Chicungunya

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Contents lists available at ScienceDirect

Antiviral Research journal homepage: www.elsevier.com/locate/antiviral

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Review

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Cellular and molecular mechanisms of chikungunya pathogenesis

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Fok-Moon Lum a,b, Lisa F.P. Ng a,b,c,⇑ a

Singapore Immunology Network, Agency for Science, Technology and Research (A⁄STAR), Singapore Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore c Institute of Infection and Global Health, University of Liverpool, United Kingdom b

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 6 May 2015 Revised 27 May 2015 Accepted 16 June 2015 Available online xxxx

Chikungunya virus (CHIKV) is an arthropod-borne virus that causes chikungunya fever, a disease characterized by the onset of fever and rashes, with arthralgia as its hallmark symptom. CHIKV has re-emerged over the past decade, causing numerous outbreaks around the world. Since late 2013, CHIKV has reached the shores of the Americas, causing more than a million cases of infection. Despite concentrated efforts to understand the pathogenesis of the disease, further outbreaks remain a threat. This review highlights important findings regarding CHIKV-associated immunopathogenesis and offers important insights into future directions. This article forms part of a symposium in Antiviral Research on ‘‘Chikungunya discovers the New World.’’ Ó 2015 Published by Elsevier B.V.

Keywords: Chikungunya virus Immune response Immunopathogenesis Pathology

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Introduction . . . . . . . . . . . . .

The virus . . . . . . . . . . . . . . . .

Replication cycle . . . . . . . . .

Route of infection. . . . . . . . .

Target cells . . . . . . . . . . . . . .

Apoptosis and autophagy . .

Importance of Type I IFN . . .

Cytokines and chemokines .

IL-6 and osteoclastogenesis .

Monocytes/macrophages . . .

NK cells . . . . . . . . . . . . . . . . .

Dendritic cells. . . . . . . . . . . .

T cells . . . . . . . . . . . . . . . . . .

B cells . . . . . . . . . . . . . . . . . .

Future perspectives . . . . . . .

Uncited references . . . . . . . .

Acknowledgements . . . . . . .

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1. Introduction

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Chikungunya virus (CHIKV) is an alphavirus belonging to the Togaviridae family that was first isolated from a human patient in

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⇑ Corresponding author at: Singapore Immunology Network, Agency for Science, Technology and Research (A⁄STAR), Singapore.

E-mail address: lisa_ng@immunol.a-star.edu.sg (L.F.P. Ng).

Tanzania in 1952. It is transmitted mainly by the Aedes agypti and Aedes albopictus mosquitoes. Infection causes a self-limiting febrile illness known as chikungunya fever (CHIKF) with symptoms such as myalgia, fever and rashes. Patients also typically exhibit polyarthralgia, which is a hallmark of the disease. Symptoms usually appear after an incubation period of 4–7 days. While many of the symptoms disappear within the following week, arthralgia may persist in some patients for up to a few years (Her et al., 2009; Kam

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Please cite this article in press as: Lum, F.-M., Ng, L.F.P. Cellular and molecular mechanisms of chikungunya pathogenesis. Antiviral Res. (2015), http:// dx.doi.org/10.1016/j.antiviral.2015.06.009

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et al., 2009). In some cases, CHIKF has been associated with neurological complications such as myeloradiculopathy and meningoencephalitis (Borgherini et al., 2007; Chandak et al., 2009). However, direct neurovirulence and neuroinvasiveness remain to be investigated.

In this article, we discuss interactions between CHIKV and the host immune response, with a focus on the balance between protection and pathology in defining CHIKV pathogenesis. Our paper forms part of a symposium in Antiviral Research on ‘‘Chikungunya discovers the New World.’’ Readers interested in a general review of the disease should refer to the article ‘‘Chikungunya: Evolutionary history and recent epidemic spread’’ (Weaver and Forrester, 2015) published in this symposium.

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2. The virus

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CHIKV is an enveloped virus that is approximately 70 nm in diameter in neutral pH and contains a 11.8 kb single-stranded, positive-sense RNA genome (Strauss and Strauss, 1994; Leung et al., 2011; Rashad et al., 2014). The genome consists of a 50 methylated terminal cap untranslated region (UTR), followed by RNA coding for 4 non-structural proteins (nsP1–4) and 5 structural

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proteins (C–E3–E2–6K–E1), and a 30 terminal poly-A tail (Strauss and Strauss, 1994; Leung et al., 2011; Teng et al., 2011). The non-structural proteins and structural proteins, governed by 2 separate open-reading frames (ORFs), contribute to the propagation of new virions (Schwartz and Albert, 2010; Leung et al., 2011; Rashad et al., 2014).

