VECTOR-BORNE AND ZOONOTIC DISEASES Volume 15, Number 4, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2014.1710

Chikungunya Virus Pathogenesis and Immunity Philippe Gasque,1 Therese Couderc,2,3 Marc Lecuit,2,3,4 Pierre Roques,5 and Lisa F.P. Ng 6

Abstract

Chikungunya virus (CHIKV) is an arbovirus associated with acute and chronic arthralgia that re-emerged in the Indian Ocean islands in 2005–2006 and is currently responsible for the ongoing outbreaks in the Caribbean islands and the Americas. We describe here the acute and chronic clinical manifestations of CHIKV in patients that define the disease. We also review the various animal models that have been developed to study CHIKV infection and pathology and further strengthened the understanding of the cellular and molecular mechanisms of CHIKV infection and immunity. A complete understanding of the immunopathogenesis of CHIKV infection will help develop the needed preventive and therapeutic approaches to combat this arbovirosis. Key Words: Chikungunya—Target cells—Innate immunity—Cytokines and chemokines—Inflammation— Adaptive immunity.

Background of Chikungunya Fever

C

hikungunya fever (CHIKF) is an arthropod-borne viral disease transmitted by the Aedes (Ae.) aegypti and Ae. albopictus mosquitoes. It characterized by fever, headache, rashes, and debilitating arthralgia (Robinson 1955, Pialoux et al. 2007). Chikungunya means ‘‘to walk bent over’’ in the Makonde language; this is the classic posture adopted by CHIKF patients during the acute phase. Caused by the chikungunya virus (CHIKV), an alphavirus belonging to the Togaviridae family, the disease has an incubation period of 3–7 days. The virus is usually detectable in peripheral blood between 1–2 weeks and the peak of viremia (up to 108 pfu/mL of plasma) is associated with the onset of the disease (Powers and Logue 2007). Although there can be 5–15% asymptomatic patients, CHIKF is mainly an incapacitating and nonfatal disease. However, since the Indian Ocean islands outbreaks due to the A226V Ae. albopictus East-Central-South-African (ECSA)-adapted strains, severe forms and deaths, often associated with co-morbidities, have been reported (Mavalankar et al. 2007, Lemant et al. 2008). Reports have chronicled the new wave of CHIKV outbreaks in the Americas since November of 2013 due to the adaptation of the Asian Ae. aegypti strain in the French West Indies and

Caribbean islands (Leparc-Goffart et al. 2014). The virus has since spread to several parts of Central and Latin America (Morens and Fauci 2014). But until now, there have been no reports that describe the disease severity in these countries. Disease Manifestations in Adults and Children

Typical disease symptoms in most patients ( > 85%) include abrupt febrile illness (temperature usually > 38.9C) and a maculopapular rash with articular pains. Other symptoms include myalgia, headache, edema of the extremities, ocular manifestations, and gastrointestinal symptoms (Borgherini et al. 2007, Lakshmi et al. 2008), and may be linked to direct or indirect effects of viral replication in these tissues (Ozden et al. 2007). Mother-to-child transmission of CHIKV was first reported during the 2005–2006 outbreaks in La Re´union (Robillard et al. 2006, Ramful et al. 2007, Ge´rardin et al. 2008) and later in India and Sri Lanka (Rao et al. 2008, Senanayake et al. 2009). These rare cases have been associated with 50% of neonates born from mothers having detectable CHIKV peripartum viremia (Ge´rardin et al. 2008). Infected neonates endured encephalopathy and seizures, thrombocytopenia, hypotension, ventricular dysfunction, pericarditis, and hyperechoic coronary

1 University of La Reunion, GRI/IRG EA4517, and Centre Hospitalier Universitaire (CHU North Felix-Guyon), Saint-Denis, La Reunion, France. 2 Institut Pasteur, Biology Infection Unit, Paris, France. 3 Inserm U1117, Paris, France. 4 Paris Descartes University, Sorbonne Paris Cite´, Division of Infectious Diseases and Tropical Medicine, Necker-Enfants Malades University Hospital, Institut Imagine, Paris, France. 5 CEA, Division of Immuno-Virology, iMETI, Fontenay-aux-Roses, France and University Paris-Sud XI, UMR-E1, Orsay, France. 6 Singapore Immunology Network, A*STAR, Singapore.

