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Chikungunya Fever: CNS Infection and Pathologies of a Re-Emerging

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 Progress in Neurobiology 91 (2010) 121–129

Contents lists available at ScienceDirect

Progress in Neurobiology
journal homepage: www.elsevier.com/locate/pneurobio

Chikungunya fever: CNS infection and pathologies of a re-emerging arbovirus

Trina Das, Marie Christine Jaffar-Bandjee, Jean Jacques Hoarau, Pascale Krejbich Trotot, Melanie Denizot,
Ghislaine Lee-Pat-Yuen, Renubala Sahoo, Pascale Guiraud, Duksha Ramful, Stephanie Robin,
Jean Luc Alessandri, Bernard Alex Gauzere, Philippe Gasque *
Immunopathology Infection Research Grouping, University and CHR Gelix Guyon of La Reunion, Saint Denis, 97400, Reunion

A R T I C L E I N F O A B S T R A C T

Article history: Chikungunya virus (CHIKV) is transmitted by Aedes mosquitoes and causes an acute symptomatic illness
Received 18 April 2009 with fever, skin rash, and incapacitating arthralgia, which can evolve into chronic rheumatoid arthritis in
Received in revised form 18 August 2009 elderly patients. This is a tropical disease originally described in central/east Africa in the 1960s, but its
Accepted 14 December 2009
2004 re-emergence in Africa and rapid spread in lands in and around the Indian Ocean (Reunion island,
India, Malaysia) as well as Europe (Italy) led to almost 6 million cases worldwide. The risk of importation
Keywords: and spreading diseases with long-term sequelae is even greater today given the global distribution of the
Alphavirus
vectors (including in the Americas), increased tourism and the apparent capacity of CHIKV to produce
Neuroinfection
Innate immunity
high levels of viremia (109–1012 virus/ml of blood) and new mutants. CHIKV-associated neuropathology
Neurodegeneration was described early in the 1960s, but it is the unprecedented incidence rate in Indian Ocean areas with
efficient clinical facilities that allowed a better description of cases with severe encephalitis,
meningoencephalitis, peripheral neuropathies and deaths among newborns (mother-to-child infection),
infants and elderly patients. Death rates following CHIKV infection were estimated at 1:1000 cases in la
Reunion’s outbreak. These clinical observations have been corroborated by experimental infection in
several mouse models, leading to CNS pathologies. We further describe in this review the capacity of
CHIKV to infect neurons and glial cells, delineate the fundamental innate (intrinsic) immune defence
mechanisms to protect from infection and argue about the possible mechanisms involved in the
encephalopathy.
ß 2010 Elsevier Ltd. All rights reserved.

Contents

1. Chikungunya, epidemiological and clinical data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

2. Neurological manifestations of Chikungunya in newborns, infants and adults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
3. CHIKV neuroinfection and possible pathological mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
4. CNS innate immune protection against viral neuroinfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
5. Conclusion: exploring novel anti-viral therapies to fight against CHIKV infection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Abbreviations: APAF-1, apoptotic protease activating factor; CHIKV, Chikungunya virus; CNS, central nervous system; FADD, Fas-associated death domain; IAP, inhibitor of
apoptosis; IFN-a/b, type-I interferons; IFNAR, interferon receptor; ISGs, IFN-stimulated genes, e.g. IFN-stimulated protein of 15 kDa; IPS-1, IFN-b promoter stimulator-1, also
known as MAVS, CARDIF and VISA; IRF, interferon (IFN)-regulatory factor; MDA-5, melanoma differentiation-associated gene 5; Mx, myxovirus resistance () protein; NFkB,
nuclear factor-kB; OAS, oligoadenylate synthetase; PARP, poly(ADP-ribose) polymerase; PKR, IFN-inducible dsRNA-dependent protein kinase; PRR, pattern-recognition
receptors; RIG-I, retinoic-acid-inducible gene I-(RIG-I)-like helicases (RLHs).; RIP1, receptor-interacting protein 1; RNaseL, ribonuclease L; STAT1, signal transducer and
activator of transcription; SVZ, subventricular zone; TLR, Toll-like receptors (TLRs); TRADD, tumour-necrosis factor receptor-associated via death domain.
* Corresponding author at: IRG, University of la Réunion, St Denis, 15 Avenue René Cassin, BP7151, 97715, Ile de la Réunion. Tel.: +262 262 938836; fax: +262 262 938801.
E-mail addresses: gasque@univ-reunion.fr, gasquep@hotmail.com (P. Gasque).

