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Chikungunya virus: recent advances in

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 Pathogens and Disease, 74, 2016, ftv119

doi: 10.1093/femspd/ftv119
Advance Access Publication Date: 10 December 2015
Minireview

MINIREVIEW

Chikungunya virus: recent advances in epidemiology,

host pathogen interaction and vaccine strategies
Farah Deeba1 , Asimul Islam1 , Syed Naqui Kazim1 , Irshad Hussain Naqvi2 ,

Downloaded from http://femspd.oxfordjournals.org/ by Shama Parveen on February 4, 2016

Shobha Broor3 , Anwar Ahmed4 and Shama Parveen1,
1
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India,
2
Dr. M. A. Ansari Health Centre, Jamia Millia Islamia, New Delhi 110025, India, 3 Department of Microbiology,
SGT University, Gurgaon 122001, Haryana, India and 4 Protein Research Chair, Department of Biochemistry,
King Saud University, Riyadh 11451, Saudi Arabia

Corresponding author: Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India. Tel: +91 011 26984167 ext.
4492; E-mail: shamp25@yahoo.com
One sentence summary: This review provides a comprehensive outline of the Chikungunya viral infection with emphasis on the epidemiology, host
pathogen interaction and vaccine stratagies.
Editor: Alfredo Garzino-Demo

ABSTRACT
The Chikungunya virus is a re-emerging alphavirus that belongs to the family Togaviridae. The symptoms include fever,
rashes, nausea and joint pain that may last for months. The laboratory diagnosis of the infection is based on the serologic
assays, virus isolation and molecular methods. The pathogenesis of the Chikungunya viral infection is not completely
understood. Some of the recent investigations have provided information on replication of the virus in various cells and
organs. In addition, some recent reports have indicated that the severity of the disease is correlated with the viral load and
cytokines. The Chikungunya virus infection re-emerged as an explosive epidemic during 200409 affecting millions of
people in the Indian Ocean. Subsequent global attention was given to research on this viral pathogen due to its broad area
of geographical distribution during this epidemic. Chikungunya viral infection has become a challenge for the public health
system because of the absence of a vaccine as well as antiviral drugs. A number of potential vaccine candidates have been
tested on humans and animal models during clinical and preclinical trials. In this review, we mainly discuss the
hostpathogen relationship, epidemiology and recent advances in the development of drugs and vaccines for the
Chikungunya viral infection.

Keywords: Chikungunya virus; disease dissemination; hostpathogen interactions

INTRODUCTION infection is underestimated because of its overlapping symp-

toms with dengue fever.
The name Chikungunya was derived from a Makonde word that
The Chikungunya virus has caused many outbreaks
means one which bends up; it refers to the posture of the pa-
worldwide in the last decade, including the Indian Ocean
tient due to extreme joint pain. The symptoms of Chikungunya
region, African countries and American continent. During
virus (CHIKV) infection are high fever, rashes, nausea, arthral-
the 200409 epidemic the CHIKV was found to infect another
gia, myalgia etc. (Schuffenecker et al. 2006). The Ades aegepti
species of mosquito, i.e. Aedes albopictus due to a single point
mosquito is the main vector involved in the transmission of
mutation in the genome (Schuffenecker et al. 2006). This severe
the disease. However, the actual burden of Chikungunya viral

Received: 11 September 2015; Accepted: 5 December 2015


C FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

1
 2 Pathogens and Disease, 2016, Vol. 74, No. 3

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Figure 1. Chikungunya virus replication cycle. The CHIKV enters into the host cell through receptor-mediated endocytosis forming an endosome. The endosome enters
the host cell and genome of the virus then replicates in the cytoplasm. The CHIKV genome carries non-translated region (NTR) at both 3 and 5 ends and non-coding
junction region (J region) between the genes coding for structural and non-structural proteins. The replicated negative strand of RNA is transcribed into positive sense
subgenomic RNA, also known as 26S RNA, that serves as mRNA for structural proteins. The genomic and subgenomic RNAs are translated into polyprotein precursors
that finally cleave into non-structural proteins (nsp1, nsp2, nsp3 and nsp4) and structural proteins (capsid, E1 and E2). The positive RNA strand, structural proteins
and capsid proteins finally assemble into the mature virion. ER, endoplasmic reticulum.