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arteries. Moreover, white matter lesions, parenchymal hemorrhages, and early cytotoxic edema have been observed by magnetic resonance imaging (MRI), and viral RNA was also detected in the spinal fluid. Long-term follow-up of the infected neonates confirmed poor outcome with a reduced mean developmental quotient (Pellot et al. 2012, Ge´rardin et al. 2014). CHIKF is usually benign in children. However, atypical manifestations with subsequent sequelae have been described, and these include neurologic manifestations ranging from simple and complex febrile seizures to meningeal syndrome, acute encephalopathy, diplopia, aphasia, acute disseminated encephalomyelitis, and encephalitis (Le Bomin et al. 2008, Robin et al. 2008, Lewthwaite et al. 2009, Valamparampil et al. 2009). Severe skin blistering was been described with intraepidermal vesiculobullous lesions (Robin et al. 2008, Valamparampil et al. 2009). Conversely, persistent arthralgia and exacerbation of underlying medical conditions are rare in children. Severe Clinical Manifestations

The 2005–2006 epidemic in La Re´union was the first time that severe adult cases and deaths due to CHIKF were documented (Economopoulou et al. 2009). These severe cases occurred in those with underlying medical conditions (cardiovascular, neurological, and respiratory disorders). Most patients presented a glycemic impairment that revealed diabetes mellitus in 20% of the severe cases, often associated with elevated levels of liver enzymes. Notably, there was a 22% increase in adult patients with Guillain–Barre´ syndrome that required respiratory support during the La Reunion outbreak (Lebrun et al. 2009). Particularly in the elderly over 60 years of age, CHIKV had profound acute arthritogenic activities that could have contributed to chronic incapacitating arthritis described in other alphaviral diseases (Tesh 1982, Levine et al. 1994, Harley et al. 2001, Suhrbier and La Linn 2004, Toivanen 2008). Rheumatic manifestations in up to 50% of the adult patients (6 months to several years postinfection) typically consisted of a febrile arthritis mainly affecting the extremities (ankles, wrists, phalanges) (Fourie and Morrison 1979, Brighton and Simson 1984, Simon et al. 2007, Borgherini et al. 2008, Sissoko et al. 2009, Manimunda et al. 2010, Schilte et al. 2013). Moreover, patients with post-CHIKV rheumatoid arthritis (RA)-like illnesses were also reported (Chopra et al. 2008). The development of progressive erosive arthritis was also reported in some studies (Brighton and Simson 1984, Malvy et al. 2009, Manimunda et al. 2010). However, in contrast to what is known in canonical autoimmune RA, the levels of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibodies were not elevated (Manimunda et al. 2010), thereby suggesting that post-CHIKV arthritis was a chronic inflammatory erosive arthritis (Ribera et al. 2012). Mechanisms of Disease Pathogenesis Infection and early events

A plethora of cutaneous manifestations, including erythematous maculopapular or morbiliform eruption, which subsided within 3–4 days without any sequelae, have been reported (Prashant et al. 2009). This eruption could be a hallmark of the inflammatory response of the skin (the portal of entry of the virus after the mosquito’s bite) that mobilized resident cells, such as keratinocytes, melanocytes, and der-

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mal fibroblasts (Couderc et al. 2008, Puiprom et al. 2013, Thon-Hon et al. 2012, Thangamani et al. 2013). CHIKV has been proposed to interact with resident dendritic cells (DCs), including Langerhans cells, and these cells contribute to virus spread to other target organs, such as muscles, liver, kidney, heart, and brain (Kam et al. 2009). It is now well established that both hematopoietic and nonhematopoietic cells are engaged in the control of CHIKV infection by the innate immune system (Her et al. 2010, Schilte et al. 2010). Nonhematopoietic fibroblast cells have been reported to be susceptible to in vitro CHIKV replication and to be the main cell type infected in target tissues (muscle, joint, and skin) in mice, and as well as in humans (Sourisseau et al. 2007, Couderc et al. 2008). Monocytes and macrophages have also been reported to be targeted by CHIKV and to be involved in virus-induced pathogenesis in both CHIKF patients and in animal models (Her et al. 2010, Hoarau et al. 2010, Labadie et al. 2010, Teng et al. 2012). One of the most frequent ocular manifestations associated with CHIKV infection is uveitis. In patients and in mouse models, CHIKV has been shown to target fibroblasts of eye tissues, including cornea, sclera, ciliary body, iris, and ocular motor muscles, which could provide a virological explanation for this ocular symptom (Couderc et al. 2012). Innate immunity and inflammation

