Accepted Manuscript

Molecular and phylogenetic analysis of Chikungunya virus in

Central India during 2016 and 2017 outbreaks reveal high
similarity with recent New Delhi and Bangladesh strains

Ankita Agarwal, Sudheer Gupta, Ashvini Kumar Yadav, Ram

Kumar Nema, Kudsia Ansari, Debasis Biswas

PII: S1567-1348(19)30161-3
DOI: https://doi.org/10.1016/j.meegid.2019.103940
Article Number: 103940
Reference: MEEGID 103940
To appear in: Infection, Genetics and Evolution
Received date: 24 April 2019
Revised date: 9 June 2019
Accepted date: 22 June 2019" role="suppressed

Please cite this article as: A. Agarwal, S. Gupta, A.K. Yadav, et al., Molecular and
phylogenetic analysis of Chikungunya virus in Central India during 2016 and 2017
outbreaks reveal high similarity with recent New Delhi and Bangladesh strains, Infection,
Genetics and Evolution, https://doi.org/10.1016/j.meegid.2019.103940

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 ACCEPTED MANUSCRIPT

Molecular and Phylogenetic analysis of Chikungunya virus in Central India during 2016

and 2017 outbreaks reveal high similarity with recent New Delhi and Bangladesh strains

Ankita Agarwal1, Sudheer Gupta, Ashvini Kumar Yadav, Ram Kumar Nema, Kudsia Ansari,

Debasis Biswas* [email protected]

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Regional Virology Laboratory, All India Institute of Medical Sciences Bhopal, Saket Nagar,

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Bhopal-462020, India

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*
Corresponding author at: Principal Investigator, Regional Virology Laboratory, All India

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Institute of Medical Sciences Bhopal, Saket Nagar, Bhopal-462020, India.
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1
Current Address: State Virology Laboratory, Gandhi Medical College, Sultania Road, Royal

Market, Bhopal-462001, India

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Abstract

Central India witnessed Chikungunya virus (CHIKV) outbreaks in 2016 and 2017. The

present report is a hospital based cross-sectional study on the serological and molecular

epidemiology of the outbreak. Mutational and phylogenetic analysis was conducted to ascertain

the genetic relatedness of the central Indian strains with other Indian and global strains.

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Chikungunya infection was confirmed in the clinically suspected patients by the detection of

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anti-CHIKV IgM antibody by ELISA and viral RNA by RT-PCR. A representative set of the

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RT-PCR positive samples were sequenced for E1 gene and analyzed to identify the emerging

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mutations and establish their phylogenetic relationship, particularly with other contemporary

strains. Phylogenetic analysis revealed the present strains to be of East Central South African
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(ECSA) genotype. Emergence of a variant strain was observed in the year 2016, which became

the predominant strain in this region in 2017. The strains showed significant identity with recent
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New Delhi strains of 2015 and 2016 and Bangladesh strains of 2017. The epidemic mutation
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A226V which emerged in 2006 outbreaks of India and Indian Ocean Islands was found to be
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absent in the current strains. Among the important mutations viz. K211E, M269V, D284E, I317V

& V322A observed in the recent strains. I317V is a novel mutation which has emerged very
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recently as it was found only in central Indian (2016, 2017), New Delhi strains (2015, 2016) and
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Bangladesh strains (2017). This study has identified a unique mutation E1:I317V in the Central

Indian strains, which is present only in recent New Delhi and Bangladesh strains till date. This

study highlights the need for continuous molecular surveillance of circulating CHIKV strains in

order to facilitate the prompt identification of novel strains of this virus and enable the

elucidation of their clinical correlates.

Keywords: Chikungunya, Mutations, Phylogeny, Molecular Surveillance

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Introduction

CHIKV belongs to genus Alphavirus, family Togaviridae and is transmitted through the

bite of Ae. aegypti and Ae. albopictus mosquitoes (Jupp and McIntosh, 1988). Chikungunya

symptoms appear after 2-4 days of mosquito bite, with abrupt onset of fever and severe joint

pain. Other symptoms include rashes, myalgia, nausea and headache. In some cases,

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neurological, ocular and gastrointestinal manifestations have also been observed

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(www.who.int/mediacentre/factsheets/fs327/en/). The first case of Chikungunya was reported in

