Journal of Clinical Virology 64 (2015) 144152

Contents lists available at ScienceDirect

Journal of Clinical Virology

journal homepage: www.elsevier.com/locate/jcv

Chikungunya, a paradigm of neglected tropical disease that emerged

to be a new health global risk
Virginie Rougeron a,b , I-Ching Sam c , Mlanie Caron a , Dieudonn Nkoghe a , Eric Leroy a,b ,
Pierre Roques d,e,
a

Centre International de Recherches Mdicales de Franceville, Franceville, Gabon

Unit Mixte de Recherche Maladies Infectieuses et Vecteurs: Ecologie, Gntique, Evolution et Contrle (IRD 224 CNRS 5290 UM1-UM2), Institut de
Recherche pour le Dveloppement, Montpellier, France
c
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
d
CEA, Institute of Emerging Diseases and Innovative Therapies, Division of Immuno-Virology, Fontenay-aux-Roses, France
e
Universit Paris-Sud 11, UMR E1, Orsay, France
b

a r t i c l e

i n f o

Article history:
Received 22 April 2014
Accepted 25 August 2014
Keyword:
Chikungunya

a b s t r a c t
Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family that causes chronic and incapacitating arthralgia in human populations. Since its discovery in 1952, CHIKV was responsible for sporadic
and infrequent outbreaks. However, since 2005, global Chikungunya outbreaks have occurred, inducing
some fatalities and associated with severe and chronic morbidity. Chikungunya is thus considered as
an important re-emerging public health problem in both tropical and temperate countries, where the
distribution of the Aedes mosquito vectors continues to expand. This review highlights the most recent
advances in our knowledge and understanding of the epidemiology, biology, treatment and vaccination
strategies of CHIKV.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Chikungunya virus (CHIKV) is an alphavirus from the Togaviridae family, which is transmitted by Aedes mosquitoes, and causes
epidemic arthritis or arthralgia together with fever and rash. It is
part of the Semliki Forest virus antigenic complex that also contains ONyong Nyong, Mayaro and Ross River viruses [1]. CHIKV
is a positive-sense, single-stranded RNA virus of about 11.8 kb
(Fig. 1). There are three main genotypes, West African, Central/East
African (C/EA), and Asian, the names reecting the initial geographic restriction of each type. Since CHIKV was rst isolated in
Tanzania in 1952 [2], the disease was mainly conned to localized
outbreaks in Asia and Africa, sometimes with hundreds of thousands of cases [3]. In 2005, a C/EA strain of CHIKV, which likely

Corresponding author at: Service dImmuno-Virologie/UMR E1, Paris XI, Institut

des Maladies Emergentes et Thrapeutiques Innovantes, CEA, B.P.6, 92 265 Fontenay
aux Roses, France. Tel.: +33 01 46 54 91 67; fax: +33 01 46 54 77 26.
E-mail address: pierre.roques@cea.fr (P. Roques).
http://dx.doi.org/10.1016/j.jcv.2014.08.032
1386-6532/ 2014 Elsevier B.V. All rights reserved.

originated in coastal Kenya, spread to islands of the Indian Ocean

and India [4]. Since then, CHIKV spread worldwide to cause large
outbreaks in Southeast and East Asia, as well as many imported
cases in travelers returning to non-endemic regions in Europe and
North America.
This global epidemic affecting millions was unprecedented in
its scale and was likely driven by several factors. These include the
increased volume of travelers, the widening geographic distribution of the mosquito vectors and the adaptation of the epidemic
strain to Aedes albopictus [5]. The susceptibility of mosquitoes in
non-endemic regions such as Australia [6] and North America [7],
and the occurrence of autochthonous outbreaks in Italy [8] and
France [9], have shown that CHIKV can no longer be considered
as a problem of tropical countries. A recent work has also pointed
that CHIKV may be present in severe forms, particularly neurological, which may be fatal [10]. It is also increasingly recognized that
there is a signicant burden of long-term morbidity due to chronic
arthralgia [11]. Consequently, from being a relatively neglected and
understudied pathogen prior to 2004, CHIKV has in recent years
been the focus of intense study.