Fever experienced by all CHIKF patients is associated to the synthesis of cytokines such as interleukin-1b (IL-1b, IL6, and tumor necrosis factor-a (TNF-a), which are known pyretics (Ng et al. 2009). These cytokines have been detected at high levels in acutely infected patients (Wauquier et al. 2011, Chow et al. 2011, Kelvin et al. 2011), and the levels returned to normal after fever and viremia disappeared (Wauquier et al. 2011, Chow et al. 2011, Kelvin et al. 2011). Arthralgia experienced by CHIKF patients closely resembles the symptoms induced by other arthritogenic alphaviruses (Surhbier 2004, Pialoux et al. 2007, Powers and Logue 2007). It is characterized by severe joint pain associated with inflammation and tissue destruction and inflammatory cytokines such as IL-1b, IL-6, and TNF-a (Ng et al. 2009, Hoarau et al. 2010, Chow et al., 2011). It has also been shown that interferon (IFN) produced by CHIKV-infected fibroblasts induced high expression of prostaglandins in these cells (Fitzpatrick and Stringfellow 1980). This may contribute to mechanisms of nociceptor activation and sensitization as described in osteoarthritis joints (Fitzpatrick and Stringfellow 1980, Malfait and Schnitzer 2013). Thus, it is plausible that CHIKV infection induces a self-perpetuating proinflammatory reaction that causes arthralgia, accounting for the frequency of persistent joint-associated CHIKV, even years after recovery from the initial febrile phase (Hoarau et al. 2010). In addition, it has been demonstrated that osteoblasts could be infected by CHIKV and drive osteoclastogenesis in vitro (Noret et al. 2012). This was confirmed by patient cohort studies where high levels of RANKL/osteoprotegerin (OPG) detected in CHIKV patients could be associated to macrophage-derived osteoclasts (Her et al. 2012, Chen et al. 2014a, b). Osteoclasts are known to cause bone erosion, indicating the importance of these cells in bone destruction in alphavirus-induced pathology (Noret et al. 2012, Phuklia et al. 2013, Chen et al. 2014a,b).

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IL-1b, IL-6, and RANTES (Regulated on Activation, Normal T Expressed and Secreted) levels have been associated with disease severity, enabling the identification of patients with poor prognosis and monitoring of disease (Ng et al. 2009). Higher concentrations of proinflammatory factors such as IFN-a, IL-6, and induced protein 10 kDa (IP-10) were also found in patients with CHIKV-induced polyarthritis than in patients without, indicating their potential causative role in chronic joint and muscle pains (Ng et al. 2009, Hoarau et al. 2010, Wauquier et al. 2011). Different patient cohorts have reported different patterns of inflammatory immune mediators, suggesting these mediators may differ according the genetic background of the populations. Nonetheless, a better understanding on the balance of pro- and antiinflammatory cytokines in CHIKF disease manifestations could potentially lead to the development of modulators to reduce disease severity and halt disease progression. The production of type I interferons, IFN-a and IFN-b, is a signature of an antiviral response in vertebrate hosts, and they are essential to the functioning of the innate immunity against the replication and spread of virus. Type-I IFNs and IFNstimulated genes (ISGs) act through diverse mechanisms against viral invasions (Akira and Takeda 2004, Stetson and Medzhitov 2006). Although CHIKV was reported to be a potent inducer of type-I IFNs during infection as early as in the 1960s (Gifford and Heller 1963), their roles in CHIKV infections were poorly characterized. Studies in patient cohorts have shed light on the interplay between type-I IFNs, Toll-like receptor 3 (TLR-3), and CHIKV during infections (Ng et al. 2009, Hoarau et al. 2010, Schilte et al. 2010, Chow et al. 2011, Her et al. 2014). On the other hand, experimental animal models have proven the critical role of type-1 IFNs sensing by nonhematopoietic cells (Schilte et al. 2010) in controlling infection and the role of retinoic acid–inducible gene I-like receptors (RIG-I like receptors), Toll-like receptors, interferon regulatory factor (IRF) 3/7, and IFN-stimulated genes (ISG15, Viperin, OAS) in sensing CHIKV replication (Couderc et al. 2008, Bre´hin et al. 2009, Rudd et al. 2012, Schilte et al. 2012, Teng et al. 2012). A recent study has shown the impact of TLR3 polymorphisms on CHIKV-induced disease outcome (Her et al. 2014). It will be important and insightful to assess the association between polymorphisms of other key host factors and disease-induced arthritis intensity in patient cohorts in the future. Adaptive immunity and protection

