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 ~ Introduction
 ~  Materials and Me...
 ~ Results
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 ~ Conclusion
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 3  |  Page : 389-393
 

Molecular and phylogenetic evidence of chikungunya virus circulating in Assam, India


1 Arbovirology Group, Entomology Division, Regional Medical Research Centre, Dibrugarh, Assam, India
2 Department of Microbiology, Gauhati Medical College and Hospital, Guwahati, Assam, India

Date of Web Publication12-Oct-2017

Correspondence Address:
Prafulla Dutta
Regional Medical Research Centre, NE Region, ICMR, Dibrugarh, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_16_127

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 ~ Abstract 

Purpose: Northeast Region of India possesses an abundant number of Aedes mosquitoes, the common vector for Dengue and Chikungunya (CHIK). Dengue is reported every year from Assam, but active surveillance for CHIK virus (CHIKV) infection is lacking in this part of India. Therefore, this present study has been undertaken to detect any CHIKV infection during a dengue outbreak in Assam. Materials and Methods: A total of 42 dengue negative samples collected from Guwahati were screened for the presence of CHIK IgM antibodies. Further, all the samples were processed for CHIKV RNA detection by reverse transcriptase-polymerase chain reaction (RT-PCR). Phylogenetic analysis was done by Maximum Likelihood method using Kimura-2 parameter model. Results: No IgM positivity was found in the processed samples; however, 7 samples were positive for CHIKV by RT-PCR. Phylogenetic analysis revealed that the circulating CHIKV belonged to Eastern, Central and Southern African genotype. Sequence analysis showed two uniform nucleotide substitutions and very less amino acid substitution. Conclusion: Silent existence of CHIKV beside dengue is reported from this study. Therefore, CHIKV diagnosis should be included as a regular practice for active surveillance of the disease and its accomplishment before commencing an outbreak.


Keywords: Assam, chikungunya, Eastern, Central and Southern African, phylogenetic analysis


How to cite this article:
Dutta P, Khan SA, Hazarika NK, Chetry S. Molecular and phylogenetic evidence of chikungunya virus circulating in Assam, India. Indian J Med Microbiol 2017;35:389-93

How to cite this URL:
Dutta P, Khan SA, Hazarika NK, Chetry S. Molecular and phylogenetic evidence of chikungunya virus circulating in Assam, India. Indian J Med Microbiol [serial online] 2017 [cited 2018 Apr 25];35:389-93. Available from: http://www.ijmm.org/text.asp?2017/35/3/389/216613



 ~ Introduction Top


Chikungunya (CHIK) is a febrile infection caused by the CHIK virus (CHIKV). First isolation of the virus was in 1952–1953, from a febrile patient during an outbreak on the Makonde Plateau in the southern province of Tanzania.[1] The name 'CHIK' was originated from a word in the Makonde language, and it means 'that which bends up'. This description refers to the development of bent posture due to the arthritic symptoms of this disease.[2] The virus belongs to the genus Alphavirus, family Togaviridae and is a spherical enveloped virus with a positive-sense single-stranded RNA molecule.

CHIK infection is often characterised by sudden onset of fever, headache, fatigue, nausea, vomiting, rash, myalgia and severe and very painful polyarthralgia, which lasts for generally one to ten days; sometimes may persist for months to years.[3],[4] In general, the infection is self-limiting. Often, the symptoms are clinically indistinguishable from dengue fever symptoms and also described as 'a clinical variant of classical dengue differing in the absence of a headache, of tenderness on pressure to the eyeballs, and of pain on eye movements'.[5] In some cases, both dengue and CHIK occur concurrently; simultaneous isolation of both viruses from the sera of the same patients has been reported indicating the presence of dual infections.[6]

CHIKV is transmitted by Aedes mosquitoes. Among Aedes, the predominant mosquito vector for CHIKV is Aedes aegypti, but an amino acid substitution (A226V) in the viral E1 envelope protein increases the susceptibility of Aedes albopictus to CHIKV, and this might be responsible for the spread of the virus to temperate areas which are inhabited by this species.[7] In the human population, CHIKV is maintained by a human-mosquito-human transmission cycle. This is dissimilar from the sylvatic transmission cycle where the virus is maintained between nonhuman primates, small mammals (e.g. bats and monkeys) and Aedes mosquitoes as observed in the African continent.[8] Mother to child transmission of CHIKV has also been reported.[9]