outbreak occurred in 2004 in the Indian Ocean region including
India. CHIKV infection is affecting the socioeconomic status of
various geographical regions because of the absence of an ef-
fective vaccine and antiviral drugs. Chikungunya viral infection
is a major health concern in India also due to the presence of
a large immunologically naive population. Therefore, regular
surveillance for the Chikungunya viral infection should be done
in the endemic regions such as India. This will assist in the
prediction and control of outbreaks. In addition, the generation
of awareness among the general public about the disease is
essential for its effective control and management.
GENOME
CHIKV is a member of the genus Alphavirus, in the family To-
gaviridae (Powers et al. 2000). The CHIKV is a small (6070 nm in
diameter) and enveloped virus. The 11.8 kb genome of CHIKV is a
linear, positive sense RNA molecule. Figure 1 shows a schematic
representation of the genome of CHIKV and its replication in
the host cell. Two-thirds of the genome from the 5 end has a
methyl guanylate cap that consists of a non-structural polypro-
tein precursor. This precursor is cleaved into non-structural pro-
teins (nsp1, nsp2, nsp3 and nsp4) that are involved in the repli-
cation of the virus. The nsp1 protein functions as a cytoplasmic
RNA capping enzyme, whereas the nsp2 protein has two sepa-
rate domains with helicase and protease activity. The nsp3 pro-
tein is essential for negative strand RNA synthesis. The nsp4
protein works as an RNA-dependent RNA polymerase (Solig-
nat et al. 2009). One-third of the genome from the 3 end has
a polyadenylate tail that consists of 26S mRNA whose prod-
ucts include the structural proteins. The structural proteins of
CHIKV are the surface glycoproteins (E1 and E2), capsid pro-
tein and small peptides (E3 and 6K) (Weaver et al. 2005; Pow-
ers and Logue 2007; Abere et al. 2012). The viral surface is cov-
ered with membrane-anchored spikes which are triplets of a
heterodimer of envelope E1 and E2 glycoproteins. The E2 is ex-
posed and has a role in viral attachment, whereas E1 is the
fusion protein that facilitates the viral entry. The epitopes in
the E2 glycoprotein are recognized by neutralizing antibodies
and therefore E2 is the vaccine candidate (Brehin et al. 2008).
The lineages of CHIKV are reported with different genotypic
characteristics. The genotypes are classified on the basis of
E1 gene sequence and the full genome of CHIKV. Three geno-
types (East, Central and South African (ECSA), West African and