CHIKF leads to a protective adaptive immunity. It has been proposed that the establishment of an anti-CHIKV immune response after a primary infection could confer complete protection against reinfection. This provided the basis of the time lapse between CHIKF epidemics (Laras et al. 2005). Anti-CHIKV immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies have been detected in the sera of infected patients during the acute phase of the infection (Panning et al. 2008, Kam et al. 2012a, b). The ability of antiCHIKV antibodies to neutralize virus infectivity has also been demonstrated by using sera from convalescent patients (Couderc et al. 2009, Kam et al. 2012a, b, c). These findings suggest that anti-CHIKV antibodies could be used as a potential prophylactic strategy against CHIKF (Bre´hin et al. 2008, Couderc et al. 2009, Lee et al. 2011, Kam et al. 2012b,

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c, Pal et al. 2013). Therefore, viremic mothers and neonates born from viremic mothers, patients with severe neurological presentation of the disease, small infants, or adults with severe underlying co-morbidities could benefit from passive immunization using anti-CHIKV immunoglobulins. Antibody-mediated protection against CHIKV has been studied extensively for vaccine development (for review, see Ahola et al. 2015), and surface viral glycoproteins have been demonstrated to be key targets for protective neutralizing antibodies against CHIKV (Bre´hin et al. 2008, Lee et al. 2011, Kam et al. 2012b, c, Pal et al. 2013). Furthermore, it was shown that immunization with CHIKV virus-like particle (VLP) vaccines and other vaccine candidates comprising key surface viral glycoproteins could induce the production of neutralizing antibodies and protect both mice and nonhuman primates against CHIKV challenge (Akahata et al. 2010, Kam et al. 2012b, Metz et al. 2013a, b, Garcı´a-Arriaza et al. 2014, Hallengard et al. 2014, Roy et al. 2014, van den Doel et al. 2014). More recently, the first CHIKV VLP vaccine (Akahata et al. 2010) was successfully demonstrated to be well tolerated and protective in human trials, making it a significant breakthrough (Chang et al. 2014). T cells are important effector cells during viral infection. Both CD4 + and CD8 + T cells can eliminate virus-infected cells. Mouse models have demonstrated that these cells are induced during experimental CHIKV infection and have a role in pathology (Teo et al. 2013). More recently, the role of these cells has also been assessed in natural CHIKV infection in humans where natural killer (NK) cells from CHIKF patients were strongly activated within the first days after infection and led to a more sustainable CD4/CD8 response against several viral proteins (Hoarau et al. 2013, Petitdemange et al. 2011, Wauquier et al. 2011). Animal Models

Several animal models have been developed to study CHIKV-associated pathologies. The mouse was the first animal model used for these studies and has been used to investigate CHIKV tissue and cell tropisms, as well as host responses to CHIKV. Mouse models to study CHIKV pathophysiology

Adult wild-type mice develop local symptoms after CHIKV inoculation via the subcutaneous route in the ventral side of each hind foot, toward the ankle (Gardner et al. 2010). Newborn and young mice have been demonstrated to be susceptible to CHIKV infection through the intradermal route in the thorax, or through the subcutaneous route either in the loose skin of the back or in the footpad (Couderc et al. 2008, Ziegler et al. 2008, Morrison et al. 2011). In contrast, adult mice with a partial or complete defect in the type-I IFN pathway developed a mild or severe infection, respectively (Couderc et al. 2008). Other studies have also demonstrated that IRF3/IRF7-deficient mice developed hemorrhagic fever and shock after CHIKV infection (Rudd et al. 2012, Schilte et al. 2012). Therefore, young age and inefficient type-I IFN signaling are risk factors for severe CHIKV disease. Studies on these mouse models have focused mainly on acute pathologies induced by CHIKV and disease severity. Regardless of age and inoculation route, susceptible mice develop viremia detectable in the peripheral blood between