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Tanzania in 1952 (Ross, 1956). Later the disease spread to Africa and Asia including India,

which experienced massive outbreaks in 1960s and 1970s. After a gap of 32 years, an enormous

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outbreak of Chikungunya was recorded in Indian Ocean islands in 2005-2006 that caused 1.2
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million cases (Schuffenecker et al., 2006). The recent spread of this disease to temperate zones

has drawn global attention. As of 2015, the presence of Chikungunya has been observed in 94
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countries belonging to Africa, Asia, Europe, Americas and Oceania and about 1.3 billion people
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are estimated to be living in Chikungunya-endemic areas (Nsoesie et al., 2016). In India also, a

large outbreak occurred in 2006-2007 that affected 1.3 million people across 16 states
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(http://nvbdcp.gov.in/Doc/Annual-report-NVBDCP-2014-15.pdf). Following these outbreaks,

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there was a sudden decline which was followed by sporadic outbreaks reported from various

parts of India (Uthappa et al., 2015; Raghavendhar et al., 2016; Kawle et al., 2017), including
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Central India (Barde et al., 2014). Though the occurrence of Chikungunya in Central India has

been in tandem with that in other parts of the country, studies on the molecular characteristics of

the virus has been rather limited (Barde et al., 2014). In 2016, Chikungunya outbreaks again

resurged in many states of India. According to data available with National Vector Borne

Disease Control Programme (NVBDCP), 64,057 cases were suspected for Chikungunya across
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the country and among them 26,364 cases were confirmed. The Central Indian state of Madhya

Pradesh accounted for 3.2 % of the confirmed cases (https://nvbdcp.gov.in).

Regardless of the potential of emerging viral mutations during outbreaks, the Central

Indian outbreaks have been relatively unexplored and most of the studies have been from other

parts of India (Afreen et al., 2014; Parashar et al., 2015; Saswat et al., 2015; Dutta et al., 2017;

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Tandel et al., 2018). Considering such scarcity of information on new mutants responsible for

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Central Indian outbreaks and in view of the need for continuous virological surveillance, we

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carried out a hospital based cross sectional study wherein febrile patients from Central India

were screened during 2016 and 2017 CHIKV outbreaks. A representative set of the RT-PCR

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positive samples were sequenced and analyzed to identify the newly emerging mutations in order
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to understand the phylogenetic relationship among 2016 and 2017 strains of Central India as well

as with other Indian and global strains.

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1. Materials and Methods

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2.1 Study design and Ethics statement

This is a hospital based cross-sectional study on Chikungunya suspected patients visiting

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the Medicine and Pediatrics Outpatient Department of our tertiary care teaching hospital between
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September 2016 and April 2018. The protocol for this study was duly approved by the

institutional human ethics committee. After recording the clinical details of the recruited
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subjects, serum samples were collected aseptically. Written informed consent was obtained from

adult participants. Parents/guardians provided consent on behalf of child participants.

2.2 Serological diagnosis and Molecular detection

Serum samples were tested by Chikungunya IgM capture ELISA (supplied by National

Institute of Virology, Pune, India) following manufacturer’s protocols. Serologically positive

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samples were subjected to RT-PCR based Chikungunya virus detection. RNA was extracted

from 140 μl of serum using QIAamp viral RNA mini kit (Qiagen, Germany). RT-PCR was

carried out in a 25 μl reaction volume using the SS III platinum one step RT-PCR kit (Invitrogen,

US). The reaction consisted of 2X Reaction Mix (0.4 mM of each dNTP, 3.2 mM MgSO 4),

SuperScript® III RT/Platinum® Taq Mix and 10 pmol of CHIKV specific primers (CHIK15F and

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CHIK16R) (Santhosh et al., 2008) targeting conserved regions of E1 gene. The thermal profile of

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the RT-PCR reaction was: 45°C for 30 min for reverse transcription, followed by an initial

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denaturation of 94°C for 2 min, subsequently followed by 40 cycles of denaturation at 94°C for

15s, annealing at 55°C for 30s, extension at 68°C for 1 min, and a final extension at 68°C for 5

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min. After amplification, PCR products were electrophoresed on 1% agarose gel and visualized
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on a Gel Documentation system (Syngene, USA).