0301-0082/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pneurobio.2009.12.006
122 T. Das et al. / Progress in Neurobiology 91 (2010) 121–129
Table 1
Main alphaviruses causing human pathologies and their geographical distribution.
1. Chikungunya, epidemiological and clinical data
CHIKV is an Alphavirus of the Togaviridae family transmitted by
mosquitoes of the Aedes (Ae) genus (Pialoux et al., 2007; Weaver
and Barrett, 2004). The alphavirus group comprises 29 viruses, 6 of
which can cause human joint disorders (arthralgia evolving to
arthritis), namely CHIKV, o’nyong-nyong virus (ONNV), Semliki
Forest virus (SFV) (Africa, Asia, Europe), Ross River (RRV)
(Australia and the Pacific), Sindbis virus (SINV) (cosmopolitan),
and Mayaro virus (MAYV) (South America, French Guyana)
(Table 1).
Three CHIKV lineages with distinct genotypic and antigenic
characteristics have been identified: isolates from the Indian
Ocean outbreak represent a distinct clade within a large east
central and southern African phylogroup distinct from the Asian
phylogroup and from the West African phylogroup. To date, several
complete CHIKV nucleotide sequences have been determined and
including the human isolates (Ross and S27) recovered during the
1952–1953 outbreaks in Tanzania. Between the 1960s and 1990s,
the virus was frequently isolated from Central and Southern Africa
including Sudan, Uganda, Democratic Republic of Congo the
Central African Republic, Malawi, Zimbabwe, Kenya and South
Africa (see Fig. 1, geographical distribution of CHIKV and Aedes
alpbopictus).
CHIKV has also been isolated in western African countries
including Senegal, Benin, the Republic of Guinea, Cote d’Ivoire and
Nigeria. In South East Asia, frequent outbreaks were reported from
the 1960s through to the 2000s in India, Malaysia, Indonesia,
Cambodia, Vietnam, Myanmar, Pakistan, Singapore and Thailand
(see comprehensive review; Powers and Logue, 2007). In 2004,
CHIKV re-emerged in Kenya and subsequently spread eastward,
causing millions of cases throughout countries in and around the
Indian Ocean. Estimated cases for India alone were in the order of
1.6–6 million infected people (Mavalankar et al., 2007). Outbreaks
occurred almost continuously during 2004–2007 with cases
reported in Europe (Italy, UK, Belgium, Germany, Czech Republic,
Norway, Spain and France), Hong Kong, Canada, Taiwan, and Sri
Lanka and these were directly associated with the return of tourists
from India and affected islands of the Indian Ocean. The risk of
emerging or re-emerging CHIKV epidemics is ever present because
of the high attack rates, the high levels of viremia in acutely
infected humans (109–1012 virus per ml of blood) (our unpublished
data and (Panning et al., 2008)), and the worldwide distribution of
the well-adapted vectors responsible for transmitting CHIKV
 T. Das et al. / Progress in Neurobiology 91 (2010) 121–129
Fig. 1. Worldwide distribution of CHIKV (endemic in yellow shade and imported cases in blue) and Aedes albopictus (before 1980 in green and after 1980 in pink) (adapted
from Powers and Logue, 2007; Charrel et al., 2007).
(Fig. 1). For instance, CHIKV has hardly been reported in Australia,
the USA and South America but this could be a matter of future
concerns given that Aedes albopictus is now found in these
countries (Fig. 1) (Charrel et al., 2007; Lanciotti et al., 2007).
The CHIKV genome is approximately 11 kb and consists of a
single-stand of positive-sense RNA with two open reading frames
(ORF). The 50 ORF encodes the virus non-structural proteins (nsp)
1–4. The nsps are multifunctional and together form the virus
replicase. The 30 ORF encodes the virus structural proteins, the
capsid and the envelope glycoproteins E1 and E2. E3 is a secreted
polypeptide of unknown functions (Chevillon et al., 2008).
Interestingly, a mutation at residue 226 of the membrane fusion
glycoprotein E1 (E1-A226 V), which was absent from the strains
isolated during the first months of the ongoing outbreak in la
Réunion, was found in more than 90% of isolates after September
2005 (Schuffenecker et al., 2006). This type of change originally
described in another close alphavirus, SFV, could be related to virus
adaptation to the mosquito vector species as recently described
(Tsetsarkin et al., 2007; Vashishtha et al., 1998). Other CHIKV
isolates are currently being sequenced to provide spatiotemporal
data on the viruses isolated from different locations and to find out
whether any molecular signatures could be associated with
unusual clinical forms (Powers and Logue, 2007). The vast scale
and sudden emergence of the la Réunion outbreak underline how
little we knew about the biology and pathology of CHIKV. Overall,
270,000 cases were reported and this outbreak was the first time
that a number of severe clinical manifestations, particularly
following infections of newborns and elderly (see below), and
rare mortality (estimated at 1:1000 of cases) were reported to be
associated with CHIKV (Economopoulou et al., 2009; Gerardin
et al., 2008; Lemant et al., 2008; Mavalankar et al., 2007; Pialoux
et al., 2007; Ramful et al., 2007; Robin et al., 2008, 2009;
Schuffenecker et al., 2006). Classically, infected people adopt a
characteristic stooped walking position which is the hallmark of
the disease and from which it derives its name Chikungunya,
meaning ‘to walk bent over’, in the Kimakonde language of
Mozambique. The classic clinical symptoms after infection by
CHIKV are abrupt febrile illness (temperature usually >38.9 8C)
and maculopapular rash, with articular pains responsible for a
stooped walk. Other symptoms include myalgia, headache, edema
of the extremities, and gastrointestinal complaints (Borgherini
et al., 2007; Lakshmi et al., 2008). The incubation period for CHIKV
123
ranges from 3 to 7 days and as few as 5% of asymptomatic cases
were generally reported. The laboratory diagnosis of CHIKV
infection is based on either specific serology or detection of viral
RNA by reverse transcription-polymerase chain reaction (RT-PCR).
RT-PCR is positive during the viremic phase lasting for 1 week after
onset of the symptoms while the levels of IgG (and surprisingly
IgM) can last for months. Interestingly, and in contrast to Dengue,
the apparition of IgG anti-CHIKV was detected in the first week
following infection and illustrating the rapid and robust adaptive
immune response (Panning et al., 2008). The acute signs and
symptoms usually resolve in less than 2 weeks, but arthralgia may
linger for weeks or months. Particularly in the elderly, CHIKV has
had rather profound acute arthritogenic activities and can
contribute to chronic incapacitating arthritis as described for
several alphaviruses (Harley et al., 2001; Levine et al., 1994;
Suhrbier and La Linn, 2004; Tesh, 1982; Toivanen, 2008).
Rheumatic manifestations in 10–20% of the patients typically
consisted of a febrile arthritis mainly affecting the extremities
(ankles, wrists, phalanges) (Borgherini et al., 2008; Brighton and
Simson, 1984; Fourie and Morrison, 1979; Simon et al., 2007;
Sissoko et al., 2009). Beneficial treatments of these chronic
conditions involved the use of paracetamol with a nonsteroid
anti-inflammatory drug, glucocorticoids and methrotrexate in the
most severe cases.
Skin lesions were also reported and included vesiculobullous
lesions with desquamation, aphthous-like ulcers and vasculitis
lesions (Pialoux et al., 2007; Robin et al., 2009) as well as unusual
complications such as fulminant hepatitis, cardiologic manifesta-
tions and neurologic diseases. Knowledge is currently limited
about neurologic forms of CHIKV and its long-term sequelae
among children. Other serious non-neurological complications
have been reported in la Réunion and elsewhere, including acute
hepatitis, bullous dermatosis, heart failure, myocarditis, pneumo-
nia, mild hemorrhage and pre-renal failure. Of critical note, these
severe cases were reported from hospitalized cohorts of patients
and may be related to several underlying medical conditions, most
commonly with hypertension, respiratory conditions and diabetes
mellitus. In a recent study from India, age >85 years was the only
risk factor for mortality although the possibility of co-infection by
dengue virus (and other pathogens such as the respiratory
syncytial or influenza viruses) may be a contributing factor to
be considered (Sankari et al., 2008; Tandale et al., 2009).
124 T. Das et al. / Progress in Neurobiology 91 (2010) 121–129