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days 1 and 5 and increased up to 105–106 plaque-forming units (pfu)/mL day 2 postinfection. Skeletal muscles exhibit severe necrotic myositis and a higher viral load. Pathological changes are also observed in joint-associated connective tissues adjacent to affected muscles. In the case of footpad injection, pathological changes are restricted to muscles of the infected foot, leg swelling, edema with evidence of arthritis, and tenosynovitis (Gardner et al. 2010, Morrison et al. 2011, Teo et al. 2013, Hallenga¨rd et al. 2014). Although CHIKV RNA is cleared from most tissues in the days following infection, viral RNA could persist in joint-associated tissues for at least 16 weeks, for example, when associated with histopathological evidence of joint inflammation (Hawman et al. 2013, Poo et al. 2014b). In the case of severe disease, viremia is high and CHIKV also disseminated to other tissues, including the skin and eye. In all of these tissues, CHIKV-antigen labeled cells were found to be fibroblasts (Couderc et al. 2008, Ziegler et al. 2008). These findings are relevant to human disease, as similar tissue and cell tropisms have been observed in biopsy samples of CHIKV-infected human patients (Couderc et al. 2008, Couderc et al. 2012). Together, these data demonstrate that infection of peripheral tissues associated with human CHIKV disease, joints, muscle, and skin, is restricted mainly to conjunctive tissues and that the fibroblast is a predominant target cell of CHIKV during acute CHIKV infection. Mice with severe disease develop a central nervous system (CNS) infection, and viral load in the CNS is inversely related with age (Couderc et al. 2008). CHIKV antigens have been detected in choroid plexus, ependymal, and meningeal cells, and are not found in the brain microvessels and parenchyma (Couderc et al. 2008, Ziegler et al. 2008). These data suggest that CHIKV infects and crosses the blood–brain barrier at the choroid plexus and leptomeningeal levels and then infects the ependyma, whereas it does not directly infect the brain microvessels and parenchyma. In agreement with these findings, CHIKV and anti-CHIKV IgM have been detected in the cerebrospinal fluid of human neonates and adult patients with encephalopathy (Grivard et al. 2007). This, with the absence of detectable tissue alteration at the brain parenchyma level, fits the natural history of CHIKV-infected patients whereby CNS symptoms are most often presented as encephalopathy. Maternal–fetal transmission of CHIKV has also been investigated in pregnant type-I IFN-deficient mice. Data have revealed that the placenta does not constitute a target for CHIKV. Indeed, no infected cells have been detected when observing placental tissue sections from infected mice. This is in line with investigations carried out in human placentas obtained from viremic mothers, where no infected cells could be detected by immunohistochemistry (Ge´rardin et al. 2008). This suggests that mother-to-child transmission, which is only observed in neonates born to viremic mothers, most likely occurs via placental breaches during birth, rather than an actual placental infection (Couderc et al. 2008, Ge´rardin et al. 2008). In contrast to adult type-I IFN-deficient mice, adult wildtype mice infected via the intradermal route were able to control CHIKV infection, because no infectious virus could be recovered from the mouse tissues or organs (Couderc et al. 2008). The study of early stage infection in adult wild-type mice has shown that CHIKV infects dermal fibroblasts at the site of injection and that infection could be rapidly controlled