2.3 Nucleotide Sequencing

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A total of 10 representative RT-PCR positive samples were selected for sequencing,

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among which 5 samples each belonged to 2016 and 2017 outbreaks. Amplification was carried

out using primers (CHIK15F and CHIK16R) (Santhosh et al., 2008) as mentioned above.
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Following amplification, PCR products were gel purified using DNA Gel extraction kit (Qiagen,
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Germany) as per manufacturer’s instructions. Double pass sequencing was carried out using

BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems,USA) as per

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manufacturer’s instructions. Reaction mixture was purified by Centri-Sep™ Spin Columns

(ThermoFisher Scientific, USA) as per manufacturer’s instructions and loaded on to the ABI

3730xl DNA Analyzer (Applied Biosystems, USA). The obtained sequences were subjected to

BLAST analysis against the entire database available in NCBI to confirm their identity.

2.4 Phylogenetic analysis

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To conduct phylogenetic analysis, nucleotide sequence of structural gene of CHIKV

representing different genotypes and lineages present worldwide and from the recent Indian

outbreaks were retrieved from NCBI. The sequences were aligned using MEGA version 7.0

(Kumar et al., 2016) to identify the variations among 2016 and 2017 strains as well as with other

Indian and worldwide strains. A Maximum-Likelihood tree was constructed based on the

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Tamura-Nei model called as GTR + G + I model (General time reversible model of nucleotide

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substitution with gamma-distribution rates among invariant sites) (Tamura and Nei, 1993). The

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tree topologies were confirmed using 1000 bootstrap values of the dataset.

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2.5 Selection pressure analysis

In order to investigate the selection pressures on the CHIKV, we performed the selection
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pressure analysis using HyPhy software of MEGA package, using Maximum Likelihood

statistical method. We computed the average numbers of non-synonymous (dN) and synonymous
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(dS) nucleotide substitutions per site (dN/dS ratio).

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2.6 Statistical analysis

Categorical variables like temporal distribution of cases, gender distribution and clinical
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manifestations between RT-PCR positive and negative cases were compared using chi-square
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test. Continuous variable like age distribution was compared by independent two-tailed t-test.

p<0.05 was considered to be statistically significant.

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2. Results

3.1 Demographic characteristics, clinical manifestations and temporal variation of CHIKV

infected patients

A total of 646 Chikungunya-suspects from 2016-2017 outbreak and 1836 from 2017-

2018 outbreak were screened in this study. Comparison of the clinical features between the RT-
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PCR positive and negative cases revealed that majority of the symptoms were similarly

distributed between these two groups except fever (p=0.039), chills (p<0.001) and bone/joint

pain (p=0.014) (Table 1). Most of the IgM and RT-PCR positive cases in both 2016 and 2017

belong to the age group of 16-30 years. Both the outbreaks were observed post-monsoon with

peaks obtained in November within the year 2016 and October within the year 2017 for both

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IgM and RT-PCR positive cases (Figure 1).

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3.2 Serological and molecular diagnosis of CHIKV

Among the Chikungunya suspects, 146 and 361 patients were serologically positive for

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anti-CHIKV IgM antibodies in 2016 and 2017 respectively. Of them, 25 and 31 patients were

positive by RT-PCR. Mean (±SD) duration of illness for IgM based diagnosis was 10 (±9) days,
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while for RT-PCR positive and negative cases was 8 (±9) days and 10 (±9) days respectively
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(Table 1). The mean duration of illness was not found to be significantly different between RT-

PCR positive and negative cases (p>0.05).

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3.3 Mutational and selection pressure analysis

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In the present study, five mutations in E1 gene viz. K211E, M269V, D284E, I317V and
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V322A have been observed (Table 2). While the first 3 mutations have been reported earlier in

ECSA strains from various parts of India, I317V mutation has been a novel emergence with
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previous reports being limited only to the New Delhi outbreak of 2015-16 and Bangladesh

outbreak of 2017. V322A mutation has been acquired by all the genotypes while ‘V’ was present

only in prototype strain.

The selection pressure analysis was done on E1 gene of CHIKV strains, where we

calculated the ratio of the number of non-synonymous nucleotide substitutions per non-
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synonymous site (dN) and the number of synonymous nucleotide substitutions per synonymous

site (dS). The value of dN/dS signifies whether the protein is under positive selection pressure

(dN/dS >1) or under negative/purifying selection pressure (dN/dS < 1 ). The E1 gene of CHIKV

strains in this study displayed a dN/dS ratio of 0.03 which suggests that the E1 gene is under

negative selection.