V. Rougeron et al. / Journal of Clinical Virology 64 (2015) 144152

Fig. 1. Genomic structure of CHIKV and expected function of the produced proteins.
CHIKV has a typical alphavirus genomic organization: 5 -nsP1-nsP2-nsP3-nsP4junction region-C-E3-E2-6K-E1-poly (A)-3 with two open reading frames (ORFs).
The 5 end of the genome has a 7-methylguanosine cap and there is a polyadenylation signal at the 3 end. The 5 ORF is translated from genomic RNA and encodes
four non-structural proteins (nsP1, 2, 3 and 4) encoding for the synthesis of RNA
capping, helicase protease, RNA and RNA dependent RNA polymerase [121]. The 3
ORF is translated from a subgenomic 26S RNA and encodes a polyprotein that is
processed as the capsid protein (C), two surface envelope glycoproteins (E1 and E2),
a small peptide designated E3, and two proteins with ion-channel activity, 6K and
the transframe protein [53,122].

2. Advances in eco-epidemiology (focus: outbreaks and

vector/virus relationship)

145

luteocephalus and Ae. neoafricanus [17]. Other mosquitoes, like Culex

annulirostris and Mansonia africana, have also been incriminated
in CHIKV transmission [14,17,19], and in vitro infection failed to
demonstrate CHIKV infection of Anopheles [21].
In Asia, previous outbreaks occurred mostly in urban areas
and virus maintenance is associated with continuous introduction of the virus in new areas and new nave human populations
[13,2224] (Fig. 2). As in Africa, the major mosquito species
involved was Ae. aegypti. However Ae. albopictus was found during
the recent worldwide outbreaks to be the major vector in several
Asian countries [25,26]. Of concern, the distribution of Ae. albopictus has expanded from South East Asia to Madagascar, the Indian
Ocean islands and Africa, as well as South Europe and USA [27].
In Asian regions, humans have long been considered as the only
reservoirs. However, the recent isolation of CHIKV from monkeys
in Malaysia suggests the potential existence of a sylvatic zoonotic
transmission cycle in Asia [28].

2.1.1. Interactions between reservoirs and vectors

2.1.2. Chikungunya outbreak history

Chikungunya is an arthropod-borne virus transmitted mainly by

Aedes species mosquitoes. Only the female are infective since they
need blood meals for egg formation. Vertical transmission within
mosquitoes is not demonstrated and thus there is no involvement
of the vector in viral persistence in a specic area. However, a recent
study found virus sequences in male mosquitoes, which indirectly
supported trans-ovarian transmission; in addition, males are able
to transmit the virus to females during mating [12]. CHIKV outbreaks often coincide with rainy periods during which increased
mosquitoes densities are observed [13].
In Africa, from where the virus originated, CHIKV transmission
cycle is sylvatic and enzoonotic (Fig. 2). Human infections are due
to occasional spillovers and happen mostly in rural zones, while
urban disease is thought to be due to human migration. Humans
are the principal reservoirs during epidemic periods. Transmission
is mainly ensured by the highly anthropophilic mosquito vectors,
Ae. aegypti and Ae. albopictus [14,15]. During inter-epidemic periods, the transmission cycle appears to be maintained by animal
reservoirs, in particular monkeys. Other reservoirs may include buffalos, rodents, and birds [16] (Fig. 2). Outbreaks occur in monkeys
and animals develop viremia but no pronounced physical manifestations have been observed in the wild [14,17,18]. The principal
sylvatic vectors are Ae. furcifer-taylori [19], Ae. africanus [20], Ae.

Chikungunya was rst identied during an outbreak in

19521953 in East Africa, along the border between Tanzania and
Mozambique (ex-Tanganyika) [2,29]. The name Chikungunya is
derived from a word in the Makonde language that means that
which bends up.
Between 1960 and 1990, Chikungunya outbreaks happened sporadically in Africa and Asia (Fig. 3). In 2004, a major epidemic of the
C/EA strain began in East Africa, specically in Lamu Island and
then in Mombasa, Kenya, before spreading to the Indian Ocean
islands, Western Africa, India, and Asia. Imported CHIKV cases
have been reported in numerous non-endemic countries, including Japan, USA, and Europe. Chikungunya is currently considered
as a real threat in these European and American countries, which
are colonized by Aedes species mosquitoes [30]. The Italian epidemic in 2007 was the rst indigenous outbreak of CHIKV outside
the inter-tropical zone [8]. The recent worldwide outbreaks and
specically the last one in December 2013 in Caribbean Islands, the
Americas, and in Micronesia highlight the important role of international travels in disease and the horizontal exchange of virus and
human populations, which means that the historical segregation
of Asian and African strains together with their ecological specicities are no longer true. The emergence of Chikungunya in mainland
North America is now a reality.
3. Advances in basic virology