2. Neurological manifestations of Chikungunya in newborns,
infants and adults
CHIKV infection was first reported to affect the nervous system
in the 1960–1970s (Carey et al., 1969; Chastel, 1963; Chatterjee
et al., 1965; Hammon et al., 1960; Jadhav et al., 1965;
Nimmannitya et al., 1969; Thiruvengadam et al., 1965). It was
found to be associated with seizures, meningoencephalopathy,
myelitis, and choroiditis (Arpino et al., 2009). The same clinical
hallmarks were reported in recent outbreaks but also included
periventricular encephalitis, meningoencephalitis and deaths in
neonates and adults hospitalized with severe CHIKV (Economo-
poulou et al., 2009; Gerardin et al., 2008; Lemant et al., 2008;
Lewthwaite et al., 2009; Ramful et al., 2007; Robin et al., 2008;
Tandale et al., 2009).
Encephalopathy (associated with the detection of the virus in
the CSF) appears to represent the most common neurologic
manifestation among CHIKV-infected neonates after mother-to-
child transmission. In la Reunion, the prevalence rate of maternal–
fetal infections was 0.25%. In contrast, during the delivery period,
the rate of transmission for viremic women was close to 50%
highlighting the intrapartum period as the critical time for
transmission to the neonate (Gerardin et al., 2008; Ramful et al.,
2007). Cesarean sections had no protective effect on transmission.
Dramatically, at least 50% of the infected neonates had pathologic
MRI findings (combination of white matter lesions and/or brain
swelling and/or cerebral hemorrhages) evolving sometimes
towards persistent disabilities and deaths (Fig. 2) (Gerardin
et al., 2008; Ramful et al., 2007; Robin et al., 2008). In acute
conditions, the supratentorial white matter lesions appeared
essentially in the corpus callosum and the frontal, parietal and
temporal lobes. These lesions evolved into vasogenic edema and
finally, in some cases, towards cavitations and subcortical
athrophy. Persistent disabilities (10–20% of the infected neonates)
after 1–2 years included seizures, cerebral palsy with ataxia and
blindness, and ocular and behavioral or postural deficiencies
(Gerardin et al., 2008).
The neurological signs were less severe in children hospitalized
following CHIKV infection but included cases of encephalitis,
febrile seizures, meningeal syndrome and acute encephalopathy.