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3.4 Phylogenetic analysis

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On the basis of phylogenetic analysis, all the 2016 and 2017 strains of this study

belonged to ECSA genotype. The strains of the present study were most similar to New Delhi

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strains of 2015 and 2016 and Bangladesh strains of 2017. Within the ECSA genotype, strains

from Gujarat (2006), Rajasthan (2006), Sri Lanka (2006, 2007), New Delhi, West Bengal, Tamil
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Nadu (2010-2012) were closest to the Central India isolates. As all the present strains harbored
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‘Alanine’ at 226 position of E1 gene, they are similar to strain from Karnataka (2006) (pre-

epidemic strain). All the strains having A226V mutation forms a distinct cluster among the
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ECSA genotype. These include strains from Mauritius, Reunion (2006), Kerala (2007),
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Malaysia, Singapore, Thailand (2008), Thailand (2009) (post-epidemic strain). Brazil (2016)

strains forms a separate group among the ECSA genotype. Asian genotype consisted of older
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strains from India (1963), Philippines (1985), Thailand (1958, 1995) and some newly emerged
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strains in Malaysia (2006) and Caribbean (2014), while strains from Nigeria (1964), Senegal

(1979, 1983), Cote d’Ivorie (1981) belonged to West African Genotype (Figure 2).

3. Discussion

Chikungunya poses a significant health threat in tropics and subtropics. The re-

emergence of CHIKV in 2016 after the massive 2005-2006 Indian Ocean Islands and Indian
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outbreaks is a matter of concern. In the present study, we have undertaken molecular

characterization of CHIKV circulating in Central India during 2016 and 2017 outbreaks.

Sequencing analysis of the E1 gene led to the identification of five important mutations viz.

K211E, M269V, D284E, I317V and V322A. Phylogenetic analysis revealed all the 2016 and

2017 strains of this study belonged to ECSA genotype. Central Indian strains showed closest

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proximity to New Delhi strains of 2015 and 2016 and Bangladesh strains of 2017.

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Among 2482 Chikungunya-suspects from 2016-2017 outbreak, only 20% were

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serologically positive for anti-CHIKV IgM antibodies. Several authors have indicated serological

cross-reactivity (Mardekian and Roberts, 2015) and high co-infection rates (Chahar et al., 2009;

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Saswat et al., 2015; Kaur et al., 2017) in Chikungunya and Dengue endemic areas (Cecilia D,
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2014; Agarwal et al., 2019). Due to the low serological positivity observed in our study, we also

screened the samples for anti-Dengue IgM antibodies and found 30% of the samples to be
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positive for anti-Dengue IgM antibodies. However, none of the samples were found to be co-
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infected. When the CHIKV sero-positive samples were subjected to RT-PCR, only 2 % of them

turned out to be positive. Low RT-PCR positivity might be due to the patients presenting to this
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tertiary care hospital in the later phase of their illness, after the viral clearance. Further, we
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compared the clinical features between the RT-PCR positive and negative cases, and found that

majority of the symptoms were similarly distributed between these two groups except for fever,
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chills and bone/joint pain. RT-PCR positivity has been shown to decline with respect to increase

in days of illness (Ray et al., 2012), but in our study, it was observed up to 8 days. This agrees

with previous studies in which viremia in CHIKV infected patients has been reported up to 8

days as detectable through virus isolation, while viral RNA has been detected as late as 17 days

of illness (Appassakij et al., 2013).

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Mutational analysis of the CHIKV E1 gene sequences revealed five mutations viz.

K211E, M269V, D284E, I317V and V322A. These mutations have been reported in 2016 strains

of New Delhi (Hisamuddin et al., 2018) along with some other mutations (Kaur et al., 2017).

