chikungunya virus infection an overview

NEW MICROBIOLOGICA, 36, 211-227, 2013

Chikungunya virus infection: an overview

Claudia Caglioti, Eleonora Lalle, Concetta Castilletti, Fabrizio Carletti, Maria Rosaria Capobianchi, Licia Bordi

Laboratory of Virology, “L. Spallanzani” National Institute for Infectious Diseases, Rome, Italy

INTRODUCTION

Chikungunya virus (CHIKV), an arbovirus trans-mitted by mosquito vectors, is an alphavirus be-longing to the Togaviridae family. Alphavirusesare small spherical enveloped viruses, with a 60-70 nm diameter. The genome is a single-strandRNA molecule of positive polarity, encoding fournon structural (nsP1-4) and three structural pro-teins (C, E1, E2). Viral replication is initiated byattachment of the viral envelope to host cell re-

Corresponding authorMaria Rosaria CapobianchiLaboratory of VirologyPadiglione BagliviNational Institute for Infectious Diseases INMI “L. Spallanzani”Via Portuense, 292 - 00149 Rome, ItalyE-mail: [email protected]

ceptors (Strauss and Strauss, 1994), followed byclathrin-mediated endocytosis of the attached par-ticle (Lee et al., 2013), low pH-mediated membranefusion and delivery of the viral nucleocapsid intothe cytoplasm (Sorisseau et al., 2007). To date noCHIKV interacting protein has been characterized,but in a very recent study, Wintachai et al. identi-fied prohibitin as CHIKV-binding protein ex-pressed by microglial cells (Wintachai et al., 2012).The replication cycle is fast, taking around 4 hours.Alphaviruses are sensitive to dissecation and totemperatures above 58°C (Strauss and Strauss,1994; Khan et al., 2002). About 30 species of arthro-pod-borne viruses are included in the alphavirusgenus, antigenically classified into 7 complexes. These viruses are widely distributed throughoutthe world, with the exception of Antarctica.Besides CHIKV, several arthropod-transmittedalphaviruses cause human disease, characterizedby similar clinical presentation: Barmah Forest

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus belonging to the Togaviridae family, first isolatedin Tanzania in 1952. The main vectors are mosquitoes from the Aedes species. Recently, the establishment of an en-velope mutation increased infectivity for A. albopictus. CHIKV has recently re-emerged causing millions of infectionsin countries around the Indian Ocean characterized by climate conditions favourable to high vector density. Importationof human cases to European regions with high density of suitable arthropod vectors (such as A. albopictus) may trig-ger autochthonous outbreaks. The clinical signs of CHIKV infection include non-specific flu-like symptoms, and a char-acteristic rash accompanied by joint pain that may last for a long time after the resolution of the infection. The deathrate is not particularly high, but excess mortality has been observed in concomitance with large CHIKV outbreaks. De-regulation of innate defense mechanisms, such as cytokine inflammatory response, may participate in the main clin-ical signs of CHIKV infection, and the establishment of persistent (chronic) disease. There is no specific therapy, andprevention is the main countermeasure. Prevention is based on insect control and in avoiding mosquito bites in en-demic countries. Diagnosis is based on the detection of virus by molecular methods or by virus culture on the first daysof infection, and by detection of an immune response in later stages. CHIKV infection must be suspected in patientswith compatible clinical symptoms returning from epidemic/endemic areas. Differential diagnosis should take into ac-count the cross-reactivity with other viruses from the same antigenic complex (i.e. O’nyong-nyong virus).

KEY WORDS: CHIKV, Arbovirus, Virus dissemination, Immunopathogenesis, Geographic distribution, Diagnosis,Treatment and prevention.