The origin of CHIKV is believed to be in Africa and later introduction to Asia.[10] Three distinct CHIKV phylogenetic groups West African, Asian and Eastern, Central and Southern African (ECSA) were reported based on the phylogenetic analysis of E1 gene/the complete genome sequences of strains isolated in Africa, Asia and islands of the Indian Ocean and in India.[10],[11],[12] The virus strain that caused the recent Indian Ocean basin outbreak (termed as Indian Ocean Lineage [IOL]) was a descendant of ECSA genotype suggesting global transmission of the virus.[13]

Northeast region of India is facing regular dengue outbreaks in recent years and holds abundant Aedes mosquitoes. During 2015, a dengue outbreak struck Guwahati, Assam where more than 500 cases occurred. However, besides dengue positive cases, there was a high number of dengue suspected cases those diagnosed dengue negative. In view of the presence of the same vector of CHIK as like dengue, we have undertaken this study to evaluate any presence of CHIKV during this dengue outbreak. In Assam, the presence of CHIKV was first reported in 2011, however, there is no active surveillance on the disease, and no further studies have been done to study the virus.[3] As clinical spectrum of CHIK is very much like dengue, the former could be misdiagnosed as dengue based on clinical features.


 ~ Materials and Methods Top


Study area

During June-September, 2015, dengue virus activity was found to be circulated in Guwahati (26.11°N 91.44°E), the Capital city of Assam [Figure 1]. Besides dengue circulation, many cases were Dengue negative but symptomatic to Dengue like illness. These cases were included in the study to detect CHIKV activity during the Dengue outbreak.
Figure 1: Map of Assam, India highlighting the study area Guwahati (star mark). The map is not to scale

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Sample collection

A total of 42 dengue symptomatic but negative blood samples were collected from Gauhati Medical College and Hospital, Guwahati during the dengue outbreak. The study has been approved by the Institutional Ethics Committee, RMRC, Dibrugarh. The Dengue diagnosis was done by either NS1 or IgM Enzyme-Linked Immunosorbent Assay (ELISA) based on the onset of symptoms. NS1 ELISA was done when the symptoms were from <5 days and IgM ELISA when symptoms were for more than 5 days. Clinical symptoms of the patients were recorded at the time of hospitalisation. Written consent was obtained from the patient to participate in the study.

Chikungunya IgM enzyme-linked immunosorbent assay

Serum was separated from the collected blood samples, and 1 μl serum was used to screen the presence of CHIK IgM antibodies using CHIK specific IgM Antibody Capture-Enzyme Linked Immunosorbent Assay (MAC) (National Institute of Virology, Pune, Maharashtra, India) as per manufacturer's instruction.

RNA extraction and reverse transcriptase-polymerase chain reaction

The RNA was extracted from 140 μl of each sample using QIAamp RNA mini kit (QIAGEN, Germany). For amplification, a conserved region of the virus, i.e., E1 glycoprotein gene region was used. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) was done using specific primers as per given protocol[14] for partial CHIKV E1 gene, namely, forward primer, CHIK/E1-S 5′TACCCATTCATGTGGGGC3′ and reverse primer, CHIK/E1-C 5′GCCTTTGTACACCACGATT3′ with modifications.

c-DNA preparation

Initially, the resulting RNA was converted to c-DNA using 2 μl of RNA in 10 μl reaction mixture containing 100U MMLV Reverse transcriptase (Promega), 1 mM dNTPs (Promega), 0.5 mM reverse primer (E1-C) and nuclease-free water. The thermal profile used for the conversion is 37°C for 1 h and 72°C for 5 min.