Fig. 2. MRI findings in a neonate (12 days of life) with encephalopathy secondary to mother-to-child transmission of CHIKV infection on the 7th day after onset of disease. (a)
Diffusion weighted imaging: hyperintensity signals in the corpus callosum, subcortical and periventricular white matter, respecting the U-fibres. Reduction of the apparent
diffusion coefficient is compatible with cytotoxic edema. (b) T2-weighted imaging: petechial hemorrhages and hematomas (nodular lesions) with hyperintensities in the
subcortical and periventricular frontal and occipitoparietal white matter. (c) Gradient echo MRI: subtentorial hematoma of the left cerebellar hemisphere. (d) T1-weighed
imaging: hyperintensities in the subcortical and periventricular with matter corresponding to areas of hemorrhage. MRI abnormalities appear as white area.
 T. Das et al. / Progress in Neurobiology 91 (2010) 121–129
The MRI scans were abnormal only in a limited number of cases
(less than 20%) but deaths were reported together with long-term
sequelae in 10–20% of the survivors (Robin et al., 2008). Deaths
were observed in previously healthy children and probably due to
massive hemorrhages associated or not with brain swellings
(Robin et al., 2008). Behavioral and communication disorders
(including autistic features) were reported in the follow-up of the
infected children (Robin et al., 2008).
Neurological manifestations described in adults requiring
hospitalization (incidence of 15–25%) involved cases of encepha-
lopathy frequently associated with the presence of IgM anti-CHIKV
in the CSF, encephalitis, Guillain Barre, encephalomyeloradiculitis
and rare deaths (Chandak et al., 2009; Economopoulou et al., 2009;
Ganesan et al., 2008; Lebrun et al., 2009; Lemant et al., 2008;
Tournebize et al., 2009; Wielanek et al., 2007). In the Ganesan’s
case studies it was found that CHIKV infection in adults was
associated to bilateral frontoparietal white matter lesions with
restricted diffusion, which is described as an early sign of viral
encephalitis (Nouranifar et al., 2003). At autopsy, the brain was
swollen and with subarachnoid cerebellar hemorrhages. Histolog-
ically, focal perivascular lymphocytic infiltrates were also present
in the area of active demyelination and some degree of microglial
activation but no astrogliosis was also noted and which may
contribute to bystander neuronal loss as described below.
Unfortunately, no staining for CHIKV was performed in this study
(Ganesan et al., 2008).
Although all the CHIKV-related neurological data are still
scarce, the number of cases with CNS involvement appears to
support the neurotropic/neuroinfectious activity of CHIKV.
3. CHIKV neuroinfection and possible pathological
mechanisms
Historically (since 1965), CHIKV has been shown to infect
mouse brain and to replicate in primary culture of glial cells
(Chatterjee and Sarkar, 1965; Precious et al., 1974). Further but
indirect evidence is supported by the observation that intracebral
injection in newborn mice can be used to amplify the virus before
preparing the hyperimmune ascitic fluid used to date in most of the
CHIKV ELISA kit (Schuffenecker et al., 2006). More convincing
evidence comes from experimental infections where mice were
inoculated subcutaneously leading to rapid (24 h) and robust
replication (106–107 PFU/ml) of CHIKV in the brain particularly of
newborn outbred mice (CD1, ICR) (Ziegler et al., 2008). Interest-
ingly, infected mice showed signs of illness such as loss of balance,
difficulty of walking, dragging of the hind limbs, and skin lesions
but rare mortality. Surprisingly, no definite histological evidence of
tropism to neurons was reported in these two mouse models and
the CNS infection seemed to be tightly controlled by ill-
characterized anti-viral mechanisms given that the viral titer
was reduced to basal levels at day 10 post-infection. In a more
recent review, Powers and Logue (2007) reported that CHIKV
injected intranasally in BALB/c mice led to neuronal infection and
tissue necrosis in the anterior olfactory lobe. A very promising
neuroinfectious model reported recently by Weaver and collea-
gues used intranasal injection of CHIKV Ross strain selected
because of its excessive mouse passage history, which may have
increased its virulence (Wang et al., 2008). Interestingly, 5-week-
old C57BL/6 mice developed encephalitis 7 days post-infection
with severe multifocal infection and liquefactive necrosis in the
cerebral cortex. Immunohistochemistry techniques revealed that
neurons were infected and induced to programmed cell death