Among these, K211E, M269V and D284E emerged earlier in 2010 outbreak of New Delhi

(Shrinet et al., 2012). Since then, circulating CHIKV has been found to be consistently

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harbouring these mutations. K211E mutation has been observed up to 2014 in all the Delhi

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samples (Singh et al., 2016). This K to E substitution (basic amino acid to acidic amino acid)

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might be responsible for enhancing low pH mediated endosomal entry of the virus within the

host cells (Voss et al., 2010). The role of this mutation in the background of 226A has been

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demonstrated in causing increased infectivity within the Ae. aegypti mosquitoes (Agarwal et al.,
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2016). In spite of the different literature supporting the emergence and sustenance of this

mutation in nature, a recent New Delhi based study on 2016 outbreak, reported the reversion of
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mutant ‘E’ (glutamic acid) to wild-type ‘K’ (Lysine) in two samples (Kaur et al., 2017). This
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underscores the need for molecular investigations for better understanding of the role of this

mutation in viral evolution and spread. M269V, D284E and V322A were previously reported in
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cases from North India during 2006-2008 outbreaks (Singh et al., 2012). It is interesting to note
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that I317V mutation has recently emerged in New Delhi outbreak strains (2015-16) (Kaur et al.,

2017; Hisamuddin et al., 2018), Bangladesh strains (2017) (Pyke et al., 2018; Rahman et al.,
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2019) and is now present in Central India strains as well, while all other strains from ECSA,

Asian and West African genotype still possess ‘I’ amino acid at E1:317 position. The

significance of this novel mutation in influencing the antigenicity, virulence and transmissibility

of the virus remains to be elucidated in future mosquito and mouse model based studies. The

selection pressure analysis of E1 gene of CHIKV strains displayed negative selection. In

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previous studies also, the protein-coding genes of viruses have been shown to display

negative/purifying selection via which the virus tends to stabilize more, by purging deleterious

mutations (Hughes, 2005; Jerzak et al., 2005; Hughes, 2007; Pybus et al., 2007).

Phylogenetically, CHIKV has been classified into three genotypes viz. ECSA, Asian and

West African genotype (Volk et al., 2010). Within the ECSA genotype, two lineages are formed,

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one with the Indian strains possessing Alanine at E1:226 and other is the IOL (Indian Ocean

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Lineage) with E1:A226V mutation. All the strains of this study do not possess the epidemic

E1:A226V mutation. Phylogenetic analysis shows that the strains of the present study belongs to

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ECSA genotype and are located closely to New Delhi strains of 2015 and 2016, Bangladesh

strains of 2017 in the phylogenetic tree. Within the 2016 strains of Central India, emergence of a
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variant strain has been observed, which became predominant strain of this region in 2017. This
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indicates emergence of the mutant virus during 2016, which gained dominance and circulated

with pre-existing strains during the outbreak of 2017.

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In conclusion, this study reflects the increasing endemicity of CHIKV in Central India.
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Our study has identified unique mutation E1:I317V in the Central Indian strains, which is present

only in New Delhi and Bangladesh strains till date. This warrants continuous surveillance for
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mutations having epidemic potential thus aiding in prediction of future outbreaks.

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Acknowledgments

This work was funded by Department of Health Research, Ministry of Health & Family

Welfare, Government of India.

Disclosure statement

The authors declare no conflict of interest.

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Figure legends

Figure 1: Temporal distribution of cases during the outbreak.

Figure 2: Molecular Phylogenetic analysis of CHIKV by Maximum Likelihood method.

Sequences obtained in this study are represented by solid blue (2016) and solid red (2017)

symbols. The tree is constructed on the basis of partial E1 sequence. Sequences used in the

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analysis are represented by GenBank accession number followed by country, year of isolation

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and state. Strains are represented as ECSA (East Central South African genotype), Asian

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genotype and West African genotype. Within the ECSA genotype, strains having novel mutation

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I317V are represented as ‘I317V cluster’. Bootstrap values (1000 replicates) are shown next to

the branches. A scale bar of 0.02 is also indicated in the tree.

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Table 1: Demographic characteristics and clinical manifestations in CHIKV infected patients

2016 - 2018

IgM positive RT-PCR positive RT-PCR negative

Variables cases cases cases
(n=507) (n=56) (n=451)

Age 37 ± 16 36 ± 15 37 ± 16

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Mean duration of illness 10 ± 9 8±9 10 ± 9
Gender

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Male 273 (53.8%) 32 (57.1%) 241 (53.4%)

Female 234 (46.1%) 24 (42.8%) 210 (46.5%)

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Clinical features
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Fever 410 (80.8%) 51 (91.0%) 359 (79.6%)

Chills 201 (39.6%) 28 (50.0%) 173 (38.3%)

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Headache 186 (36.6%) 24 (42.8%) 162 (35.9%)

Bone/ Joint Pain 275 (54.2%) 39 (69.6%) 236 (52.3%)