Asian) have been described for the Chikungunya virus (Santhosh
et al. 2008).
VECTOR AND DISEASE TRANSMISSION
CHIKV is an arbovirus that spreads mainly through Aedes ae-
gypti and Aedes albopictus mosquitoes. The CHIKV life cycle in-
volves: (i) an enzootic sylvatic cycle, and (ii) an epidemic ur-
ban cycle. The sylvatic cycle is maintained primarily in African
forests involving several arboreal Aedes mosquito species as
vectors and non-human primates like monkeys, rodents, birds,
etc. as reservoirs (Apandi et al. 2009; Caglioti et al. 2013). It is
believed that initially in Africa, the enzootic transmission cy-
cle spilled over and infected nearby human populations (Thon-
non et al. 1999; Peyrefitte et al. 2007). The mosquitoes are also
involved in inter-human transmission during outbreaks. Dur-
ing epidemics, human beings serve as reservoirs in the urban
cycle.
CLINICAL MANIFESTATIONS
The alphaviruses are clinically divided into two subgroups: en-
cephalogenic (e.g. Venezuelan equine encephalitis virus, West-
ern equine encephalitis virus), which infect neurons, and
arthritogenic (e.g. Chikungunya virus, Sindbis virus) which are
basically associated with polyarthritis and rashes. The infection
with CHIKV is followed by a silent period of incubation that lasts
24 days. The incubation period is followed by an abrupt onset
of symptoms, including fever, headache, nausea, myalgia and
rashes (Pialoux et al. 2007). The most prominent and typical clin-
ical feature is the polyarthralgia that can sometimes persist for
as long as a few months to years (Schilte et al. 2013). Chikun-
gunya viral infection is commonly not found to be associated
with mortality. The severity of disease in the case of CHIKV
infection is directly attributed to the severe and prolonged
symptoms or effects. In some cases lymphopenia, thrombo-
cytopenia and minor hemorrhagic signs such as epistaxis or
gingivorrhagia have also been reported (Schuffenecker et al.
2006). During the 2006 outbreak in Reunion Island neurological
complications such as meningoencephilitis were also reported
in a small proportion of patients (Quatresous 2006). During this
epidemic optic neuritis, anterior uveitis and dendritic lesions oc-
curred as primary acute onset inflammatory reactions (Lalitha
et al. 2007; Mittal et al. 2007). Certain follow-up studies of pa-
tients with Chikungunya viral infection have shown evidence
of cardiac complications such as myocarditis (Obeyesekere and
Hermon 1973).
Chikungunya viral infection is generally a self-limiting febrile
illness. The resolution of CHIKV infection takes place within the
first 15 days of illness after which the disease enters a stage
where chronic rheumatism remains as the major feature (Man-
imunda et al. 2010). The symptoms often mimic those of dengue
fever and its spread is also common in dengue endemic regions,
therefore the two diseases are commonly indistinguishable (Ravi
2006).
DETECTION OF CHIKUNGUNYA
VIRUS INFECTION
Diagnosis of Chikungunya viral infection is based upon the clini-
cal symptoms and laboratory diagnosis. Routinely used diagnos-
tic methods include isolation of virus in cell culture, identifica-
tion of viral RNA by reverse transcriptase (RT)-PCR/real-time PCR
Deeba et al. 3
and detection of antibodies (IgM/IgG) to the virus. The CHIKV
isolation is performed on serum samples collected within the
first 7 days of illness. The virus can be isolated in insect and
mammalian cell lines (C636, Vero). The cultured virus can be
confirmed by RT-PCR or immunofluorescence assay. Virus iso-
lation is time consuming and needs expertise therefore it is no
longer being used for the detection of CHIKV infection. The con-
ventional PCR and real-time PCR assays have been developed
targeting the envelope and the non-structural genes of CHIKV.
RT-PCR can be used for early identification of the CHIKV in-
fection before detection of the antibodies, i.e. within the first
710 days of infection. DNA sequencing coupled with the RT-PCR
technique can be used to identify the corresponding genotype.
Real-time PCR is used to determine the viral load in the clini-
cal samples. The sensitivity of real-time RT-PCR was found to be
around 10-fold higher than conventional RT-PCR and as low as
20 copies of RNA transcript can be identified by real-time RT-PCR
(Edwards et al. 2007).
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The diagnosis of the CHIKV infection can also done by de-
tection of the immune response by serological assays. The im-
munoassays for IgM/IgG (ELISA) and indirect immunofluores-
cent assay (IFA) are used for detection of antibodies to the virus.
The indirect immunofluorescent assay is also a method for the
detection of Chikungunya virus in the clinical samples (Yer-
golkar et al. 2006). The IgM antibodies develop within 23 days
after the onset of symptoms of infection and may persist for sev-
eral months (Sam and AbuBakar 2006; Malvy et al. 2009). The IgG
antibodies develop in the convalescent phase and may persist
for several months to years (Cavrini et al. 2009). The sensitivity
and specificity of serological assays are poorly established be-
cause of the possibility of false positive results due to cross re-
activity with antibodies to dengue or other arboviruses (Pialoux
et al. 2007). Therefore, limitations with detection of the Chikun-