while prominent microgliosis and perivascular cuffs were distrib-
uted throughout the parenchyma. Moreover, the authors reported
neuronal degeneration in the hippocampus and multifocal
lymphocytic leptomeningitis. In inbred mice deficient in the
125
IFN-a signaling pathway (KO for the IFNA receptor), CHIKV
neuroinfection was particularly severe, targeting the leptome-
ninges, the choroid plexus and ependymal cells lining the
subventricular zone (SVZ) also recognized as the neural stem cell
niche (Hauwel et al., 2005a,b). This area of the CNS seems to be
particularly targeted by several RNA viruses and with the key
observation that ependymal cells lining the SVZ express a plethora
of receptors (coxsackie adenovirus receptor (CAR), junction-
associated molecules (JAM), complement regulators CD55 and
CD46) for viruses such as coxsackie and adenovirus, reovirus,
echovirus, and measles (Antar et al., 2009; Hotta et al., 2003;
Kemper and Atkinson, 2009; Lindahl et al., 2000). Is the SVZ really
considered more immune-privileged then the rest of the brain? We
hypothesize that CHIKV targets progenitor and stem cells in the
SVZ as well as ependymal cells, possibly affecting neurogenesis
and neuronal migration which in turn could lead to demyelinating
encephalomyelitis, as recently reported in children and neonates
infected by CHIKV (Gerardin et al., 2008; Ramful et al., 2007; Robin
et al., 2008). CNS infections involving subventricular white matter
lesions and intraparenchymal hemorrhages have been described
experimentally and in clinical settings for other alphaviruses such
as SFV, RRV, EEV and SINV (Deresiewicz et al., 1997; Fazakerley
et al., 2006; Jackson et al., 1987; Mims et al., 1973). Interestingly, it
has recently been shown that flavivirus infection of the SVZ can
affect neurogenesis and have long-term effects with the depletion
of the neural stem cell pool, but whether this is also the case for the
alphavirus CHIKV remains to be ascertained (Das and Basu, 2008).
The capacity of alphaviruses to interact with platelets/endothelial
cells and hence contribute to hemorrhagic complications is still a
matter of debate (Couderc et al., 2008; Dropulic and Masters, 1990;
Larke and Wheelock, 1970; Scott et al., 1978).
The cell receptor for CHIKV is unknown but CHIKV was shown
to have profound cytopathic effects in vivo and in vitro (Couderc
et al., 2008; Ozden et al., 2007; Sourisseau et al., 2007); there is a
rapid shut-off of host cell transcription and translation followed by
apoptotic death as assessed by the presence of numerous active
caspase-3 and CHIKV double-positive cells (Sourisseau et al.,
2007). These data were obtained using the HeLa epithelial cell
model and it remains to be ascertained whether this is also the case
in neuronal and glial cells. To begin to address some of these issues,
we have infected mouse brain mixed cultures with low multiplicity
of infection (MOI) of CHIKV. Original data presented in Fig. 3 clearly
indicate that neurons (Tuj1 bIII tubulin+ cells) and astrocytes (glial
fibrillary acidic protein, GFAP+ cells) were extremely efficient at
replicating CHIKV. The events leading to apoptosis remain to be
fully determined although our data clearly indicate that both,
intrinsic (mitochondrial) and extrinsic (Fas/TRAIL-like) apoptosis,
pathways are involved consequently to CHIKV infection (pathways
illustrated in Fig. 4). The intrinsic apoptosis pathway is engaged by
cytochrome c released by the mitochondria to activate caspase 9
and sub sequentially caspase 3 (for review Clarke and Tyler, 2009;
Griffin, 2005) (see illustration Fig. 4). In the IFNAR/ mouse
model, it was shown that CHIKV induced severe vacuolization of
choroid plexus epithelial cells and ependymal cells, but failed to
affect endothelial cells of the blood–brain barrier (Couderc et al.,
2008). In the Ziegler study (Ziegler et al., 2008), glial cells and
neurons were rarely immunopositive for CHIKV antigens, but were
nevertheless found to be affected plausibly as a consequence of the
robust anti-viral innate immune and inflammatory responses as
described for many alphaviruses (Amor et al., 1996; Rowell and
Griffin, 2002; Schoneboom et al., 2000; Sharma et al., 2008; Vogel
et al., 2005). Gene profiling studies have clearly indicated that
alphavirus CNS infection is accompanied by an upregulation of
multiple pro-inflammatory molecules including inducible nitric
oxide synthase (iNOS), tumor necrosis factor (TNF-a), and several
cytokines and chemokines which ultimately would have direct or
126 T. Das et al. / Progress in Neurobiology 91 (2010) 121–129