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Joint Swelling 19 (3.7%) 3 (5.3%) 16 (3.5%)

Myalgia 182 (35.8%) 20 (35.7%) 162 (35.9%)

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Malaise 147 (28.9%) 16 (28.5%) 131 (29.0%)

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Rigors 121 (23.8%) 10 (17.8%) 111 (24.6%)

Rash 36 (7.1%) 5 (8.9%) 31 (6.8%)

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Retro-Orbital Pain 38 (7.4%) 4 (7.1%) 34 (7.5%)

Jaundice 4 (0.7%) 0 (0%) 4 (0.8%)

Thrombocytopenia 5 (0.9%) 0 (0%) 5 (1.1%)

Haemorrhagic manifestations 25 (4.9%) 1 (1.7%) 24 (5.3%)

Altered sensorium 1 (0.2%) 0 (0%) 1 (0.2%)
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Table 2: Amino acid mutations in E1 protein of CHIKV

GenBank Genotype Country/State/City 211 226 269 284 317 322
Accession no. with Year
MH379096 ECSA Central India 2016 E A V E V A
MH379097 ECSA Central India 2016 . . . . . .
MH379098 ECSA Central India 2016 . . . . . .
MH379099 ECSA Central India 2016 . . . . . .
MH379100 ECSA Central India 2016 . . . . . .

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MH379101 ECSA Central India 2017 . . . . . .

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MH379102 ECSA Central India 2017 . . . . . .
MH379103 ECSA Central India 2017 . . . . . .

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MH379104 ECSA Central India 2017 . . . . . .
MH379105 ECSA Central India 2017 . . . . . .

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MF773566 ECSA Bangladesh 2017 . . . . . .
LC364266 ECSA Bangladesh 2017 . . . . . .
KY039475 ECSA Delhi 2015 . . . . . .
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KX619426 ECSA Delhi 2016 . . . . . .
KR706558 ECSA Delhi 2012 . . . . I .
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JN048826 ECSA Delhi 2010 . . . . . .

KF818473 ECSA West Bengal 2012 . . . . . .
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JX276650 ECSA West Bengal 2011 . . . . . .

HM159386 ECSA Tamil Nadu 2010 . . . . . .
EF210157 ECSA Karnataka 2006 K . . . . .
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EU564335 ECSA Rajasthan 2006 . . . . . .

JF274082 ECSA Gujarat 2006 . . . . . .
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GU189061 ECSA Sri Lanka 2006 . . . . . .

HM045799 ECSA Sri Lanka 2007 . . . . . .
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KY704954 ECSA Brazil 2016 . . . D . .

KX228391 ECSA Brazil 2016 . . . . . .
FJ959103 ECSA Mauritius 2006 . V . E . .
MF696157 ECSA Reunion 2006 . . . . . .
FJ807899 ECSA Malaysia 2008 . . . . . .
FJ445506 ECSA Singapore 2008 . . . . . .
EU372006 ECSA Kerala 2007 . . . . . .
GQ428215 ECSA Kerala 2008 . . . . . .
GU908223 ECSA Thailand 2009 . . . . . .
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MF696158 ECSA Thailand 2008 . . . . . .

AF369024 ECSA Tanzania 1953 . A . . . V
HM045796 Asian Thailand 1995 E . M D . A
HM045790 Asian Philippines 1985 . . . . . .
HM045813 Asian India 1963 . . . . . .
HM045810 Asian Thailand 1958 . . . . . .
KP164567 Asian Caribbean 2014 . . . . . .
EU703762 Asian Malaysia 2006 . . . . . .

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HM045818 West Cote d’Ivorie 1981 K . V . . .

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African
HM045786 West Nigeria 1964 . . . . . .

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African
HM045815 West Senegal 1979 . . . . . .
African

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AY726732 West Senegal 1983 . . . . . .
African
*Dot symbol (.) represent similar amino acid, as mentioned in the immediately preceding row
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Highlights
 First report from Central India undertaking clinical and molecular characterization of
circulating CHIKV strains
 A variant strain emerged in this region in 2016, which became the predominant strain in
2017
 The strains showed significant identity with recent New Delhi and Bangladesh strains
 The mutations observed in E1 protein are K211E, M269V, D284E, I317V and V322A
 I317V, identified in our strains, is a relatively novel mutation found only in recent New
Delhi and Bangladesh strains till date

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