The non-structural genes encode for nsP1 and nsP3, helicase (nsP2) and polymerase (nsP4). These proteins associate to form the viral replication complex needed for downstream viral genome replication (Solignat et al., 2009; Schwartz and Albert, 2010). On the other hand, the structural capsid protein is involved in the formation of the icosahedral fenestrated nucleocapsid of a mature virion, in which the viral RNA genome will be contained (Solignat et al., 2009; Schwartz and Albert, 2010; Rashad et al., 2014). The E1 and E2 glycoproteins associate as a heterodimer before being incorporated onto the surface of the mature virion as trimeric spikes and are involved in the attachment and entry of the virion into susceptible target cells during subsequent infection. A total of 80 trimeric spikes are present on the surface of a mature virion (Voss et al., 2010).

The role of the 6K protein remains ambiguous, but it is thought to be involved in virus assembly and budding (Leung et al., 2011;

Fig. 1. CHIKV replication cycle. The virus enters susceptible cells through endocytosis, mediated by an unknown receptor. As the endosome is acidic, conformational changes occur resulting in the fusion of the viral and host cell membranes, causing the release of the nucleocapsid into the cytoplasm, The RNA genome is first translated into the 4 nsPs, which together will form the replication complex and assist in several downstream processes (depicted by dashed arrowed line). Subsequently, the genome is replicated to its negative-sense strand, which in turn will be used as a template for the synthesis of the 49S viral RNA and 26S subgenomic mRNA. The 26S subgenomic mRNA will be translated to give the structural proteins (C–pE2–6K–E1). After a round of processing by serine proteases, the capsid is released into the cytoplasm. The remaining structural proteins are further modified post-translationally in the endoplasmic reticulum and subsequently in the Golgi apparatus. E1 and E2 associate as a dimer and are transported to the host plasma membrane, where they will ultimately be incorporated onto the virion surface as trimeric spikes. Capsid protein will form the icosahedral nucleocapsid that will contain the replicated 49S genomic RNA before being assembled into a mature virion ready for budding. During budding the virions will acquire a membrane bilayer from part of the host cell membrane.

Please cite this article in press as: Lum, F.-M., Ng, L.F.P. Cellular and molecular mechanisms of chikungunya pathogenesis. Antiviral Res. (2015), http:// dx.doi.org/10.1016/j.antiviral.2015.06.009

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Teng et al., 2011). In addition, it has also been reported that alphavirus 6K protein is part of a cation-selective ion channel (Melton et al., 2002), which has the potential to affect membrane permeability. Finally, E3 protein is important in directing the structural proteins to the endoplasmic reticulum (ER) for proper assembly and spike formation via its cognate interactions with E2 glycoprotein (Snyder and Mukhopadhyay, 2012). It has also been reported that alphavirus E3 protein could play a role in pH protection during virus formation and budding (Uchime et al., 2013).

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3. Replication cycle

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Upon entry into target cells (Fig. 1), conformational changes in the virus envelope take place, due to the acidic endosomal environment. With these conformational changes, the E1 glycoprotein will be exposed and this mediates fusion of the CHIKV membrane with the host endosomal membrane, with the nucleocapsid being released into the cytoplasm in the process. The free viral RNA then gets translated into a polyprotein that gets cleaved into the nsPs 1– 4. These nsPs associate to form a functional viral replication complex, which will then go on to generate the full-length negative sense RNA intermediate. This RNA intermediate will be used as a template for downstream synthesis of the 49S genomic RNA as well as the 26S subgenomic mRNA.

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The 26S subgenomic mRNA is translated to give the Capsid–p E2–6K–E1 polyprotein that is further processed by serine proteases to release the capsid into the cytoplasm. The remaining proteins are then directed to the ER in which they will undergo further post-translational modifications. pE2 will then be cleaved in the Golgi to give E2 and E3 glycoproteins. E1 and E2 glycoproteins will heterodimerize and get transported to the host cell plasma membrane where they would be incorporated onto the virion surface as trimeric spikes. Capsid protein associates in the cytoplasm, forming the icosahedral nucleocapsid that contains the 49S genomic RNA. Finally, mature virions are assembled at the plasma membrane, and bud out of the infected host cell, during which the mature virions will acquire a membrane bilayer from the host cell plasma membrane (Strauss and Strauss, 1994; Leung et al., 2011; Rashad et al., 2014). Typically, CHIKV replication occurs between 8 to 16 h post-infection (Teng et al., 2011), while in vertebrate cells, virions are typically produced less than 8 h after infection at 37 °C (Sourisseau et al., 2007).