gunya viral infection by IgM capture ELISA exist. Recently a study
has tried to improve the ELISA by developing antigen for the
assay using Eilat virus instead of the Chikungunya virus (Eras-
mus et al. 2015). The antigen that was developed does not re-
quire biosafety containment facilities and can be used for ELISA
without inactivation. However, this assay needs to be tested and
evaluated before its implementation on clinical samples.
HOSTPATHOGEN INTERACTIONS
The Chikungunya virus first replicates in the skin fibroblasts af-
ter inoculation into the skin of humans through the mosquito
bite. Only fibroblasts of the skin have been shown to be sus-
ceptible to CHIKV infection amongst various types of cells
(Talarmin et al. 2007; Robin et al. 2009). Once replicated in the
skin, it further disseminates through blood to liver, muscle tis-
sues, joints, lymph nodes and brain (Fig. 2). On reaching muscles,
the Chikungunya virus replicates in muscle satellite and fibrob-
last cells and not in muscle fibres (Ozden et al. 2007). The CHIKV
replicates in joint fibroblasts and the synovial tissues (Hoarau,
Jaffar Bandjee and Krejbich Trotot 2010). In addition, the Chikun-
gunya virus RNA and proteins have been identified in the syn-
ovial tissues during severe and persistent arthralgia (Parola et al.
2006; Manimunda et al. 2007; Ozden et al. 2007; Hoarau, Jaffar
Bandjee and Krejbich Trotot 2010).
When the virus disseminates to brain it seems to infect the
lining of the choroid plexus of stromal cells of the nervous sys-
tem. The CHIKV tissue tropism studied in a mouse model was
found to match with the tissue tropism reported in the in vitro
system. The onset of symptoms coincides with a rise in viral
 4 Pathogens and Disease, 2016, Vol. 74, No. 3
Figure 2. A schematic representation of the systemic Chikungunya virus dissemination. The Chikungunya virus enters the host cell by the mosquito bite. The virus
then replicates into fibroblasts of skin and enters nearby lymph nodes through which it disseminates into the blood. The Chikungunya virus further migrates to
different organs and tissues through blood vessels.
Figure 3. A graphical representation showing levels of various cytokines and
chemokines after CHIKV infection. The graph shows an increase and decrease
in levels of different cytokines with respect to the onset of symptoms and its
resolution.
titer. Thus activation of the innate immune response occurs,
which results in the production of type I interferons as the pri-
mary response. The type I interferons are mainly involved in
the innate immune responses against viral infections. These
interferons are known to induce the expression of interferon-
stimulated genes (ISGs) having antiviral, antiproliferative and
immune-modulatory functions (Kumar et al. 2012). The immune
response elicited by CHIKV involves the production of many cy-
tokines, chemokines and growth factors. The levels of many cy-
tokines (INF , CXCL9, CXCL10, CCL2 (MCP-1), IL6 and INF ) were
found to be higher in the acute phase and their level decreased
with the convalescent phase (Fig. 3) (Rulli et al. 2011; Wauquier
et al. 2011). Interferon (INF ) is detectable even on the first
day of infection and its level correlates with the plasma viral
load (Dupuis-Maguiraga et al. 2012). The high level of CXCL9,
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CXCL10, CCL2 and IL6 in the acute phase indicates inflammation
initiating the adaptive T cell immune response. The CXCL9 and
CXCL10 are also part of the chemokine program regulating the
migration of monocytes/macrophages, natural killer cells and
memory T cells (Deshmane et al. 2009). The chemokines are cru-
cial mediators of innate as well as adaptive immune responses
against various viral infections (Rot and von Andrian 2004). Both
CXCL9 and CXCL10 are induced by INF and work as a chemo-
attractant for T cells. Both these chemokines signal through a
common receptor, i.e. CXCR3. Therefore, they may have a role
in activation of T cells also, as CXCR3 identifies the T cell sub-
sets (Qin et al. 1998). The CCL2 is also referred to as monocyte
chemotactic protein 1 (MCP-1) that leads monocytes, memory T
cells and dendritic cells to the sites of inflammation.
IL6 is an immune mediator of fever that activates muscle
metabolism so as to increase body temperature (Cronstein 2007).
The levels of granulocyte macrophage colony-stimulating fac-
tor (GM-CSF) receptor and IL6 levels in plasma were significantly
higher in patients with persistent arthralgia (Chow et al. 2011).
IL6 is a pleotropic cytokine and has a destructive role in rheuma-
toid arthritis due to which joint inflammation and pain occur
(Cronstein 2007; Schaible et al. 2010). The increased levels of IL6
may also contribute to the severe joint pain in the CHIKV infec-
tion. The IL6 is also a major B cell growth factor (Hirano et al.
1985) and a known inducer of IgG3 (Kawano, Noma and Yata
1994). The high IgG3 titer is induced by high viremia and is also
correlated with the higher IL6 level. An early increase of IgG3
levels was found to be associated with efficient viral clearance in
vivo (Kam et al. 2012). The higher level of C-reactive protein (CRP)
is activated by higher IL6 levels. Higher CRP levels, in turn, ac-
tivate macrophage-mediated phagocytosis, lymphopenia (very