SUMMARY

Received May 26, 2013 Accepted May 30, 2013

(BFV) and Ross River viruses (RRV) (Oceania),O’nyong-nyong (ONNV) and Semliki Forest virus-es (SFV) (Africa), Mayaro (South America),Sindbis (SINV) and Sindbis-like viruses (Africa,Asia, Scandinavia and Russia) (Taubiz et al.,2007). Chikungunya fever (CHIKF) derives itsname from Makonde, a language spoken in southTanzania, and means “that which bends up”, re-ferring to the posture of patients afflicted withsevere joint pain characterizing this infection.First isolated in Tanzania in 1952 (Robinson,1955), CHIKV attracted worldwide attentionwhen it caused a massive outbreak in the IndianOcean islands (Enserik, 2006). Since 1952,CHIKV has caused a number of epidemics, bothin Africa and in Southeast Asia, many of them in-volving hundreds of thousands of people. After afew years of relative dormancy in La RéunionIsland, CHIKV transmission has restarted, re-newing concerns about the possibility of renewedautochthonous transmission in Mediterraneancountries.

GEOGRAPHIC DISTRIBUTION

CHIKF has an epidemiological pattern with bothsporadic and epidemics cases in West Africa,from Senegal to Cameroun, and in many otherAfrican countries (Democratic Republic ofCongo, Nigeria, Angola, Uganda, Guinea, Malawi,Central African Republic, Burundi, and SouthAfrica). Moreover, many epidemics occurred inAsia (Burma, Thailand, Cambodia, Vietnam,India, Sri Lanka, Timor, Indonesia, and thePhilippines) in the 1960s and in the 1990s(Pialoux et al., 2007; Jain et al., 2008). Major epidemics appear and disappear cyclical-ly, usually with an inter-epidemic period rang-ing from 7 to 20 years. The huge outbreak thatincreased concern about CHIKV started inKenya in 2004, where the seroprevalence ratesreached 75% in Lamu island (Pialoux et al.,2007), before reaching the Comores, Seychelles,and Mauritius islands. The virus reached LaRéunion island in March-April 2005, probably

212 C. Caglioti, E. Lalle, C. Castilletti, F. Carletti, M.R. Capobianchi, L. Bordi

FIGURE 1 - Geographic distribution of CHIKV shown in the most recent map (May 2012) retrieved from the CDCwebsite (http://www.cdc.gov/chikungunya/map/index.html, last accessed May 2013).

as a result of importation of cases among immi-grants from the Comores and rapidly spread toseveral countries in the Indian Ocean and India(Enserik, 2006; Mavalankar et al., 2007).Compared to earlier outbreaks, this episode wasmassive, occurred in highly medicalized areassuch as La Réunion, and had very significanteconomic and social impact. Since the beginningof the outbreak in the Indian Ocean region, morethan 1,000 imported CHIKV cases have been de-tected among European and American travellersreturning from the affected areas (Fusco et al.,2006; Taubiz et al., 2007), giving rise, in 2007, tothe first autochthonous (human-to-mosquito-to-human transmission) European outbreak in Italy(Rezza et al., 2007; Charrel and de Lambellerie,2008). During the period December 2006-July2009, no confirmed cases were detected on LaRéunion and Mayotte Islands, but new outbreakswere reported in Madagascar. After a few yearsof relative dormancy in La Réunion, CHIKVtransmission restarted in 2009 and 2010, lead-ing to re-importation to Europe (May 2010)(D’Ortenzio et al., 2011). During the last three years (2011-2013) concernsabout Chikungunya outbreaks arose again dueto increasing number of CHIKV infections, start-ing from 2011, when a massive outbreak withmore than 11,000 cases occurred in the Republicof Congo (Brazzaville) (ProMED-mail:20110613.1806). During 2012, 29 cases of CHIKVinfection were reported in India (Rajasthan)(ProMED-mail: 20120716.1203694), and two ad-ditional outbreaks were recorded: one inCambodia, with almost 1,500 cases (ProMED-mail: 20120920.1303166) and one in the main is-land of Papua New Guinea, with a total of 633suspected cases (ProMED-mail: 20121010.1335814); Bali has also had sporadic outbreaks(ProMED-mail: 20130320.1594512). In Samar(Philippines) 600 cases were recorded in 2012,but in 2013 the infection rate has been increas-ing, with 500 cases recorded until March; thesenumbers appear to be increasing day by day(ProMED-mail: 20130128.1518853). Consideringthe capacity of CHIKV to emerge, re-emerge, andquickly spread in novel areas, heightened sur-veillance and preparedness seem to be a priori-ty. In particular, travellers act as carriers who in-advertently ferry pathogens between countries.They can thus serve as a sentinel population pro-

viding information on the emergence or re-emer-gence of an infectious pathogen in a source re-gion, and can be used to map the location, dy-namics and movement of pathogenic strains(Pistone et al., 2009). The geographic range of CHIKV is mainly inAfrica, Asia and Australia (Figure 1).