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4. Route of infection

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CHIKV infects the host through inoculation at the skin via bites from infected mosquitoes. It can infect skin resident cells such as

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Fig. 2. Pathogenesis of CHIKV infection: viral dissemination and impact on host immune response. (1) CHIKV infection occurs through the bite of an infected mosquito, resulting in a (2) dermal infection phase. Skin resident cells become infected, allowing for the initial rounds of replication. (3) Direct inoculation of virus into the circulation can also occur via mosquito bites. (4) Subsequently, the virus disseminates to draining lymph nodes, where it further replicates before being (5) released into the circulation.

(6) It then disseminate to peripheral organs, such as the spleen and muscles, where further replication takes place. The figure is adapted from (Ong et al., 2014) with permission.

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the skin fibroblasts and dermal macrophages (Sourisseau et al., 2007; Couderc et al., 2008; Kam et al., 2009). This will lead to the first round of virus replication and trigger an initial host immune response to contain the virus at the skin (Kupper and Fuhlbrigge, 2004; Schilte et al., 2010). Despite the immune response, the virus rapidly disseminates into the lymph node before further dissemination to other tissues (e.g., muscles) via the circulatory system (Kam et al., 2009). Virus replication occurs at these peripheral tissues, resulting in the viremic phase of the disease (Fig. 2). During this phase, virus can be easily transmitted to mosquitoes via a blood meal. Viremia is normally cleared within 2–10 days postinfection (Panning et al., 2008; Kam et al., 2009).

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5. Target cells

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The identification of susceptible cell types is important for understanding the pathogenesis of CHIKV infection. Studies of cellular tropism have reported that the virus can infect and replicate in a variety of cell lines and adherent cells, such as endothelial, epithelial and fibroblastic primary cells (Sourisseau et al., 2007; Salvador et al., 2009; Wikan et al., 2012). It was also demonstrated that CHIKV was capable of infecting human muscle satellite cells, but not in the differentiated myotubes (Ozden et al., 2007). On the other hand, various groups have reported contrasting results regarding the infectability of peripheral blood mononuclear cells (PBMCs) (Sourisseau et al., 2007; Her et al., 2010; Teng et al., 2012).

Animal models have been developed to aid in the investigation of CHIKV infection. Adult C57BL/6 wildtype animals inoculated at the ventral side of the footpad recapitulated the rheumatic manifestation commonly observed in infected humans (Gardner et al., 2010; Lum et al., 2013; Teo et al., 2013). In these animals, virus was also identified in the spleen, lymph nodes, liver and muscles, complementing observations obtained in in vitro studies. The profile of systemic viremia and blood count reflected that of an infected patient (Gardner et al., 2010; Lum et al., 2013; Teo et al., 2013).

Studies in non-human primates have also reported the presence of fever, rash and pronounced joint pain during the acute phase of the disease, with the peak of viremia at 1–2 days after infection (Chen et al., 2010; Labadie et al., 2010; Messaoudi et al., 2013).

These symptoms were accompanied by transient acute lymphopenia and neutropenia. Virus was detected in numerous organs and tissues such as the skin, brain, liver, muscle, lymph nodes, spleen and joint-connective tissues. Persistent infection was also detected in the joints, muscles, and splenic macrophages, as well as in endothelial cells lining the liver sinusoids (Chen et al., 2010; Labadie et al., 2010; Messaoudi et al., 2013). However, it remains to be further investigated if this observation is a true reflection of an infected patient, although there has been a report of CHIKV persistence in the perivascular synovial macrophages of a chronically infected patient some 18 months after illness onset (Hoarau et al., 2010).

The effect of age on CHIKV persistence should also be investigated as it has been shown that the virus persists in elderly rhesus macaques due to a defective antiviral immune response (Messaoudi et al., 2013). Moreover, given the increased episodes of neurological complications and the presence of viral antigen in the cerebrospinal fluid of patients (Chandak et al., 2009; Economopoulou et al., 2009; Kashyap et al., 2010), further development of animal models would serve as valuable research tools to investigate neurotropism and neuroinvasiveness. In fact, it has been shown using neonatal mice and mice with an abrogated Type I Interferon (IFN) signaling pathway, that the virus can disseminate into the central nervous system, targeting the choroid plexuses and leptomeninges (Couderc et al., 2008).

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6. Apoptosis and autophagy

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Apoptosis has been demonstrated to increase the spread of virus from apoptotic infected cells to neighboring uninfected cells (Krejbich-Trotot et al., 2011a). The virus has also been shown to hide in apoptotic blebs as an evasion strategy, escaping cellular host responses such as extracellular antibodies (Long and Heise, 2015).

