected dengue samples. Dengue viruses were detected in 18.7% of the samples confirming that the fever outbreak was caused by dengue viruses. Molecular typing and phylogenetic analysis revealed that the outbreak was mainly caused by DENV-1. DF outbreaks are generally dominated by a particular serotype as reported from different parts of the world.[19],[20] DENV-2-dominated fever outbreaks were reported in 2011 from Pakistan,[21] in 2013 from Madhya Pradesh, India,[22] in 2010 from Delhi, India, in 2014 from Mozambique [23] and in 2013 from Nepal.[24] DENV-3-dominated outbreaks have been reported in 2013 in Angola,[25] in Yunnan, China.[26] DENV1 dominated outbreak was first reported from Vellore in South India in 1956 and later in 1962-64 from Delhi and Gwalior followed by many DENV1 outbreaks in various parts of India in 1970,1982,1997-1998, 2002-2006,2010 and 2013.[13] DENV1 outbreaks were also reported in 2014 from Japan [27] and Southern Taiwan [28] and in 2011 from Singapore.[29] DENV-4-dominated outbreak was reported in 2009 in French Polynesia.[30] A cross-border surveillance program, UNITE Dengue, revealed that the 2013 outbreaks in Singapore and Malaysia were associated with the replacement of predominant serotype.[31],[32] While the predominant virus in Singapore switched from DENV2 to DENV1, DENV2 became predominant in neighbouring Malaysia.[31]
The year 2015 was characterised by large dengue outbreaks worldwide, with the Philippines reporting more than 169,000 cases and Malaysia exceeding 111,000. Brazil alone reported over 1.5 million cases in 2015, approximately 3 times higher than in 2014. India too had a major dengue outbreak in 2015 reported from various parts of India, with the highest number of dengue-positive cases from New Delhi. The predominant serotype circulating in New Delhi in 2015 was DENV-2 and 4 as reported by investigators from All India Institute of Medical Sciences. DENV-2-dominated outbreak was also reported from the tribal villages of Mandla district of Madhya Pradesh in 2015.
While the dengue outbreak in rest of India was DENV-2 dominated, the dengue outbreak in Pasighat, Arunachal Pradesh, was predominantly due to DENV-1. The role of import of DENV-2 serotype by the student community or the migrant population in Pasighat is therefore cynical. The last dengue epidemic occurred in Arunachal Pradesh in 2014 in which 17.5% samples detected positive for dengue virus by RT-PCR, and all four DENV serotypes were detected almost equally. Our results show a change in serotype dominance from DENV-3 in 2012 to dengue virus Type 1, 2, 3 and 4 in 2014 to DENV-1 in 2015 in Pasighat of Arunachal Pradesh. Interestingly, the DENV-1 strain of 2014 outbreak in Arunachal Pradesh had three unique mutations which were absent in the present outbreak. Close clustering of DENV-1 strains from the present study with the isolates from China indicates that these strains might have predominantly been introduced from China in the recent years.
DENV-2 genotype IV has a wide distribution in tropical and subtropical localities in the world. In India, genotype IV was first isolated during the 1996 dengue epidemic in New Delhi.[33] In the present outbreak, DENV2 strains circulating in Arunachal Pradesh too belonged to genotype IV which has been reported from earlier outbreaks in Gwalior 2001, Delhi 2007–2009 and Kerala 2008–2010.[33],[34] This indicates that the genotype IV of DEN-2 is still the predominant genotype involved in the major outbreaks in India.[35]
Further, it is established that, in secondary infection, particularly sequential infections i.e. DENV-1 followed by DENV-2/DENV-3 or DENV-3 followed by DENV-2 may result in acute vascular permeability syndrome or dengue shock syndrome. Thus, future outbreak with DENV-2/DENV-3 serotype may result in a higher morbidity or mortality in the region. Therefore, enhanced surveillance operations should be deployed, especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in this dengue-endemic region of Arunachal Pradesh in Northeast India.
Genetic diversity amongst the DENV strains of Arunachal Pradesh was observed with unique mutations differing from those circulating strains in Delhi and other parts of India. This increased genetic diversity of the dengue virus may be due to the simultaneous infection of Aedes aegypti/Aedes albopictus by different viruses during outbreaks and epidemics, resulting in a high rate of viral replication resulting in the emergence of genetic changes.[36]
Evolutionary studies worldwide have shown that the genetic diversity of the dengue virus is increasing and is mainly due to mutations and recombinations. The role of each base substitution mutation has not been studied elaborately. However, molecular characterisation of the circulating dengue virus serotypes is essential to monitor the genomic changes occurring in virus as well as its spread.[37] Further study of different gene regions of this strain may be required to detect important mutations and their role in pathogenesis, virulence and outbreak potential, but till then close monitoring of circulating strains along with clinical correlation will be essential to diagnose major epidemics and the geographical migration of these strains.
Financial support and sponsorship
This work was supported by the Regional Virus Research Diagnostic Laboratory, Dibrugarh, Assam, a scheme under Department of Health Research, Government of India.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
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