low levels of lymphocytes), lower monocyte level, neutrophilia
and high viral load (Chow et al. 2011). Eighty percent of the indi-
viduals infected with CHIKV were found to have lymphopenia.
These patients experienced a significantly lower level of circu-
lating B and T lymphocytes (Borgherini et al. 2007). Such low lev-
els of lymphocytes cannot be directly due to the infection, but
it is the higher levels of type I INFs that induce cell death in
lymphocytes (Schwartz and Albert 2010). The elevated level of
RANTES (Regulated on activation, normal T cell expressed and
secreted) is observed during many viral infections. Likewise, the
elevated level of RANTES is also reported during dengue viral in-
fection. An investigation by Ng and colleagues observed that the
level of RANTES decreased significantly during the Chikungunya
virus infection. Therefore, a decrease in the level of RANTES can
be considered a biomarker to differentiate between the Chikun-
gunya and dengue infections since both these viral infections
have overlapping symptoms (Kelvin et al. 2011).
The onset of the adaptive immune response through activa-
tion and proliferation of CD8+ T cells occurs in the early stage
of infection. The percentages of CD3+ and CD8+ lymphocytes
were found to be significantly higher in patients infected with
CHIKV on days 0, 1 and 2 in comparison with healthy controls
(Wauquier et al. 2011). The level of cytokines such as TNF-, IL-
1, IL2, IL5 and IL12 were found to increase with patient recov-
ery (Fig. 3) (Ng et al. 2009; Chow et al. 2011). TNF and IL-1 play
a major role in inflammatory diseases and contribute mainly to
the joint pain in diseases like rheumatoid arthritis. However, in
Chikungunya viral infection the levels of TNF and IL-1 were
found to increase in the convalescent phase and did not signifi-
cantly differ in patients reporting mild or severe conditions (Ng
et al. 2009). This indicates no or a mild contribution of TNF and
IL-1 in persistent arthralgia in CHIKV patients.
EPIDEMIOLOGY
The CHIKV is believed to have originated in Africa and then
spread to all over the world, causing many epidemics and af-
fecting millions of people (Powers et al. 2000). The geograph-
ical distribution of CHIKV commonly includes tropical coun-
tries, but the viral infection also extended into the temper-
ate regions after the 2004 epidemic (Robinson 1955). It was
first reported from East Africa around Tanzania during an epi-
demic of fever in 195253. This fever was later described in
1955 as a dengue like disease (Lumsden 1955; Sergon et al.
2008). After the first report, the Chikungunya virus infection
was documented worldwide involving many countries including
Tanzania, Ghana, Mozambique, Burkina Faso, Comoros, Kenya,
Central African Republic, Benin, Uganda, Nigeria, Zimbabwe,
Senegal, South Africa, Cameroon, Congo, Guinea, Mayotte, Bu-
rundi, Sudan, Malawi, Namibia, Gabon, Sri Lanka, Myanmar,
Thailand, Vietnam, Indonesia, Malaysia, Taiwan, Laos, Philip-
pines, Pakistan, Singapore, Cambodia, India, China, Sri Lanka,
etc. Indian Ocean regions with reported cases are La Reunion,
Madagascar, Mauritius, Seychelles etc. (Hammon, Rudnick and
Sather 1960; Shah, Gibbs and Banerjee 1964; Thaung et al. 1975;
Thonnon et al. 1999; Lam et al. 2001; Muyembe-Tamfum et al.
2003; Laras et al. 2005; Schuffenecker et al. 2006; Lanciotti et al.
2007; Renault et al. 2007; Cavrini et al. 2009). Chikungunya has
been reported to re-emerge unexpectedly in many countries
with a gap of 720 years (Powers et al. 2000). The first Asian
outbreak was documented in 1958 in Bangkok and Thailand
(Jupp and McIntosh 1988), after which there were outbreaks in
many parts of India in 196365. Later on re-emerging epidemics
Deeba et al. 5
were documented in 19992000 in Gabon affecting around
50 000 people (Pastorino et al. 2004). A subsequent epidemic was
reported from Indonesia in 200103 (Laras et al. 2005).
The most recent re-emerging epidemic was reported from
the Indian Ocean region, including India, from 2004 to 2009 that
affected millions of people in various countries. This epidemic
in the Indian Ocean region was believed to have originated from
Kenya. Strains of this outbreak formed a separate clade in the
ECSA genotype that is known as Indian Ocean Lineage (IOL) (Yer-
golkar et al. 2006; Volk et al. 2010; Weaver and Forrester 2015).
Thus, this epidemic provided a genotypic shift of the CHIKV
from Asian to ECSA genotype in the Indian Ocean region. This
ECSA genotype has been reported in the subsequent epidemio-
logical studies from this region (Vashishtha et al. 1998; Arankalle
et al. 2007). A unique molecular signature of this outbreak was
the E1 A226V mutation that was absent in the initial phase of
the epidemic, but later on was found in almost 90% of the iso-
lates (Schuffenecker et al. 2006). Due to this mutation the virus
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was able to infect A. albopictus, which was not the usual host of
CHIKV until this epidemic. The mutation A226V in the E1 pro-
tein was shown to provide cholesterol independence to a Sem-