More recently, autophagy (Glick et al., 2010) was reported to be induced during active infection, further increasing viral replication (Krejbich-Trotot et al., 2011b). However, while autophagy was shown to increase CHIKV replication in a human cell line (Krejbich-Trotot et al., 2011b), a decrease in replication was observed in mouse embryonic fibroblasts (Joubert et al., 2012).

This species-specific difference was later demonstrated to be due to interaction of the human autophagy receptor NDP52 with the viral nsP2 to promote viral replication, which was not observed with the mouse orthologue of NDP52 (Judith et al., 2013). The cross-talk between autophagy and apoptosis to mediate CHIKV protection and pathogenesis remains to be further established.

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7. Importance of Type I IFN

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The first line of host defense against pathogen infection is the innate immune response. Given that the adaptive immune response requires over a week to develop, the innate immune response would thus be crucial in defending against CHIKV infection. The type I IFN pathway is a vital innate anti-viral pathway, and it has been well-reported that CHIKV infection elicits the type I IFN response alongside the production of other pro-inflammatory cytokines (Gardner et al., 2010; Labadie et al., 2010; Chow et al., 2011; Wauquier et al., 2011; Teng et al., 2012). Particularly, IFNa and IFNb have been reported to play crucial anti-viral roles in both CHIKV infected humans and mice, as well as in a series of cell lines derived from both species (Sourisseau et al., 2007; Couderc et al., 2008; Gardner et al., 2010; Schilte et al., 2012; Teng et al., 2012).

The importance of Type I IFN response was further demonstrated where CHIKV infection was lethal in mice deficient in Type I IFN receptors (Couderc et al., 2008). Further experiments in chimeric mice demonstrated that Type I IFN produced by non-hematopoietic cells is vital in the effective clearance of CHIKV (Schilte et al., 2010). It has also been revealed that the nsP2 protein of CHIKV has the ability to shut off Type I IFN signaling leading to a decreased expression of anti-viral mediators (Fros et al., 2010).

Induction of Type I IFN is triggered through pattern recognition receptors (PRRs) following the recognition of structurally conserved pathogen-associated molecular patterns (PAMPs) (Medzhitov and Janeway, 2000; Janeway and Medzhitov, 2002).

Cellular PRRs such as the Toll-like receptors (TLRs) and cytosolic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), such as the melanoma differentiation-associated protein 5 (MDA5) and RIG-I have been reported to be play crucial roles in mediating Type I IFN production during CHIKV infection (Stetson and Medzhitov, 2006; Schilte et al., 2010; Teng et al., 2011; Her et al., 2015).

Upon infection and replication, double-stranded RNA (dsRNA) intermediates picked up by both MDA5 and RIG-I will kick-start a downstream signaling cascade which further involves the CARD adaptor inducing IFNb adaptor protein (CARDIF) and crucial interferon-regulatory factors (IRF) 3 and 7 (Kawai et al., 2005; Honda et al., 2006; Stetson and Medzhitov, 2006). The activation of this pathway ultimately leads to the production of Type I IFNs, which act either as a paracrine or autocrine signal to further amplify the anti-viral signaling cascade mediated through Janus

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Please cite this article in press as: Lum, F.-M., Ng, L.F.P. Cellular and molecular mechanisms of chikungunya pathogenesis. Antiviral Res. (2015), http:// dx.doi.org/10.1016/j.antiviral.2015.06.009

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kinases (JAK) and signal transducers and activators of transcription (STAT) (Decker et al., 2002; Stetson and Medzhitov, 2006). This is followed by the production of interferon-stimulated genes (ISG), such as Viperin, GADD44, Tetherin (BST-2), ISG15, Mx proteins, protein kinase R (PKR) and 20 ,50 -oligoadenylate synthase 3 (OAS3), which have reported anti-CHIKV properties (Bréhin et al., 2009; Schoggins and Rice, 2011; Schoggins et al., 2011; Werneke et al., 2011; White et al., 2011; Clavarino et al., 2012; Teng et al., 2012; Jones et al., 2013).

Anti-viral ISGs can also be induced through TLR signaling (Kawai and Akira, 2010). The importance of TLR3 in hematopoietic cells in controlling CHIKV infection has recently been revealed (Her et al., 2015). Given that TLR3 recognizes dsRNA, it was shown in the absence of TLR3 that CHIKV-infected mice suffered a more pronounced disease accompanied with higher viremia. This complemented an earlier report that the loss of TRIF (Toll/IL-1 resistance domain-containing adaptor inducing IFNb), an essential adaptor molecular for TLR3 signaling (Kawai and Akira, 2010), induced a more severe disease outcome in mice (Rudd et al., 2012). In PBMCs obtained from clinically CHIKV-infected patients, it was reported that high levels of IFNa, IFNb and TLR3 transcripts were detected when compared to non-infected controls (Teng et al., 2012; Her et al., 2015). These observations further substantiate the importance of these pathways in controlling CHIKV progression, which is likely to be controlled through the combined effects of two or more sensor pathways, deriving from both hematopoietic and non-hematopoietic cells (Schilte et al., 2010; Her et al., 2015).