PHYLOGENESIS

Three lineages of CHIKV, with distinct genotyp-ic and antigenic characteristics, have been iden-tified. Isolates that caused the 2004-06 IndianOcean outbreak form a distinct cluster within thelarge eastern/central Africa (ECSA) phylogeneticgroup, in addition to the Asian and west Africanphylogenetic groups (Powers et al., 2000;Schuffenecker et al., 2006). The divergence of each distinct lineage reflects,to some extent, the path of global transmissionand occasional outbreaks. According to phyloge-netic analysis performed by Volk and colleagues(2010), the currently circulating CHIKV strainshave an ancestor that existed within the last 500years. Interestingly, despite their close geographicdistance, the two African lineages did not clustertogether, indicating limited genetic exchange be-tween the two lineages in Africa. The only ex-ception was a 1963 bat isolate from Senegal,which grouped in the ECSA clade. This findingis the first to suggest that the main West Africanand ECSA lineages may overlap spatially in theenzootic cycle, at least occasionally (Volk et al.,2010).Moreover, phylogenetic analysis of CHIKV strainscirculating in A. albopicus-human transmissioncycles, obtained during outbreaks, identified theindependent acquisition of a common mutationin E1 glycoprotein (E1gp), namely A226V, instrains isolated from different geographic regions(Schuffenecker et al., 2006; de Lambellerie et al.,2008a). This mutation, together with M269V andD284E E1gp mutations, have been described asmolecular signatures of the Indian Ocean out-break (Arankalle et al., 2007; Tsetsarkin et al.,2007; Vazeille M, 2007). In particular, the A226Vmutation, which was absent in the strains isolat-ed during the initial phases of the outbreak in LaRéunion, appeared in >90% of the isolates afterDecember 2005. This change could be related to

Chikungunya overview 213

virus adaptation to the mosquito vector species(see below). Together with the lack of herd im-munity, this might explain the abrupt and esca-lating nature of the La Réunion outbreak. TheA226V mutation was clearly demonstrated to in-crease viral fitness in the A. albopictus vector(Vazeille et al., 2007; Tsetsarkin et al., 2007) that,in turn, may expand the potential for CHIKV todiffuse to the Americas and Europe, due to the

widespread distribution of this vector, particu-larly in Italy (Knudsen, 1995). In a previous pa-per we characterized 7 viral isolates (5 importedand 2 autochthonous cases) with respect to themolecular E1 signatures of the Indian OceanOutbreak, particularly the A226V mutation. Theseisolates had been obtained from 3 travellers re-turning from Mauritius in 2006, 2 returning fromIndia in 2006 and 2007, and 2 autochthonous cas-


FIGURE 2 - Phylogenetic tree of CHIKV strains performed on partial E1 gene. Sequences of a 1013 bp fragment ofE1 gene (nucleotide positions 10145-11158, with respect to the reference strain S27). The CHIKV strains isolated fromhuman cases in Italy (3 strains deriving from patients returning to Italy from Mauritius, 2 strains from patients re-turning from India, 2 strains from patients involved in the 2007 Italian outbreak) are indicated with the strain namein bold. Their GenBank accession numbers are: EU188924 for ITA1_TAM_06; EU190879 for ITA2_BMI_06; EU190881for ITA3_CGO_06; EU190884 for ITA4_MRA_06; EU272130 for ITA5_JEM_07; EU272132 for ITA7_BI_07; EU272133for ITA8_VEN_07 (Bordi et al., 2008). The sequences used for comparison are indicated with their GenBank acces-sion number. CHIKV strains carrying the A226V mutation are underlined.