Fig. 2. Biological life cycle of Chikungunya virus with interconnection between rural
and urban cycles. In Africa, the CHIKV transmission cycle is considered as sylvatic
and enzoonotic, and human infections are due to occasional spillovers and happen
mostly in rural zones. Urban CHIKV introduction is thought to be due to human
migrations. In Asia, outbreaks occur mostly in urban areas, and virus maintenance
is associated with continuous introduction of the virus into new areas and new
nave human populations. In relation to type of cycle (urban vs. rural), the mosquito
vectors involved are different.

As all alphaviruses (Togaviridae), the chikungunya virion has an

icosahedral capsid enclosed by a lipid envelope and a diameter of
6070 nm. As an enveloped virus, CHIKV is sensitive to desiccation,
temperatures >60 C and detergent.
Beside their role in the viral replication complex, non-structural
proteins are involved in cell control and avoidance of innate
response. Alphaviruses are highly sensitive to the antiviral activity of type I interferons (IFN) [31] and several IFN-stimulated
genes (ISG) including ISG15, ISG20, P56, ZAP, viperin, and 2 ,5 oligoadenylate synthetases (OAS) exert antiviral activity against
them [32,33]. However, alphaviruses have also developed strategies to escape these blocks, and are able to inhibit cellular mRNA
translation. Indeed, their genomic-mRNA contains stem loop structures that allow initiation of translation without requirement of
the cellular translation initiation factor component eIF2a. At the
late stages of the alphavirus life cycle (6 h post-infection), shutoff
of mRNA translation is followed by induction of apoptosis and cell
death [34]. Recent studies showed that balance between autophagy
and apoptosis might play a major role in the capacity of the virus

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V. Rougeron et al. / Journal of Clinical Virology 64 (2015) 144152

Fig. 3. Geographical distribution of reported outbreaks of chikungunya virus infection in African and Asian regions. Each country presenting CHIKV outbreaks is colored
differently in relation to the outbreaks dates: in green the old outbreaks countries (corresponding to the countries presenting outbreaks only before 2004), in blue countries
touched by outbreaks before and after 2004 and in red the new outbreaks countries (corresponding to the countries where outbreaks occurred only since or after 2004).

to spread during the early stages of the disease [35]. Viral protein recruitment of cell components is prominent, as highlighted
in the selection of ubiquinated capsid by the autophagy receptor p62 promoting cell survival, and the binding of human NDP52
(but not mouse) to nsP2, which promotes viral replication [36].
This may explain the exquisite differences observed in the nonhuman primate and mouse models despite a general involvement
of macrophages in viral persistence [31,3739].
In contrast to new-world alphaviruses, CHIKV RNA packaging
signal was found in the nsP2 sequence, not in nsP1, and is recognized by chikungunya capsid [40]. Moreover, a mutation in nsP2
protease (P718S) showed a role in inhibition of JAK-STAT signaling following IFN type 1 cell activation [41], probably by inhibiting
nsP2 nuclear localization [42]. Another in vitro study, comparing
CHIKV and Sindbis, showed that for CHIKV nsP4, but not Sindbis, the RNA dependent RNA polymerase might specically inhibit
phosphorylation of cellular eIF2a and thus modulate the unfolded
protein response to control the host cells fate [43]. The nsP3 protein
seems to be the most exible protein of the viral genome, as it is the
only place where protein markers can be stably inserted without
modifying CHIKV viral replication [44]. Evolution however seems
to maintain a role in intracellular structure which may vary largely
between alphaviruses and support the need to explore the CHIKV
nsp3 [45]. Specically, CHIKV nsP3 blocks stress granule assembly
by recruitment of G3BP into cytoplasmic foci, and amphiphysin2, which is involved in efcient viral RNA synthesis [46,47]. Some
of these functions are shared within the SFV alphavirus subgroup
[45,47]. Furthermore, nsP3 may play a role in host cell specicity, as
introducing the Onyong nyong nsP3 gene within a CHIKV backbone
allowed a vector shift to Anopheles [21].
In addition to nsP3, motifs in the viral envelope are involved in
the capacity to bind and infect various host cells. Concerning the
vector shift, from Ae. aegypti to Ae. albopictus, so important in the
current outbreaks, the E1-E226V mutation is major, [4851] but
epistatic mutations in E2 (E2-D60G and E2-I211T) may modulate