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8. Cytokines and chemokines

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Extensive studies have been performed to characterize the immune response of CHIKV-infected patient cohorts. In these studies, pro-inflammatory cytokines such as IL-6, monocyte chemotactic protein-1 (MCP-1) and IFNc were found to be elevated during the acute phase of the disease in several patient cohorts (Ng et al., 2009; Hoarau et al., 2010; Chow et al., 2011; Kelvin et al., 2011; Wauquier et al., 2011). Positive correlation was also observed between the expression of IL-6 or MCP-1 and the high viral load in CHIKV-infected patients (Chow et al., 2011).

Interestingly, IL-6 and GM-CSF were also observed to associate with persistent arthralgia (Hoarau et al., 2010; Chow et al., 2011).

More recently, a meta-analysis comparative study demonstrated that pro-inflammatory cytokines such as IFNa, IFNc, IL-2, IL-2R, IL-6, IL-7, IL-12, IL-15, IL-17 and IL-18; anti-inflammatory cytokines such as IL-1Ra, IL-4 and IL-10; chemokines: granulocyte colony-stimulating factor (GM-CSF), IP-10, MCP-1, monokine induced by gamma interferon (MIG), macrophage inflammatory protein (MIP) 1a and MIP-1b; and growth factor: basic fibroblast growth factor (FGF) formed a generic acute CHIKV signature in all the different patient cohorts around the world (Teng et al., 2015).

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9. IL-6 and osteoclastogenesis

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The exact role of IL-6 in CHIKV infection has yet to be fully elucidated. However, it has been shown using primary osteoblasts prepared from the knee-joint of a healthy male subject that these cells could be infected by the virus, and upon infection produced high levels of IL-6 and RANKL that were maintained up to 40 days post-infection (Noret et al., 2012). This was further supported by the detection of IL-6 in the infected joint of a chronically ill patient, further indicating that IL-6 could drive persistent arthralgia (Hoarau et al., 2010). Given that the IL-6 receptor is expressed on osteoblasts, IL-6 may act in an autocrine manner to induce more of its own production in a positive feedback manner (Li et al.,

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2008). It was also shown that there was a repression of osteoprotegerin (OPG) production in infected osteoblasts (Noret et al., 2012). The high level of RANKL coupled with a decreased presence of OPG activates osteoclasts and drives osteoclastogenesis (Hofbauer et al., 1999), a mechanism that has been proposed to be responsible for CHIKV-induced rheumatic symptoms (Noret et al., 2012).

Using a closely related alphavirus, the Ross River Virus (RRV), it was demonstrated that RRV infection in primary human osteoblasts led to the production of high levels of pro-inflammatory cytokines, including IL-6 and MCP-1. This was further coupled with a disrupted RANKL/OPG ratio in the synovium of RRV patients. The importance of IL-6 in driving bone loss was then determined in a RRV mouse model, where neutralization of IL-6 prevented abnormality of the RANKL/OPG ratio (Chen et al., 2014).

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IL-6 is also known to strongly induce the expression of MCP-1 (Romano et al., 1997), a monocyte/macrophage chemo-attractant (Lu et al., 1998). As shown by numerous studies, MCP-1 is strongly associated with the acute phase of CHIKV infection both in infected humans and animals (Chen et al., 2010, 2015; Gardner et al., 2010; Hoarau et al., 2010; Teng et al., 2015). In CHIKV-infected animals, this elevated level of MCP-1 was accompanied by increased infiltration of monocytes into the site of inflammation (Gardner et al., 2010; Labadie et al., 2010; Poo et al., 2014a).

The importance of monocytes/macrophages in driving CHIKV-induced pathology was further illustrated in mice treated with liposomes containing clodronate, which depletes macrophages (Gardner et al., 2010). In these treated mice, CHIKV-associated disease pathology was ameliorated, which suggests the critical role of macrophages in driving CHIKV pathology.

However, it should be noted that the effect of clodronate is not specific to macrophages as it is known to deplete all phagocytic cells including dendritic cells and neutrophils (Zhang et al., 2002).