liki Forest virus population (Tsetsarkin et al. 2007). The ability
of a virus to infect insect cells by providing a better adaptation
to the lipid composition of these cells could be improved by the
mutation that affects cholesterol dependency. The experimen-
tal demonstrations showed that viruses without the E1 A226V
mutation were not as successful at replicating in A. albopictus
(Vazeille et al. 2007). On the other hand, the mutated ones had a
higher efficiency of replication and dissemination in A. albopic-
tus (Parola et al. 2006). Therefore, this mutation was attributed to
the higher epidemic potential of the virus during this epidemic.
Since A. albopictus has a long life span and is active all day long,
the spread of CHIKV infection became rapid and reached many
more geographical regions than expected. The overall geograph-
ical distribution of the Aedes mosquitoes has changed since this
outbreak. The Aedes albopictus is now spreading to regions that
were earlier inhabited by the Aedes aegypti. Consequently, the
circulation patterns of the Chikungunya virus, as well as the dy-
namics of the epidemics caused by this virus, are expected to be
modified in the future.
The first outbreak of Chikungunya virus infection in India
took place in 196364 in Kolkata (Shah, Gibbs and Banerjee 1964).
Subsequent epidemics were reported from many parts of India,
including Chennai, Pondicherry, Vellore, Visakhapatnam, Raj-
mundry, Kakinada and Nagpur in 196465 (Rao 1966; Rodrigues
et al. 1972) and Barsi in 1973 (Padbidri and Gnaneswar 1979)
affecting millions of people. The virus was postulated to have
disappeared from India and South East Asia after 1973 (Burke,
Nisalak and Nimmannitya 1985; Neogi et al. 1995; Bhatia and
Narain 2009), but during 2004 it suddenly re-emerged as an ex-
plosive epidemic in India affecting around 1.3 million people
in 13 different states after a gap of almost 32 years (Wauquier
et al. 2011). Several states in India experienced massive out-
breaks of Chikungunya viral infection during 200409, including
Andhra Pradesh, Karnataka, Maharashtra, Rajasthan, Gujarat,
Tamilnadu, Orissa and Madhya Pradesh (Cavrini et al. 2009).
Subsequent smaller outbreaks of Chikungunya viral infection
have been reported from many parts of India after the massive
200409 epidemic. The main affected regions were Delhi,
Haryana, Kolkata, Kerala, Andaman and Nicobar Island (Pialoux
et al. 2007; Niyas et al. 2010; Singh et al. 2010; Shrinet et al. 2012;
Afreen et al. 2014). These studies have reported 845% CHIKV
positivity using different diagnostic methods, including PCR,
real-time PCR and serological assays.
 6 Pathogens and Disease, 2016, Vol. 74, No. 3
A detailed investigation of the evolution of Chikungunya
virus strains in India from 1963 to 2006 was carried out dur-
ing the massive outbreak. The phylogenetic characterization
showed that the strains identified during 196373 clustered
into the Asian genotype. All the isolates identified during
the 200409 epidemic clustered into the ECSA genotype mak-
ing a separate group, IOL (Indian Ocean Lineage) (Arankalle
et al. 2007). Another investigation clustered the CHIKV strains
in the ECSA genotype by phylogenetic analysis of the E2
protein gene during another small outbreak in Odisha in
201011 (Sahu et al. 2013). Various mutations were also identi-
fied in the E2 protein. The T cell and B cell epitopes were also
predicted for the E protein and two of them were found to be
associated with antigenicity. Structural characterization by ho-
mology modeling revealed three domains (A, B and C) in the
E2 protein. Most of the mutations were identified in the do-
main B, which modulates the binding of virion with the host
cell, and domain C is the transmembrane domain. The most
probable B and T cell epitopes were identified in the conserved
domain A.
An investigation of full genome analysis of five isolates from
Pune during the 200409 epidemic identified numerous muta-
tions throughout the genome along with the typical A226V mu-
tation (Arankalle et al. 2007). Comprehensive full genomic anal-
ysis of two outbreak strains from Gwalior revealed the pres-
ence of three unique mutations, two in the E1 protein and
one in the nsp1 protein (Santhosh et al. 2008). These unique
molecular features, including the A226V mutation in the E1
protein, were found to be associated with high evolutionary
and epidemic potential of these CHIKV strains. Full genome
analysis of the six isolates of these outbreak strains from Ker-
ala revealed the presence of 37 novel mutations (Sreekumar
et al. 2010). At least 20 mutations mapped to the functionally
significant domains of the corresponding viral proteins. They
also concluded that 18 mutations in the structural proteins af-
fected the T cell epitope immunogenicity. Another investiga-
tion from Pune studied the evolution of the CHIKV during the
massive epidemic using 27 full genomes and partial E1 gene
datasets. The mean substitution rate of the CHIKV was calcu-
lated to be 8.8 104 substitutions site1 year1 (Cherian et al. mice experiments with favipiravir protected mice from severe