this effect and even explain the absence of adaptation of the Asian
virus strain to Ae. albopictus [49]. Surprisingly other E1E2 mutant
viruses obtained by truncation of the trans-membrane part of E2
(TMB position AA 374 to 383) remained infectious in mosquito cells
but are attenuated in the vertebrate susceptible host, showing a
complex relationship between infection and pathogenic capacity
[52]. A recent study identied a transframe protein related to the
structural protein 6K which plays a role in particle release in addition to E3 [53,54]. Recently, it was demonstrated that mutants E2
(E2-E166K) in CHIKV viruses, probably found in virus quasi-species,
are able to counteract IFN-induced genes like OAS-3 and ZAP and
in addition trigger a quasi-pure apoptosis phenotype in vitro ([32];
P. Desprs unpublished). Of note, because the packaging of viruses
not only involves the structural proteins but also some of the nonstructural ones, compensatory mutations were observed in nsP
genes as recently described for VEEV nsP2 [42,55].
4. Advances in immuno-virology: mechanisms of
pathogenesis
Following the bite of an infected mosquito, CHIKV is injected
into blood capillaries and dermis. During this intradermal stage,
CHIKV infects the most common cells of connective tissues, epithelial cells and dermal broblasts (Fig. 4). These cells have been shown
to be susceptible to infection and allow viral production [56,57].
Thus viruses produced by both host broblasts and mosquitoes
enter the blood vessels. During the acute phase of infection, CHIKV
antigens can be detected in vivo in blood monocytes [58]. However, it seems that monocytes become a signicant target for CHIKV
only when high levels of virus are present in the circulation. Even
if these cells might be infected, they did not produce substantial
levels of new viruses [58]. This step corresponds to the acute stage
of the CHIKV infection, which can last up to 12 days [59]. At this
stage, viral load can be extremely high, at >108 copies/mL [60]
(Figs. 4 and 5). Because of their extensive distribution among the

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147

Fig. 4. Chikungunya infection pathogenesis model. Following the bite of an infected mosquito, CHIKV is injected into blood capillaries and dermis. During this intradermal
stage, CHIKV infects human epithelial cells and dermal broblasts. Blood monocytes and macrophages then become infected with CHIKV and viral replication happens
(>108 copies/m). Monocytes are then responsible for viral dissemination and systemic infection. The principal secondary infection sites are muscles and joints, endothelial
cells of the liver and the brain and macrophages and stroma cells of the spleen and lymph nodes. Immune responses are also induced, with high production of IFN- and
liberation of several cytokines and chemokines.

different organs and tissues and their migration capacity after

activation through blood and lymphatic systems, monocytes are
thought to be responsible for viral dissemination and systemic
infection (Fig. 4). The principal secondary infection sites are muscles and joints, where endothelial cells, broblasts and satellite cells
are infected. Endothelial cells of the liver and the brain are also
infected, leading to possible hepatitis and encephalopathy. Finally,
macrophages and stroma cells of the spleen and the lymph nodes
are infected too, and, together with joint tissues, may be sites of
virus persistence [38,39].
During viral infection, innate immunity is rst activated to suppress virus propagation, replication and dissemination. There is a
large and rapid production of several pro-inammatory cytokines,
chemokines and growth factors [61] (Figs. 4 and 5). IFN-/ play a
key early role in controlling the severity of viral infections including
alphavirus infections [6265]. IFN-/ sub-types trigger signaling
pathways including the expression of hundreds of interferonstimulated genes, resulting in the synthesis of multiple antiviral
proteins and the establishment of an overall antiviral state. IFN has been shown to be produced readily in vitro and after CHIKV
infection in humans [58,66], particularly by CHIKV-infected broblasts [67]. IFN- levels have been shown to be normal or marginally
increased [38,66]. During the acute phase, in parallel with this