MCP-2 and MCP-3 were also reported to be present in high amounts in the joints of CHIKV-infected animals (Chen et al., 2015). Treatment with Bindarit, a MCP inhibitor (Bhatia et al., 2005) was demonstrated to completely abolish CHIKV-induced pathology (Rulli et al., 2011; Chen et al., 2015). This is further complemented by studies in the related arthritogenic RRV, where monocytes/macrophages were shown to be the main drivers of pathology (Lidbury et al., 2000, 2008; Herrero et al., 2011, 2013; Taylor et al., 2013). Monocytes/macrophages have also been suggested to be the cellular vehicle for virus dissemination as well as being a cellular reservoir for persistent CHIKV infection (Her et al., 2010; Hoarau et al., 2010; Labadie et al., 2010). CHIKV infection in CCR2 / animals resulted in a more severe, prolonged and erosive arthritis, with no effect on viral replication (Poo et al., 2014a). Loss of CCR2, which is the receptor for MCP-1, caused a drastic change in the profile of infiltrating immune cells, coupled with a dysregulation of both pro- and anti-inflammatory pathways.

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Alongside the increase of monocyte/macrophage infiltration into the inflamed joints of infected mice, the presence of NK cells was also significantly elevated (Gardner et al., 2010). IL-12, which stimulates NK cell activity (Orange and Biron, 1996), was also present in high quantities, suggesting that activated NK cells play significant roles during CHIKV infection (Nakaya et al., 2012; Teo et al., 2015).

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In clinical settings, it was previously reported that activated NK cells expressing CD69 were detected in the synovial fluid obtained from acutely infected CHIKF patients (Hoarau et al., 2010). Given that the functions of NK cells are tightly controlled by the concerted effects of activating and inhibitory receptors, it was revealed using PBMCs obtained from CHIKV-infected patients that a transient expansion of the highly cytotoxic NKG2C+ NK cells may function to decrease their own activity and limit overall immunopathology (Petitdemange et al., 2011). Nevertheless, the specific role of NK cells in CHIKV infection should be further explored with NK cell deficient animals, especially since NK cells adopted a pathogenic role in Venezuelan Equine Encephalitis virus (VEEV)-mediated central nervous system infection (Taylor et al., 2012).

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Although rapid activation of plasmacytoid dendritic cells (pDCs) has been reported in patients during acute infection (Hoarau et al., 2010), little work has been done to understand the role of DCs. pDCs are major producers of Type I IFNs during viral infections (Siegal et al., 1999) that could initiate and activate NK cell mediated cytolysis (Colonna et al., 2004). However, CHIKV infection of pDCs isolated from healthy PMBCs did not secrete detectable amounts of both IFNa and IFNb (Schilte et al., 2010).

More recently, the dendritic cell immunoreceptor (DCIR), a type of C-type leptin receptor (CLR), was demonstrated to play an important role in host protection against CHIKV infection in DCIR / mice (Long et al., 2013). This is in line with earlier studies where DCIR was reported to be one of the few CLRs that contain an intracellular immune receptor tyrosine-based inhibition motif (ITIM) (Meyer-Wentrup et al., 2008). Given that DCIR is expressed across other immune subsets such as monocytes and B cells (Bates et al., 1999), efforts to elucidate its role in these immune cells and in CHIKV immunopathology should be emphasized.

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While innate immunity plays a role in the early phase of CHIKV infection, adaptive immunity comes in later for prolonged virus-specific memory responses (McCance and Huether, 2014).

There are limited studies on the role of T cells in patients, except that CD8+ T cells dominate the early stages of the disease, with CD4+ T cells appearing at a later time to aid in the production of CHIKV-specific humoral response (Wauquier et al., 2011).

However, it has also been reported that CD4+ T cells, but not CD8+ T cells were detected in the synovium of a patient suffering from chronic inflammation (Hoarau et al., 2010).

To further decipher the roles of the adaptive immune response, RAG2 / mice were used (Teo et al., 2013). Interestingly, these immune-compromised animals did not exhibit any CHIKV-associated joint footpad pathology upon infection despite persistent viremia. However, a pathogenic role was elucidated for CD4+ T cells in driving joint inflammation when CD4 / animals were utilized (Teo et al., 2013). Cytokine profiling further indicated the involvement of a Th1-related pathway (Gardner et al., 2010; Nakaya et al., 2012; Teo et al., 2013, 2015).

The pathogenic role of CD4+ T cells was further supported by another study where vaccination of B cell deficient lMT mice with whole inactivated virus was able to prime and activate CD4+ T cells to a level comparable to WT animals (Poo et al., 2014b).

Subsequent challenge in these animals with CHIKV resulted in exacerbated joint footpad pathology, further confirming the pathogenic role of CD4+ T cells, albeit in a B cell deficient environment.

Intriguingly, T cells were demonstrated to be protective in the

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mouse models of other alphaviruses such as RRV and VEEV (Soden et al., 2000; Yun et al., 2009). Currently, the role of CD8+ T cells in CHIKV infection remains to be determined, although it has been shown in CD8 / mice that CD8+ T cells could be redundant in CHIKV infection (Teo et al., 2013).