2009). The evolutionary time scale of the CHIK viruses was es-
timated to be 300 years old. In addition, the progenitor of the
200507 viruses was identified 9 years ago that originated in
Central Africa.
Numerous phylogenetic studies have also provided insights
into the molecular evolution of Chikungunya virus in India
(Santhosh et al. 2008). Comparison of synonymous and non-
synonymous substitution rates in different genomic regions
suggested purifying selection pressures (Arankalle et al. 2007;
Sahu et al. 2013). This analysis also revealed the presence of
many positively selected sites in the genome. An investigation
from Pune identified 10 positively selected sites throughout the
genome (Arankalle et al. 2007). Five positively selected sites were
identified in the E2 region in most of the isolates in a study from
Odisha (Sahu et al. 2013). However, the affect of these positively
selected sites on the pathogenesis of Chikungunya virus infec-
tions should be investigated further. The phylogenetic investi-
gations from different parts of India have thus contributed to
the understanding of the molecular evolution and epidemiology
of this re-emerging viral pathogen. Therefore, regular surveil-
lance for the Chikungunya viral infection should be carried out
in endemic regions like India. The data generated from the
surveillance will assist in the formulation of control measures to
prevent outbreaks in this region.
PREVENTION AND CONTROL
Vector control is the only way to reduce CHIKV infections due
to the absence of an effective vaccine and antiviral drugs. Pre-
vention of mosquito breeding can be achieved by elimination
of stagnant water in drains, room air coolers, broken bottles,
tin cans, etc. Insecticides should be used to control the adult
and larval mosquito population. Protection from mosquito bites
can be achieved by using mosquito-repellent creams, wearing
clothes which cover the body, use of mosquito nets over the
beds and the use of wire mesh on doors and windows. There-
fore, vector surveillance should be carried out in the endemic
areas for effective control of infection. In addition, the general
public should be educated on various aspects of the disease that
will assist in better management of this viral infection.
THERAPEUTICS AND CURE
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No antiviral drugs are available for CHIKV infections, therefore
the treatment is completely symptomatic. Antipyretics, anal-
gesics and anti-inflammatory drugs are usually given for the
treatment of fever, pain, etc. Attempts are being made to develop
therapeutics and drugs for the treatment of CHIKV infection
by various scientists all over the world. Chloroquinone has an-
tiviral activity against many viruses, including CHIKV, but a re-
cent study from India showed that chloroquinone was not effec-
tive in the treatment of early musculoskeletal pain and arthri-
tis in acute CHIKV infection compared with the non-steroidal
anti-inflammatory drug (NSAID) e.g. meloxicam (Chopra, Saluja
and Venugopalan 2014). Ribavarin also has antiviral activity
against Chikungunya virus. Ribavarin in combination with INF-
2b showed a synergistical antiviral effect against Chikungunya
viral infection in in vitro conditions (Briolant et al. 2004). Another
study of the combination of ribavarin with doxycycline showed
a decrease in CHIKV replication in cell culture and decreased vi-
ral load and inflammation in mice models (Rothan et al. 2015).
Another broad spectrum antiviral drug, favipiravir, was shown
to inhibit replication of different Chikungunya virus strains in
cell cultures in a recent investigation (Delang et al. 2014). The
neurological disease and a decrease in mortality rate was also
observed.
The use of human immunoglobulins purified from convales-
cent phase CHIKV patients proved to have a good prophylactic
and therapeutic efficacy against CHIKV infection in mouse mod-
els (Couderc et al. 2008). In one particular study two different
monoclonal antibodies, 5F10 and 8B10, were prepared against
CHIKV. These antibodies were formulated by activation, immor-
talization, amplification and cloning of B cells from a donor with
a history of CHIKV infection. Both these antibodies specifically
neutralize CHIKV infection strongly in in vitro conditions (Warter
et al. 2011). The level of MCP-1 was found to be elevated dur-
ing the CHIKV infection. Therefore, one particular study demon-
strated that treatment of infected mice with bindarit, which is
an inhibitor of MCP-1 synthesis, showed symptoms that were
mild and of short duration. The histology of joint and muscle tis-
sue of infected mice treated with bindarit also showed very low
inflammation compared with the untreated mice (Hoarau, Jaf-
far Bandjee and Krejbich Trotot 2010). Trials of antiviral therapy
using RNA interference-mediated inhibition of viral replication
are promising but sequence dependent. A reduction of CHIKV
titer up to 99.6% in specially designed small interfering (si)RNA
for conserved regions of nsp3 and E1 gene was reported in an
in vitro study. Complete inhibition of viral replication targeting