IFN-type 1 liberation, a strong inammatory response is observed,

at rst with production of IL-6, MCP-1, IP-10 and RANTES. These
chemokines are involved in the recruitment of leucocytes to the
infection sites and in the deployment of efcient antiviral defenses
as well as high inammatory response although levels vary with
the cohort studied [6870]. There is also induction of a strong cellular immune response, during which IFN-, IL-4, IL-7 and other
cytokines involved in adaptive immunity are released [61]. The proinammatory cytokine IL-12 has also been detected rapidly after
infection with CHIKV [58,71]. Both IFN- and IL-12 activate natural
killer (NK) cells, a major component of the innate immune defense.
Specic subsets of NK cells are able to sense CHIKV from the beginning of the infection and may thus contribute to viral clearance
[61,72]. The B-cell promoting cytokines IL-4 and IL-10 are upregulated during the rst days after symptoms onset, and may initiate
CHIKV-specic IgG production [61,73] (Fig. 5). This is followed by
the activation of adaptive responses through CD8+ T cells. Most of
these observations were conrmed in non-human primate model
of the infection [39]. Finally, a switch of CD8+ T cells to CD4+ T cells
activation and the production of IL-1R and IL-2RA [61] characterize the late acute stages. CD4+ T cells are the major mediator of
inammation in the joint swelling and tissue damage in an acute
mouse model [74].

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Fig. 5. Chikungunya virus infection evolution and clinical diagnosis. After transmission of CHIKV through an infected mosquito bite, a period of incubation begins for
24 days on average. Then, an abrupt clinical onset is observed which lasts between
7 and 10 days. During this time, viral load may be extremely high, exceeding 108 viral
copies/ml of blood, and is followed by activation of innate immunity leading to high
levels of interferon (IFN). Anti-CHIKV antibodies can be detected in patients shortly
after symptoms onset, usually from 4 to 6 days for IgM, and remain detectable for
several weeks to several months. IgG are detectable only a few days later (715
days), and persist for life.

The role of cytokine in chronic stage is far less understood [38],

but the role of infected osteoblasts and joint macrophages in the
persistence of the disease might be associated with a vicious loop
of IL6/RankL production [68,75,76].
5. Advances in clinical virology
5.1.1. Clinical presentation
Following an incubation period of 24 days (range 112 days)
after an infective mosquito bite, Chikungunya infection is characterized by the classical and self-limiting symptoms of an abrupt
onset of fever (38 C), macular rash, and arthralgia [77,78].
However, the recent outbreaks enabled close scrutiny of large
numbers of patients, leading to reports of atypical and severe manifestations, sometimes fatal (reviewed by Rajapakse [79]). These
include myocarditis [10], extensive bullous skin lesions [80], and
encephalopathy [10]. Neonatal encephalopathy was also described
following vertical transmission at birth [81]. Chronic arthralgia,
which may be incapacitating, causes long-term morbidity and have
high economic impact. This may be due to defective clearance and
subsequent persistence of CHIKV antigen in macrophages in joints,
stimulating ongoing inammation [68]. Reported rates of chronic
arthralgia vary between countries, from 0 to 4.1% at 3 months in
Gabon [80] and Malaysia [82], to 60% at 3 years in La Runion [83],
which may reect differences in underlying population genetics,
healthcare practices, or CHIKV strain virulence. The most commonly reported risk factor for chronic arthralgia is older age [83,84].
In endemic countries, acute Chikungunya disease may be difcult to distinguish clinically from other febrile illnesses such as
dengue and malaria [80,84], although certain specic features such
as acute red ears have been described [85]. Therefore, laboratory
diagnosis remains important.
5.1.2. Laboratory diagnosis
There have been notable advances in CHIKV diagnostics over
the last few years. The gold standard methods for diagnosis are
virus isolation and nucleic acid amplication tests, although these