Fig. 3. CHIKV infection and replication in neurons and astrocytes drive programmed cell death and innate immunity. Cells were either mock-infected or infected for 24–48 h
with CHIKV (MOI of 1). Cells were then double stained using antibodies against CHIKV (in green), markers of astrocytes (GFAP, red), of neurons (Tuj1, not shown), markers of
innate immunity (ISG15, in red), or markers of late apoptosis (cleaved PARP, in red). Of note, none of these antibodies stained mock-infected cells (not shown). (a) Cluster of
mouse embryonic neurons (E16–19) were strongly stained for CHIKV. (b) Primary astrocytes from 1- to 2-day-old mice were stained for CHIKV. CHIKV infection was also
evidenced in the CLTT mouse astrocyte cell line model stained for CHIKV (c), ISG15 (d), and cleaved PARP (e–f). Magnification 400.

indirect neurotoxic activities (Labrada et al., 2002; Sharma et al.,
2008). The innate immune and inflammatory cascade is an attempt
by resident cells to eliminate the challenge imposed on the brain,
clear the dead neurons, and rescue the normal functioning of the
tissue. However, during the process of microglial/astroglial
activation, the proinflammatory molecules may exert certain
detrimental effects and neurons/neural stem cells are likely to be
affected (Poluektova et al., 2005).
4. CNS innate immune protection against viral neuroinfection
Viruses are the most abundant pathogens that the host innate
immune system is challenged by particularly when they find a
privileged niche inside the CNS tissue (Griffin, 2003). Alpha-
viruses like many other RNA viruses are known to persist in
tissue sanctuaries but our understanding of the mechanisms
involved is still in its infancy (Levine and Griffin, 1992; Levine
et al., 1994; Precious et al., 1974). In most tissues, the loss of
infected cells preferably by apoptosis is accompanied by
enhanced cell division to replenish the cell population but this
may not be achievable in tissues with little capacity for renewal,
such as the CNS. The balance would be to favor non-cytolytic
clearance of the virus to preserve neuronal connections even if
this could promote latent viral infection (Binder and Griffin,
2001). In this case, a robust and rapid innate immune response
needs to be mobilized to limit viral replication in neuronal cells.
For instance, CNS viral infection rapidly results in the induction
of expression of the type-I interferons (IFNs) IFNa and IFNb, and
of pro-inflammatory cytokines by resident cells (for comprehen-
sive review see Paul et al., 2007). Type-I IFNs are essential for the
anti-viral CHIKV response as demonstrated in vitro and in vivo
(Couderc et al., 2008; Gardner et al., 2008; Sourisseau et al.,
2007). Accumulating evidence indicates that several key cyto-
plasmic cellular detection systems (RIG-I, MDA-5) are involved in
innate immune sensing of RNA viruses (Bowie and Unterholzner,
2008; Takeuchi and Akira, 2007) to initiate the protective IFN
response (Fig. 4). RNA viruses such as CHIKV can also activate
TLR-7 and TLR-8 localized into endosomes together with TLR3
and TLR9 (Fig. 4). This activation step is dependent on the
acidification of the endosomes which is blocked by chloroquine
(Lund et al., 2003, 2004), a drug that was tested for the treatment
of CHIKV but, hence found unlikely to protect from infection
(Coombs et al., 1981; de Lamballerie et al., 2009; Maheshwari
et al., 1991; Seth et al., 1999).
 T. Das et al. / Progress in Neurobiology 91 (2010) 121–129 127