Polymerase chain reaction

Subsequently, PCR was carried out using the resulting cDNA as template. Five microlitter of cDNA is added to 50 μl PCR reaction mixture with constituents used for the amplification, namely, 2.5U GoTaq DNA polymerase, 0.2 mM dNTPs, 0.6 mM of both forward (E1-S) and reverse (E1-C) primers and nuclease-free water. The primers amplify a 294 bp region of the E1 gene of the virus. African CHIKV (S27) isolated from human serum in Newala, Tanzania and maintained at National Virus Repository (National Institute of Virology, Pune, India) was used as a positive control while performing RT-PCR. The PCR product was electrophoresed in 1.5% agarose gel.

Sequencing and phylogenetic analysis

The amplified products were outsourced to IDT, Malaysia for sequencing by Sanger's method. The obtained sequences were analysed for both directions using BioEdit Sequence Alignment Editor Software (BioEdit, California, USA) and consensus sequences were obtained for each sequence. The sequences were submitted to GeneBank (NCBI) to obtain accession number. The sequences were compared with 47 other sequences selected from detailed literature search and a phylogenetic tree was constructed using maximum likelihood method based on the Kimura 2-parameter model in MEGA version 6 (Pennsylvania State University, USA). As references, strains of all the three genotypes, namely, Asian, West African and ESCA along with IOL strains among ECSA were taken.


 ~ Results Top


All the collected samples were processed for IgM antibody detection, however, no IgM antibody positivity was seen in the samples. Following RT-PCR, the amplification in seven samples (band size 294 bp) confirms their positivity for CHIKV RNA [Figure 2]. Most common symptom observed was fever (100%) followed by headache (42.86%) and backache (28.57%). The symptoms such as arthralgia, vomiting, photophobia and skin rash were observed in only 14.29% cases.
Figure 2: Reverse transcriptase-polymerase chain reaction-based detection of Chikungunya virus in human serum sample. Lane 1: Positive control, Lanes 2: Negative control, Lane 3, 4, 5, 8, 9: Positive samples, Lane 6, 7, 10: Negative samples, Lane 11: 100bp DNA Ladder

Click here to view


After sequencing of all the samples, three sequences were not found satisfactory for further analysis. Therefore, we considered a total of four samples for sequence analysis. These four sequences after obtaining consensus sequence were submitted to NCBI. The accession numbers of the samples are KU216362-KU216365. The BLAST search was done and the sequences of CHIKV from this region were found close to a strain isolated in Calcutta (Now Kolkata) (GenBank accession no.: KJ679577.1) with 99% homology. The similarity-based on partial E1 gene within the CHIKV circulating in Assam was 99.4%. The studied gene region of the virus was also found AG-rich with an average percentage of 29.4% for A and 23.2% for G. The phylogenetic analyses using MEGA6 revealed that the sequences belonged to ECSA genotype [Figure 3]a and did not belong to the IOLs of CHIKV [Figure 3]b responsible for causing massive outbreak in Indian Ocean Island countries.
Figure 3: (a and b) Molecular phylogenetic analyses by using the Maximum Likelihood method based on the Kimura 2-parameter model. The sequences from this study are marked by black circles. WA: West African, ECSA: East/Central/South African, IOL: Indian Ocean Lineage

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The comparison of the entire CHIKV partial E1 gene regions sequenced during the study with the S27 African prototype (GenBank accession no. AF369024.2) revealed the nucleotide substitutions as shown in [Figure 4]. All the substitutions were a transition in nature, and two transitions (T → C at positions 10512 and 10539) were uniform in all CHIKV sequences of this region.
Figure 4: Nucleotide substitution when compared with Chikungunya S-27 African prototype (GenBank accession no. AF369024.2) nucleic acid sequence

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Further, the obtained nucleotide sequences were translated using the Expasy translation tool, Bioinformatics Resource Portal (Swiss Institute of Bioinformatics). Translated results showed that this region is rich in amino acid Alanine with an average of 14.28% followed by almost equal proportion of valine (8.89%) and serine (8.63%). Comparison of amino acid sequences of samples from this region with the prototype CHIKV revealed nonuniform point mutations in KU216363 (A→V at position 936 and 957) and KU216365 (V→M at position 987).