IL-17, a cytokine produced by Th17 T cells, has been detected in several cohorts of CHIKF patients (Ng et al., 2009; Chow et al., 2011; Teng et al., 2015). Th17 cells have been shown to play pathogenic roles in other alphaviral infections (Kulcsar et al., 2014), and in rheumatoid arthritis (RA) patients (Kotake et al., 1999; Chabaud et al., 2000). Given that the disease outcome of RA is quite similar to CHIKV infection (Nakaya et al., 2012), it would be interesting to elucidate the role of Th17 in defining CHIKV pathogenesis. It has been shown that a subpopulation of T cells called regulatory T cells (Tregs) is present to maintain immune tolerance, and Tregs are often studied in autoimmune diseases (Fessler et al., 2013; Dhaeze et al., 2015). But recent studies have shown increasing involvement of Tregs in infections (Belkaid and Rouse, 2005; Belkaid, 2007). More recently, Tregs were demonstrated in mice to protect against CHIKV-induced pathology by driving CHIKV-specific T cells into a state of anergy (Lee et al., 2015).

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14. B cells

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B cells and CHIKV-specific antibodies have been characterized extensively during the past 5 years (Kam et al., 2012a,b,c, 2014, 2015; Lum et al., 2013; Poo et al., 2014b). Studies using patient plasma samples showed that CHIKV infection induced a robust anti-CHIKV IgG antibody response dominated by the IgG3 isotype (Kam et al., 2012a). Further characterization of these anti-CHIKV antibodies led to the finding that viral load is a driving force in inducing the production of these neutralizing antibodies (Kam et al., 2012a). Likewise, in CHIKV-infected animals, high titers of neutralizing CHIKV-specific antibodies were produced after infection and their epitopes within the E2 glycoprotein has been mapped (Table 1). Furthermore, the virus-like particle (VLP) vaccine was demonstrated to induce the production of neutralizing antibodies in vaccinated non-human primates that protected the animals against challenge (Akahata et al., 2010). Likewise, a measles virus-based vaccine, which expresses CHIKV VLPs, protected susceptible mice from lethal challenge (Brandler et al., 2013).

Recently, these promising vaccine candidates underwent small clinical trials, in which vaccinated healthy adults produced neutralizing antibodies, with no reports of adverse effects (Chang et al., 2014; Ramsauer et al., 2015). The specific role of CHIKV-specific antibodies in viremia clearance was also demonstrated using lMT mice, whereby viremia persisted in these animals for more than a year (Lum et al., 2013; Poo et al., 2014b).

The importance of specific antibodies in resolving viremia was further proven by the passive transfer of human anti-CHIKV antibodies into IFNAR / or neonatal mice, which would otherwise succumb to infection (Couderc et al., 2009). It was further revealed that anti-CHIKV antibodies from patients and animals targeted a dominant epitope, E2EP3, which sits predominantly exposed on the CHIKV E2 glycoprotein (Kam et al., 2012b,c, 2014; Lum et al., 2013). The importance of protective antibodies was further demonstrated with the development of CHIKV-specific monoclonal antibodies (mAbs) from either mouse or human origin. These mAbs targeted epitopes located on CHIKV E2 (Warter et al., 2011; Fric et al., 2013; Pal et al., 2013; Selvarajah et al., 2013; Chua et al., 2014; Goh et al., 2015), E1 (Warter et al., 2011; Pal et al., 2013; Masrinoul et al., 2014), Capsid (Goh et al., 2015) and nsP2 (Chattopadhyay et al., 2014), and were protective against CHIKV infection in mice (Fric et al., 2013; Goh et al., 2013; Pal et al.,

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Regions of epitopes found common to humans, mice and nonhuman primates are highlighted in bold type.

The numbers correspond to the amino acid positions along the CHIKV viral genome. The first amino acid from nsP1 is annotated as 1. a

3121–3146 3177–3210 PTEGLEVTWGNNEPYKYWPQLSTNGT LLSMVGMAAGMCMCARRRCITPYELTPGATVPFL

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2013; Selvarajah et al., 2013). However, it has also been shown that these protective antibodies were not necessarily CHIKV-isolate specific. In fact, some of these neutralizing antibodies have been shown to cross-react with closely related alphaviruses, such as O’Nyong Nyong virus (ONNV) and Sindbis virus (SINV) (Warter et al., 2011; Partidos et al., 2012; Goh et al., 2015).