Fig. 4. Innate immune sensing, apoptosis signaling pathways and anti-viral responses in the CNS. Several innate immune receptors also called ‘pattern-recognition receptors
(PRRs)’, such as RIG-I (retinoic-acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5) and TLR (Toll-like receptors) are expressed by neurons and glial
cells (microglia and astrocyte) to initiate signaling pathways that converge at the activation of the transcription factors interferon (IFN)-regulatory factor 3 (IRF3), IRF7 and/or
nuclear factor-kB (NFkB). These key events lead to the expression of type-I interferons (IFN-a/b). IFNs binding to the receptor (IFNAR) then initiate an anti-viral effector
program in the infected cell and neighboring cells, which involves the expression of numerous IFN-stimulated genes (ISGs) and pro-inflammatory cytokines and chemokines.
ISGs such as oligoadenylate synthetase (OAS) and ribonuclease L (RNaseL), IFN-inducible dsRNA-dependent protein kinase (PKR), myxovirus resistance (Mx) protein, are
involved in anti-viral mechanisms that interfere with the life cycle of individual viruses. OAS and PKR are further activated by double-stranded RNA (dsRNA). IFNAR1,
interferon-a receptor; IPS1, IFNB promoter stimulator 1; ISG15, IFN-stimulated gene of 15 kDa; PPP, 5 triphosphate; ssRNA, single-stranded RNA; STAT, signal transducer and
activator of transcription. The innate immune signaling pathways are intimately linked to apoptosis through the mobilization of TRADD, RIP1 and FADD. The intrinsic
apoptosis pathway is initiated by the release of cytochrome C from the mitochondria which promotes the formation of the apoptosome, including APAF-1 (apoptotic protease
activating factor) and caspase 9 which in turn activates the effector caspase 3. The extrinsic pathway mobilized notably by TNF-a, TRAIL or FASL involves death-receptor
signaling pathways linked to FADD and caspase 8. Some viruses such as CHIKV (our unpublished data) can escape apoptosis by inducing the expression of anti-apoptotic
proteins such as IAP and Bcl2.