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by IFN-b secreted locally (Schilte et al. 2010). This early control of infection by type-I IFN at the site of injection in the skin explains the absence of susceptibility to CHIKV infection of wild-type mice upon intradermal infection. The role of type-I IFN in CHIKV pathogenesis has been investigated further in mouse models and in human cells. Data showed that infected nonhematopoietic cells sense viral RNA in a mitochondrial antiviral-signaling (MAVS)dependent manner (MAVS is also known as Cardif or IPS1) and participate in the control of infection through their production of type-I IFN. Although the MAVS pathway contributes to the immune response both in cell culture of human fibroblasts and in mice, evidence for a MyD88dependent sensor in preventing viral dissemination has been revealed in mice. Importantly, it has been shown that IFN-a/b receptor (IFNAR) expression is required in nonhematopoietic cells but not in hematopoietic cells, as IFNAR - / - /wildtype (WT) bone marrow chimeras are able to clear the infection, whereas WT/IFNAR - / - chimeras succumb to disease. These data define an essential role for type-I IFN acting directly on nonhematopoietic cells, most likely fibroblasts, for the control of CHIKV (Schilte et al. 2010), although treatment with type-I IFN is not efficacious when given after virus infection (Gardner et al. 2010). Autophagy is another host factor possibly involved in the species specificity of CHIKV. It has been shown that the human autophagy receptor NDP52 interacts with the nonstructural protein nsP2, thereby promoting viral replication in human cell cultures, whereas the NDP52 mouse ortholog is unable to bind to nsP2 and to promote CHIKV infection in mouse cell cultures ( Judith et al. 2013). Thus, the absence of the proviral effect of NDP52 in the mouse may contribute to the lower permissiveness of mice to CHIKV relative to humans. Whereas it is clear that an increased neonatal susceptibility is also observed in humans, the relevance of a type-I IFN defect and autophagy receptor NDP52 as a basis for severe infection in humans remains to be demonstrated. The roles of T cells were also explored in adult RAG2 - / - , CD4 - / - , CD8 - / - , and wild-type C57BL/6 CHIKV-infected mice. Interestingly, results indicated that CHIKV-specific CD4 + but not CD8 + T cells are essential for the development of joint swelling without effect on virus replication and dissemination (Hawman et al. 2013, Teo et al. 2013). These observations indicate that mechanisms of joint pathology induced by CHIKV in mice resembles that in humans and may differ from infections caused by other arthritogenic alphaviruses, such as Ross River virus (Morrison et al. 2006). Furthermore, using mice deficient for major histocompativbility complex II (MHC II) and IFN-c, gene set enrichment analysis showed a significant overlap in differentially expressed genes from CHIKV arthritis and rheumatoid arthritis (Nakaya et al. 2012). The importance of B cells was also explored in B cell deficient (lMT) knockout mice infected with CHIKV in the footpad, and viremia in these animals persisted for over a year, indicating a direct role for B cells in mediating CHIKV clearance (Lum et al. 2013, Poo et al. 2014b, Poo 2014). These animals also exhibited a more severe disease than wild-type mice during the acute phase. The role of the monocyte-macrophage infiltration into the joint, bone erosion, and ankle swelling in CHIKV-infected animals by footpad inoculation was demonstrated by the

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Bindarit treatment. An inhibitor of monocyte chemoattractant protein-1 (MCP-1) chemokine (CCL2) production, Bindarit was shown to block monocytes recruitment and reduced joint inflammation (Rulli et al. 2011, Chen et al. 2014). However, CHIKV-infection in CCR2 - / - -deficient mice with a defective CCL2 receptor resulted in more severe disease due to an excessive recruitment of neutrophils to the inflamed joint (Poo et al. 2014a). In short, these data highlight the power of inbred mutant mice to identify key elements in the physiopathology of CHIKV infection. However, the relevance of these findings has not been assessed in human or in nonhuman primates. Nonhuman primates as a CHIKV infection model

Roques and collaborators have infected the cynomolgus macaque (Macaca fascicularis) using the La Reunion CHIKV isolates (Labadie et al. 2010). Performing in vivo titrations, they demonstrated that virus inoculation of as little as 10 pfu via the intravenous route could produce infection in macaques, with viremic levels of up to 108 pfu/mL. Although CHIKV could be detected in the cerebrospinal fluid at day 4 postinfection from all virus-infected macaques, clinical neurological disease was only detected in macaques inoculated with the highest infectious doses. Interestingly, acute infection was rapidly controlled given that the viral titer in peripheral blood was below detection levels by day 10 postinfection, similar to reports described in patients or in mice (Ziegler et al. 2008). Virus replication profiles were also recorded in rhesus macaques (Akahata et al. 2010, Chen et al. 2010, Roy et al. 2014). Similar to patients, early leukopenia was observed (Borgherini et al. 2007, Borgherini et al. 2009, Akahata et al. 2010, Labadie et al. 2010) together with markers of IFN-a/b antiviral response, inflammation, and cell immune activation (Higgs and Ziegler 2010, Labadie et al. 2010, Messaoudi et al. 2013). Whereas virus has been found in cerebrospinal fluid samples during the acute phase of infection, no virus and no neuronal tissues destruction were detected. Thus, neurological disease could be associated with nonspecific inflammation, and high levels of cytokines associated with encephalopathy (shivering and loss of central temperature control) in animals infected with high virus titer (Ge´rardin 2010, Ge´rardin et al. 2008, Labadie et al. 2010). However, even in animals without clear encephalopathy, a loss in temperature control could be observed after the onset of the disease when temperature significantly dropped below the normal level (Labadie et al. 2010, Roy et al. 2014). Experimental infection of newborn macaques remains to be explored, and these studies will be able to assess the capacity of CHIKV to infect and replicate within fetal brain tissues. Our studies in macaques demonstrated that CHIKV persists in target tissues after virus clearance from the blood, as demonstrated by immunohistochemistry and by viral RNA detection using PCR and in situ hybridization assays (Labadie et al. 2010). CHIKV antigens were detectable between 7 and 9 days postinfection in all organs, including joints, secondary lymphoid organs, and, to a lesser extent, muscles up to 3 months postinfection. CHIKV was also shown to replicate in several cell types during the acute phase (Higgs and Ziegler 2010, Labadie et al. 2010), but thereafter were detected mainly in macrophages by immunohistochemistry. CHIKV-infected monocytes and macrophages could be de-