are not widely available in most endemic countries. Both methods are most useful in the rst 7 days of acute symptoms, when
viremia is present before IgM becomes detectable (Fig. 5). Numerous conventional and real-time RT-PCR assays have been described
[60,86]. Peak viremia levels are seen at days 13 of symptoms and
RNA from non-viable virus can still be detected in the presence of
antibodies as late as day 12 [60]. Apart from increased sensitivity
compared to conventional PCR, real-time PCR also allows viral load
quantication, which warrants further study as a potential indicator of prognosis. At present, conicting data exist as to whether
higher viral loads are associated with increased [38] or reduced risk
of persistent arthralgia [87], or if there is no link [83]. An alternative
for diagnosis during the early stages of infection is antigen detection, of which there are two reports, [88], [89] although further
evaluation of performance is required.
CHIKV patients often do not present early in infection; in Sri
Lanka and Malaysia, patients presented at a median of 45 days [90]
(Sam, unpublished). The serological detection of IgM is thus important. However, the limited data available on use of commercial kits
in a clinical setting are disappointing. A commercial immunouorescence assay had a sensitivity of 76% at day 5 and 100% at
day 7 [91], but most diagnostic laboratories in endemic areas are
unlikely to have a uorescence microscope. Other ELISA and rapid
immunochromatographic tests (ICT) performed poorly, with sensitivity rates of 022% before day 7, and 1783% after day 7, when
retrospective diagnosis is of less clinical value [90,92], [93]. Furthermore, performance of the tests is affected by the match between
the antigen used in the assay and the circulating CHIKV strain, presumably leading to differences in IgM binding. In Singapore, an ICT
using recombinant E1-226A had a sensitivity of 77% for samples collected at days 57 during an outbreak of CHIKV bearing E1-226A;
during a later outbreak of CHIKV with E1-226V, sensitivity fell to
0% [91]. The use of CHIKV antigens of C/EA origin in ICTs may also
explain the poor sensitivities of 2053% in Indonesia, where the
Asian genotype of CHIKV was circulating [92]. Currently available
CHIKV IgM kits therefore are either unsuited, or cannot be solely
relied upon for diagnosis of acute infection.
There remains a need for simple, affordable, sensitive and specic diagnostic tests for CHIKV in developing countries, for the
different periods over which a patient may present, preferably at
the point of care. For early presentations of <7 days, an RNA/antigen
detection assay would be appropriate. Loop-mediated isothermal
amplication is a possible option for resource-limited settings,
as it can be performed in a single tube at a single temperature
in a water-bath, with greater sensitivity than conventional PCR
[94]. For presentations after >5 days, an IgM assay is necessary
[86]. Recently, patients antibody responses to CHIKV proteins have
been characterized, showing that linear epitopes at the N terminus
of E2 glycoprotein induce early antibodies that persist, although
binding may be affected by a K252Q change which can occur
naturally [95]. Further work is needed to identify and validate wellconserved antigens that may be reliably used for serological assays.
All new assays will then require subsequent testing with wellcharacterized samples in different settings with genetically diverse
circulating strains.

6. Advances in prophylaxis and therapy

Development of Chikungunya vaccine began as early as 1967
but after the relative failure of the US-Army vaccine in 1980,
interest and efforts waned until the recent re-emergence of the
disease. [68]. Currently, no licensed vaccine against Chikungunya is
commercially available, but numerous candidates are under study
(reviewed by Thiberville [96]). The virus-like particle (VLP) candidates are of most interest [9799], one of which is currently

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149

undergoing safety evaluation [100]. Only few vaccines have been

tested in non-human primates or phase 1/2 studies in humans
[101], but most seem efcient in a mouse model as showed by our
European group [102,103], and future deployment depends more
on economic measures rather than technical barriers [52,98].
Passive immunotherapy was historically used against viral disease. Some studies showed that extracted IgG against CHIKV
might protect against infection [97,104]. This protection may
also be achieved with human recombinant monoclonal antibodies [105,106]. Although these proofs of concept were obtained in
mice, such strategies might be very valuable to treat acute severe
diseases such as infection in babies [104].
As CHIKV disease was previously relatively rare, specic drugs
acting against CHIKV are not available. Molecules, known to have
non-specic antiviral action like chloroquine and arbidol, showed
some efciency in vitro [107,108], but no clinical efcacy [109]. In
addition, using the non-human primate model that we have developed in CEA, we demonstrated that chloroquine administration
during the acute phase or before infection seems to enhance viral
replication and may have adverse effects on the number of chronic
cases [110].
Screening of crude extracts of tropical plants revealed molecules
(trigocherrins, trigowiin) with good selectivity index in vitro against
CHIKV. However, their in vivo toxicity, tumorigenicity, and mechanism of action still remain to be dened [111,112]. Using a similar
method to screen a library of 502 highly puried natural product
compounds, harringtonine, a cephalotaxine alkaloid, was found as
acting as a CHIKV inhibitor [113]. In a high throughput screen using
uorescent CHIKV replicons of 356 clinically approved drugs, 5
potential inhibitors of CHIKV were also discovered [114].
Previously the demonstration of the role of cellular furinprotease in the processing of envelope proteins of Chikungunya
was reinforced by specic inhibition of these proteins [115]. Nevertheless, usage in clinical assay does not seem relevant as targeting
the viral protease (nsP2- or capsid-associated protease) should
be more specic and hopefully will induce fewer side effects.
Thus by combining structure-based specic computing, an in silico screen of molecules able to interfere with the nsP2 protease site
allowed selection of 4 putative candidates [116], for future in cellulo
experiments. Hence, a screen of 3040 molecules against the nsP2
mediated shut-off using a luciferase-based reporter gene identied only one natural product able to inhibit specically CHIKV
replication in cellulo [117]. Recently, targeting the natural cell
response against alphavirus [67] by inducing RIG-1 using an optimized 5 triphosphorylated RNA in vitro inhibited viral replication
[118].
Finally, few experimental treatments targeting host response
or host components involved during Chikungunya cell infection
have been tested in animal models. However, in a mouse model of
Ross River virus-induced arthritis, the demonstrated effects of an
inhibitor of macrophage migration inhibitory factor action [119]
deserve to be evaluated in pre-clinical assay, especially because
of the analogy of the CHIKV pathophysiology in both mouse and
macaque models [120].
To date, none of these treatments have been used in humans
either in the acute or chronic phases of disease, due to lack of effort
to develop relevant animal models of the chronic disease despite
our rst promising results [39].