Despite their protective role, B cells and antibodies have also been suggested to enhance virus infections in a phenomenon known as antibody-dependent enhancement (ADE) (Takada and Kawaoka, 2003). ADE has been most well reported in Dengue virus (DENV) infections, whereby antibodies against a particular serotype of DENV can cross-react with another serotype, leading to an elevated level of infection and a more severe disease (Chareonsirisuthigul et al., 2007; Balsitis et al., 2010; Zellweger et al., 2010; Halstead, 2014). In DENV-infected patients, ADE can lead to severe dengue vascular permeability syndrome upon heterotypic DENV re-infection (Guzman et al., 2002; Alvarez et al., 2006; Guzman and Kouri, 2008).

ADE is therefore a concern for CHIKV infections, especially with the emergence of possible quasi-species harboring mutations (Stapleford et al., 2014) that might render neutralizing antibodies enhancing at sub-neutralizing levels. In this scenario, severe disease due to re-infection could become a reality. In addition, CHIKV-specific antibodies have been suggested to be maternally transferred to their offspring (Watanaveeradej et al., 2006). Thus, while these maternally acquired antibodies are degraded with time, there exists a window during which these antibodies would become present in sub-neutralizing quantities, and expose the offspring to enhanced CHIKV infection, in a manner similar to DENV infection (Ng et al., 2014). Although an enhancement in the severity of disease was reported during a prime-boost immunization study (Hallengärd et al., 2014b), it remains to be determined if ADE can occur in CHIKV infections.

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Despite numerous studies, defining the precise functional role of immune cells in CHIKV-infected humans remains a challenge.

Researchers could further explore alternative approaches to better understand immunopathogenesis. Among the approaches, the development of viable mouse models has assisted in addressing some questions. However, as the immune systems of mice and humans are not identical, new research tools must be designed to aid in future studies. The development of humanized mice (Shultz et al., 2007) with immune cells of human origin may offer a possible research direction to define immunopathogenesis.

The dream of having a chikungunya vaccine could be near with the success of the VLP vaccine (Chang et al., 2014). Nevertheless, caution should still be exercised during vaccine design, so that vaccines generated do not prime a sub-par humoral response, which might lead to an exacerbated disease and pathology in patients.

Likewise, given the proposed pathogenic roles of CD4+ T cells, vaccine formulations targeting T cells could be further manipulated to prime protective CD4+ T cells. In addition, given that both monocytes and CD4+ T cells are potentially pathogenic during CHIKV infection, drugs targeting these subsets could be explored.

Furthermore, with Tregs shown to play an important immune-regulatory role, it would be interesting to explore whether Tregs could also serve as a plausible point of intervention.

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16. Uncited references

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Lumsden (1955) and Rezza et al. (2007).

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b

3177–3210

3097–3146 3073–3098

Region on E2 glycoprotein

Part of the acid-sensitive region 2985–3002 3025–3066 GNVKITVNGQTVRYKCNC HAAVTNHKKWQYNSPLVPRNAE LGDRKGKIHIPFPLANVTCR 2985–3002 3025–3081

GNVKITVNGQTVRYKCNC HAAVTNHKKWQYNSPLVPRNAEL GDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQ RNMGEEPNYQEEWVMHKKEVVLT VPTEGLEVTWGNNEPYKYWPQLSTNGT LLSMVGMAAGMCMCARRRCITPYELTPGATVPFL 3009–3034 3025–3062

2800–2818 2961–2978

Amino acid Macaque B cell epitopes

STKDNFNVYKATRPYLAHC ATTEEIEVHMPPDTPDRT 2800–2818 2929–2954

Amino acid Mouse B cell epitopes

STKDNFNVYKATRPYLAHC HHDPPVIGREKFHSRPQHGKELPCST 2800–2818 2841–2882

STKDNFNVYKATRPYLAHC TDGTLKIQVSLQIGIKTDDSH DWTKLRYMDNHMPADAERAGL LTTTDKVINNCKVDQCHAAVTNHKKW HAAVTNHKKWQYNSPLVPRNA ELGDRKGKIHIPFPLAN PTVTYGKNQVIMLLYPDHPTLLSYRN

Amino acidb Human B cell epitopesa

Table 1 Comparison of identified CHIKV B cell epitopes in humans, mice and macaques, within the E2 proteome.

E2EP3 region

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Acknowledgements

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We acknowledge Wendy Lee and Kai Er Eng from SIgN for their critical comments of this manuscript. Fok-Moon Lum is supported by the President Graduate Fellowship from the Yong Loo Lin School of Medicine, National University of Singapore (Singapore).

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Please cite this article in press as: Lum, F.-M., Ng, L.F.P. Cellular and molecular mechanisms of chikungunya pathogenesis. Antiviral Res. (2015), http:// dx.doi.org/10.1016/j.antiviral.2015.06.009