ns1 and E2 protein genes in in vivo conditions was reported in
another investigation (Dash et al. 2008; Parashar et al. 2013).
VACCINES
The recurring epidemics, re-emergence and widespread global
distribution of CHIKV have necessitated the development of an
effective vaccine. Challenges to vaccine development for the
CHIKV infection are enormous. The vaccine should induce high
levels of antibodies to completely protect against the CHIKV in-
fection. In addition, the vaccine should be economical so that
developing countries can easily afford them since most of the
epidemics occur in these regions. Many clinical and preclinical
trials for development of an effective and economical vaccine
are being carried out across the globe (Singh et al. 2013).
WHOLE VIRUS-INACTIVATED VACCINES
The initial vaccine candidate that was developed was a whole
virus grown in a green monkey kidney cell line inactivated by
formalin (Harrison et al. 1971). The preliminary clinical trials
showed good immune response with few side effects. Three dif-
ferent CHIKV strains grown in cell culture that were inactivated
by Tweenether showed good immune response in preclinical
trials (Eckels, Harrison and Hetrick 1970).
The formalin-inactivated whole virus vaccine was generated
from the ECSA genotype of the 2006 Indian outbreak strain.
This vaccine had a very good immunogenic potential to neutral-
ize the infectivity of the virus by enhancing both the humoral
and cell-mediated immune response in a mouse model (Tiwari
et al. 2009). Another vaccine candidate was also derived from the
ECSA genotype of CHIKV. The whole virus was inactivated by
-propiolactone (BPL)formalin, which proved to be a promis-
ing vaccine candidate in the mouse model (Kumar, Sudeep and
Arankalle 2012). The inactivated whole virus vaccine derived
from an Asian isolate completely protected against mice viremia
and arthritis in another study (Gardner et al. 2010).
LIVE ATTENUATED VACCINES
The first live attenuated vaccine strain for CHIKV was developed
by Levitt and colleagues in 1986 by passaging a Thailand strain of
CHIKV in cell culture (Levitt et al. 1986). The vaccine was able to
replicate in both species of the vector A. aegepti and A. albopictus.
The live attenuated vaccine was developed by serially passaging
plaque-purified CHIKV in the MRC-5 (Human fetal lung fibrob-
last) cell line. This vaccine was safe and highly immunogenic
with side effects well within the tolerable limits. In the phase II
trial, investigators observed sero-conversion in 98% of the vac-
cines by day 28 and the presence of neutralizing antibodies in
85% of volunteers for 12 months (Edelman et al. 2000). CHIKV
with point mutations at amino acid numbers 12 and 82 in the
E2 glycoprotein proved to be a good candidate for development
of live attenuated vaccine in another investigation (Gorchakov
et al. 2012).
SYNTHETIC VACCINES
Three different chimeric alphavirus vaccines were developed
by using naturally attenuated strains of Venezuelan equine en-
cephalitis virus, Eastern equine encephalitis virus and Sind-
bis virus as a backbone expressing structural protein genes of
CHIKV (Mallilankaraman et al. 2011). An efficient replication of
Deeba et al. 7
these vaccine candidates was achieved in the cell cultures. In ad-
dition, all the vaccine candidates generated a robust immune re-
sponse in mice. Wang and colleagues developed another recom-
binant vaccine candidate comprising the non-replicating aden-
ovirus vector expressing the structural proteins of CHIKV (Wang
et al. 2011). The vaccine induced a good humoral immune re-
sponse in mice. In an effort to develop a vaccine against CHIKV
infection, Plante and colleagues replaced the subgenomic pro-
moter in a CHIKV clone by internal ribosome entry site (IRES)
from encephalomyocarditis virus and thus altered the levels
and host-specific mechanism of structural protein gene expres-
sion (Muthumania et al. 2008). This vaccine was found to be
highly immunogenic and efficacious in mice. In addition, the
vaccine was found to be incapable of replicating in the mosquito
cells. Another potential vaccine candidate is MVA-CHIKV that is
based on modified vaccinia virus Ankara (MVA) expressing all
the structural genes of Chikungunya virus (capsid, E3, E2, 6K and
E1) (Plante et al. 2011). The trials of the above-mentioned vaccine
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candidate were carried out on mice that showed both potent
and broad cellular immune responses even on administration
of high viral doses.
DNA VACCINES
Another approach for the design of a vaccine against Chikun-
gunya virus includes the development of DNA vaccines. During
an investigation on DNA-based vaccine, several modifications
were made in the consensus sequences of capsid and envelope
protein genes of CHIKV that included codon optimization, addi-
tion of a Kozak sequence, and a substituted immunoglobulin E
leader sequence inserted in a plasmid (Wanga et al. 2008). The
vaccine was able to induce both humoral and cellular immune
response in mice. Vaccination using envelope-based synthetic
DNA vaccine using a combined E1, E2 and E3 gene construct
was found to generate a humoral as well as a cell-mediated im-
mune response in rhesus macaques, each of which is capable of
providing protection against the CHIKV infection (Garcia-Arriaza
et al. 2014).
VIRUS-LIKE PARTICLES
The selective expression of structural proteins (C-E3-E2-6K-E1)
of the West African strain of CHIKV forms non-infectious virus-
like particles (VLPs) in vitro and showed high titers of neutral-
izing antibodies both in monkeys and immunodeficient mice
(Akahata et al. 2010). The recombinant baculoviruses were used
to generate CHIKV VLPs in insect cells in another study (Metz
et al. 2013). The VLPs provided protection against viremia and
arthritis in the mouse model. The VLPs generated in another re-
cent investigation proved to be a most promising vaccine can-
didate as the phase I clinical trials showed immunogenic, well-
tolerated and safe effects (Chang et al. 2014).
CONCLUSION
The Chikungunya virus is a re-emerging infection affecting mil-
lions of people worldwide. The Chikungunya viral infection is
associated with a number of challenges such as overlapping
clinical symptoms with dengue virus, recurring epidemics, per-
sistent arthralgia and mutations in viral genome leading to
enhanced geographical spread and inability to control the
vector-mediated transmission of the disease. The recurring and
persistent epidemics in different geographical regions have
 8 Pathogens and Disease, 2016, Vol. 74, No. 3
enhanced efforts to develop drugs and vaccines to combat
Chikungunya virus infection.
The Chikungunya viral infection is a major health concern in
India. Therefore, continuous viral surveillance should be carried
out in the regions reporting regular outbreaks in the monsoon
season. This surveillance will describe the circulating genotypes
of Chikungunya virus in these regions. The information on the
circulating strains of Chikungunya virus will assist efforts to de-
velop vaccines and antiviral drugs. These data will also facilitate
the prediction and control of the epidemics. In addition, an in-
crease in the general awareness of the disease by educating the
general public can be beneficial for a better management of this
emerging viral infection.
FUNDING
Work in our laboratory is supported by grants from the Univer-
sity Grants Commission and the Council of Scientific and Indus-
trial Research, Government of India.
Conflict of interest. None declared.
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