 ~ Discussion Top


The first Indian outbreak of CHIKV was reported in 1963 in Kolkata[15] following epidemics in Chennai, Pondicherry and Vellore in 1964.[16] Subsequently, epidemics occurred in Vishakapatnam, Rajmundry, Kakinada and Nagpur in 1965 and Barsi in 1973.[16] During 2005–2006, severe epidemic of CHIKV infection occurred in Réunion Island, France with approximately 244,000 cases and extending to neighbouring islands such as Mayotte, Madagascar, the Seychelles, Comoros and Mauritius.[17] During this epidemic in these Indian Ocean Island nations in 2005, CHIK re-emerged in India after a gap of 32 years[18] and resulted in massive outbreak.

In Northeast India, CHIK was first reported from Assam in 2008[3] and later from Meghalaya in 2010.[19] In Assam, 10 persons out of 280 symptomatic patients were found to have IgM antibodies against CHIKV, but no further study on the genotype of the virus was done during that period.[3] During CHIK outbreak in Garo Hills, Meghalaya, 23 cases out of 64 were reported to possess IgM antibodies against the virus.[19] High prevalence of Aedes mosquitoes was also reported from both of these states.[3],[19]

During the dengue outbreak in Guwahati, a high number of people suffered. We report in this study the presence of CHIKV beside dengue during the outbreak. In India, the vector responsible for transmitting both dengue and chikungunya is same, and therefore, there is a high probability of CHIK circulation alongside dengue. Many previous studies revealed circulation of dengue and CHIK simultaneously.[20],[21],[22] Furthermore, we did not find any samples positive for IgM antibodies. Thus, screening of suspected samples only for the presence of antibodies is not preferable for active surveillance of the disease.

Phylogenetics study revealed the presence of East/Central African genotype of the virus in the region. Previous Indian isolates of CHIKV were found to belong to the Asian genotype; however, the finding of this study showed that the genotype presently circulating in this region is similar with the genotype circulated after re-emergence of CHIK after a gap of 32 years.[23] Our finding is comparable to different studies in India that showed the circulation of this genotype. The states such as Andhra Pradesh, Maharashtra, Karnataka, Goa and Madhya Pradesh, have reported the same genotype as found in this study.[23],[24] Also in Meghalaya during 2010, East/Central African genotype was responsible for CHIK outbreak.[19] However, when the sequences from this study were compared to the sequences of the monophyletic lineages that caused severe outbreaks in Indian Ocean Island, it revealed that these sequences do not belong to the lineage. Also during that CHIK endemic in India, this region was not invaded by the virus and still it is causing a nonsevere infection. Previous genomic data of the virus from this region are also not available to find out any similarities or differences with the present one.

The comparison of sequences from this region with the CHIK prototype sequence of Africa revealed two uniform synonymous substitutions in all the CHIKV partial E-1 gene sequences. However, in two sequences (KU216363 and KU216365), nucleotide substitution gave rise to amino acid changes. In KU216363, the amino acid valine at the position 957 was previously reported in a CHIK strain IndKL02 from Kerala (GenBank accession no. EU131893.1).[25] The other amino acid point mutations at position 936 of KU216363 and position 987 of KU216365 are novel for the sequences of this region only. We are unable to derive any conclusion about these changes as we do not have any previous data on a molecular level from this region. Further studies on the virus from this region will help in enhancing our knowledge about the evolution of the virus.


 ~ Conclusion Top


The silent existence of CHIKV concealed by dengue has been reported in this study. In the absence of a proper diagnostic facility, the CHIKV can undergo evolutionary changes quietly hidden by other viruses and can take a severe form in future outbreaks. Therefore, the surveillance of arboviral diseases needs to be carried out in a regular basis in the areas where their circulation is often reported, so that the effective control measures can be implemented before its accomplishment into a severe epidemic form.

Acknowledgement

We would like to thank Indian Council of Medical Research, New Delhi, India for funding the study. We are also grateful to the health authorities of the state of Assam for their kind cooperation and help during the study.

Financial support and sponsorship

This study was supported by Indian Council of Medical Research, New Delhi, India.

Conflicts of interest

There are no conflicts of interest.



 
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