reservoirs should be investigated to assess their involvement in

transmission in endemic areas and the associated risk in newly
exposed countries.
The most impressive advances have been made in basic virology. Up to 2005, most of the data for CHIKV were deduced from
studies of other alphaviruses. The role of epistatic mutations in
adaptation to host cells was an important discovery that might
help to understand specic pathogenic effects of the virus in mammalian or mosquito cells. This may aid in future viral surveillance
for potential epidemic threats. Moreover, this will assist in designing attenuated viruses for vaccination, currently the least expensive
mode of vaccine design. Another important advance was the use of
Chikungunya human clinical strains with a focus on low passaged
viruses. Finally, understanding the cellular cycle in vivo, the role
of the various cellular targets and the interplay between viral and
cellular proteins in the clinical evolution of CHIKV patients will provide tools for both diagnosis and treatment. In particular, there is
a real need for simple and affordable diagnostic tests for endemic
developing countries.
The understanding of the pathogenesis of acute CHIKV improved
these last years, and several cytokines and chemokines have been
explored and suggested as playing a key role in the interactions
between CHIKV and the host immune system. However, exploring
how the innate immune system, in particular the role of NK cells,
affects the outcome of CHIKV infection should be considered in the
future. Interestingly, the nature and the severity of CHIKV infection and of cytokine proles vary between human populations with
the percentage of chronic disease. In this context, it would also be
of great interest to focus future research on the study of human
genetic resistance.
Chikungunya fever is currently symptomatically treated with
antipyretics and non-steroidal anti-inammatory drugs. There will
be a continued search for new antiviral candidates with a clearly
dened mechanism of viral inhibition in cell-based systems and
signicant activity in animal models. These will include therapy
based on specic immunoglobulins or molecules that modify the
inammatory response associated with CHIKV infection. Moreover,
studies on the pathogenesis of Chikungunya chronic rheumatic
manifestations and the effects of disease-modifying anti-rheumatic
drugs should be explored, as the burden of chronic disease has been
shown to be unexpectedly high. Standardization of chronic patient
follow-ups and animal models are needed for this purpose.

7. 10 years research roadmap

Acknowledgments

The recent global outbreaks of CHIKV have driven great

advances in understanding of the disease. Interestingly, CHIKV or its
specic antibodies have been detected in several mammals other
than primates. The poorly understood role of these intermediate

The authors are grateful to Dr. Philippe Desprs for helpful comments and for providing unpublished data. We are also thankful to
Caroline Petitdemange from CIRMF for help in editing the text and
gures.

Funding
PR and ICS received funding from the European Unions Seventh Framework Program ICRES (Grant Agreement No. 261202).
ICS was funded by University Malaya (UMRG grant RG526-13HTM
and PPP Grant PG030-2012B) and the Ministry of Higher Education,
Malaysia (FRGS Grant FP036-2013A).
Competing interests
The authors declare they have no competing interest.
Ethical approval
Not required.

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