These innate immune receptors are the first line of defense
against pathogens infiltrating the CNS and they are called pattern-
recognition receptors (PRRs) for their capacity to recognize a
plethora of pathogens and to drive signaling pathways to promote
anti-viral responses. These PRRs use diverse adaptor proteins and
signaling pathways resulting in activation of a distinct set of
transcription factors and include NFkB (p50/p65 subunits) and
IFN-regulatory factors (IRFs). NFkB and IRFs translocation into the
nucleus will lead to the transcription of pro-inflammatory
mediators and survival factors, including IL-6, TNF-a, IL-12, IL-8
and IFN-g. RIG-I and MDA-5 activate IPS-1 (IFN-b promoter
stimulator-1, also known as MAVS, CARDIF and VISA), a protein
located on the surface of mitochondria. Downstream this would
result in translocation of the transcription factors IRF-3 and NFkB
but could also lead to apoptosis through the recruitment of the
(TRADD, tumour-necrosis factor receptor-associated via death
domain; RIP1, receptor-interacting protein 1 and FADD, Fas-
associated death domain) complex linked to caspase 8 (Fig. 4)
(Balachandran et al., 2000; Michallet et al., 2008; Takahashi et al.,
2006; Wilson et al., 2009). The precise role of these different
adaptor proteins in the signalling cascades to promote cytokine
production and/or apoptosis following CHIKV infection remains to
be ascertained. Recent data indicate that nsP2 of an alphavirus is an
antagonist of the type-I interferon response (Breakwell et al., 2007)
and CHIKV may control innate immunity in a similar manner.
Coordinated PRR amplification involves IFN-a/b dependent path-
ways with the translocation of phosphorylated STAT1 and STAT2
molecules into the nucleus to drive the expression of several
interferon-stimulated genes (ISGs) and other anti-viral proteins.
Factors directly affecting viral replication are PKR, OAS, RNaseL,
and ISG15, ISG54 and ISG56, which all modulate translation; Mx
which inhibits viral transcription; the RNA deaminases ADAR-1
and APOBEC3G, leading to mutations in viral genomes. IFN-a/b
responsiveness in CNS cells is indicated by induction of various
ISGs, including RNaseL, ISG54 and ISG56, Mx, IRF-7 and IRF-9
following distinct infections (for review Paul et al., 2007).
Interestingly, OAS has recently been shown to be active against
CHIKV replication in HeLa cells and its role in neuronal cells will
need to be further established (Brehin et al., 2009). Of preliminary
note, we found a robust expression of ISG15 by CLTT astrocyte cell
line in response to CHIKV infection (see Fig. 3). This astrocyte
model will be invaluable to decipher the molecular signaling
pathways engaged by one or many PRR to drive anti-CHIKV
responses in the CNS.
5. Conclusion: exploring novel anti-viral therapies to fight
against CHIKV infection
A new and comprehensive description of CHIKV-induced
neuroinfection and pathology has been gained from recent
outbreaks in countries with efficient clinical facilities, including
MRI scanning. Severe encephalitis and deaths are clear testimo-
nies of the severity of the disease particularly following mother-
to-child transmission and in elderly patients with cardiovascular
diseases or diabetes. The plausible role of mutated forms of
CHIKV with high neuroinfection fitness needs to be more firmly
addressed, making use of the several mouse models reproducing
the disease and cDNA CHIKV infectious clones (Powers and
Logue, 2007). From a clinical standpoint it is critical that
physicians are educated in recognizing acute cases and that
diagnostic tools are available to perform discriminative screen-
ings from other tropical diseases. Interestingly, several cases of
CNS infection have been associated with co-infection (mainly
Dengue), which may explain some of the severe cases in recent
outbreaks. Further research is warranted to have a better
understanding of CHIKV-induced pathology and the long-term
sequelae particularly in children. Arboviruses have long been
known to persist in infected tissues, driving chronic pathologies
(Kuno, 2001).
The restriction of CHIKV diseases is undoubtedly through
prevention measures such as vaccination. A live, attenuated
vaccine was originally advanced through phase II clinical trials,
but its development was halted because of its poor tolerance
(Edelman et al., 2000). Ongoing studies will help to delineate the
immunogenicity of the different CHIKV proteins and to design a
safer and more potent vaccination regime to protect from future
128 T. Das et al. / Progress in Neurobiology 91 (2010) 121–129
outbreaks and life-threatening infection (Muthumani et al., 2008;
Wang et al., 2008).
Acknowledgements
The authors’ work is funded by the University of La Reunion,
French Ministry of Health (PRHC 2006–2009), French overseas
Ministry (MOM), the CRVOI (centre de recherche et de veille de
l’Océan Indien), the Regional council of La Réunion and Europe
(CPER/FEDER/GRII). PG is a fellow of INSERM ‘contrat d’interface’
with Pr. P. Debre and Pr. B. Autran (INSERM U945, Salpétrière,
Paris). The authors thank Mrs Claudine Vitry for help in editorial’s
management of the paper and figure.
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