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tected in the blood 6 h after infection (Roques et al. 2011), and in most tissues by the following day, as shown by in situ hybridization, immunohistochemistry, RT-PCR, and virus isolation. Significant macrophage infiltration was also detected by histology throughout the study and long after virus clearance in blood (Labadie et al. 2010). Similarly, CHIKV was demonstrated to infect primary macrophages in vitro (Rinaldo et al. 1975), resulting in the production of viruses from 103 to 106 pfu/mL (Sourisseau et al. 2007, Gardner et al. 2010, Hoarau et al. 2010, Labadie et al. 2010, KrejbichTrotot et al. 2011a, b). Although viruses could be isolated up at 44 days postinfection by co-culture or by crushed tissues from infected animals (Labadie et al. 2010), another study detected viral RNA by RT-PCR (Chen et al. 2010). To date, no animal model fully reproduces the chronic rheumatoid syndrome following CHIKF. Indeed, the disease pathology reported in mice is mainly driven by destruction of tissues with huge cell infiltration that could only be resolved 1–2 weeks after acute disease (Gardner et al. 2010, Morrison et al. 2011, Rulli et al. 2011, Her et al. 2014). Despite virus persistence, severe joint damage is not always observed in macaques (Labadie et al. 2010, Chen et al. 2010). Nevertheless, both animal models present inflammation, macrophage tissue tropism, and virus persistence in tissues (Labadie et al. 2010, Hawman et al. 2013). However, the exact mechanism in the establishment of chronic disease induced by CHIKV infection remains undefined. Conclusions

To date, most of the studies performed in patient cohorts remained observational and limited to peripheral biomarkers and X-ray imaging. Importantly, the number of patients with neurological symptoms during the acute phase is low, and therefore the mechanisms remain to be studied. Although soluble biomarkers have been shown to associate with CHIKF disease severity, their precise relationship remains unclear. Animal models have shown that the immune response from various cell types could be deleterious or beneficial depending on the route of virus inoculation, indicating that the immune response in peripheral blood is different from the site of local inflammation. In addition, chronic arthralgia observed in patients after virus clearance remains a mystery, and sampling of tissues within the joint and muscles will be an ethical challenge. Hence, there is a need to strengthen the current state of the art both in the mouse and nonhuman primate models to understand the development of chronic CHIKF further. Future human cohort studies should include disease assessment in exposed babies during acute infection and also the genetic polymorphisms associated with chronic disease. Along the same lines, it will be crucial to assess the contribution of co-morbidities in disease severity and duration. The detailed understanding of CHIKV interplay with the host in acute and chronic diseases may help in the development of therapeutic treatments for CHIKVassociated diseases. Acknowledgments

This work was supported by the European Union FP7 project ‘‘Integrated Chikungunya Research’’ (ICRES), grant agreement #261202.

246 Author Disclosure Statement

No conflicting financial interests exist. References

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Address correspondence to: Pierre Roques Immuno-Virology Department Institute of Emerging Disease and Innovative Therapy Commissariat a` l’Energie Atomique 18 route du Panorama Fontenay aux Roses, 92265 France E-mail: [email protected] and Lisa F.P. Ng Singapore Immunology Network (SIgN) 8A Biomedical Grove, Biopolis #04-06, Immunos Singapore 138648 E-mail: [email protected]