es that occurred during the 2007 Italian outbreak(Bordi et al., 2008) (Figure 2). All the strains isolated in Italy, both imported andautochthonous, displayed two molecular signa-tures of the Indian Ocean outbreak (M269V andD284E). The A226V mutation was present in allimported and autochthonous cases, with the ex-ception of the isolate imported from the Indiansubcontinent in 2006. The absence of this muta-tion in the isolate imported in 2006 from Indiawas in agreement with published data (Arankalleet al., 2007), and with available GenBank se-quence data indicating that the virus strains cir-culating in India in 2006 lacked this mutation.The presence of A226V in the isolate importedfrom India in July 2007 and in the isolates fromthe 2007 Italian outbreak (originating from a caseimported from India) supports the view that thevirus envelope sequence of strains from Indiachanged over time, acquiring the E1 mutation as-sociated with enhanced fitness in A. albopictusafter 2006. So it appears that the acquisition andfixation of the A226V mutation may be a com-mon pathway of Chikungunya explosion in epi-demic areas, in a parallel interplay with the mos-quito vector dynamics. It is noteworthy that theoutbreak in Singapore, where the A226V muta-tion was absent, was rapidly controlled.

VECTOR AND RESERVOIR

Two distinct transmission cycles have been welldocumented for CHIKV: an enzootic sylvatic cy-cle and an endemic/epidemic urban cycle. TheAfrican sylvatic cycle likely involves several ar-boreal Aedes mosquitoes species as vectors (A.furcifer, A. vittatus, A. fulgens, A. luteocephalus, A.dalzieli, A. vigilax, A. camptorhynchites) and non-human primates as reservoir/amplifying hosts. InAfrica, the enzootic transmission cycle can spillover to infect people who live nearby, and en-zootic mosquito vectors may be involved in in-ter-human transmission during small outbreaks.A. furcifer, probably a principal enzootic vector, isknown to enter human villages (Diallo, 1999),where it presumably transmits the virus frommonkeys to humans (Peyrefitte et al., 2007;Peyrefitte et al., 2008). Endemic/epidemic trans-mission cycles were established when the viruswas introduced into Asia around 1950, and into

the Indian Ocean region, India and thenSoutheast Asia since 2005. As previously stated,a mutation in the E1gp gene, that results in theA226V amino acid substitution, dramatically in-creased the infectivity of some epidemic strainsfor an alternative urban vector, A. albopictus(ProMED archive 20100926.3495). Therefore, theurban transmission cycle relies only on A. aegyp-ti and/or A. albopictus, anthropophilic vectors thatcan initiate human-mosquito-human transmis-sion, and human amplification hosts. This en-demic/epidemic cycle results in high levels of hu-man exposure to mosquito transmission, partic-ularly because these vectors live in close proxim-ity to people. The behaviour and ecology of A. ae-gypti, in particular, are ideal for epidemic trans-mission because adult females prefer to feed onhumans, often take several blood meals during asingle gonotrophic cycle, oviposit in artificial con-tainers as their preferred larval sites, and rest in-side houses with ready access to human hosts(Weaver et al., 2012).A. albopictus is zoophilic and anthropophilic, ag-gressive, silent, active all-day long, and has a lifes-pan longer than other mosquitoes (up to 8weeks). In recent decades it has expanded to sev-eral areas previously known to be Aedes-free(Charrel et al. 2007). It seems that most new in-troductions of A. albopictus have been caused byvegetative eggs contained in timber and tyres ex-ported from Asia throughout the world. Otheremerging events also contributed to the intro-duction of A. albopictus mosquitoes into previ-ously unaffected areas, such as climate changeand the increasing use of plastic containers in de-veloping countries. Indeed, climate changes mayhave several effects on vector biology: increasingtemperatures may improve survival at higher lat-itudes and altitudes, increase the growth rates ofvector populations, and alter their seasonality; in-creased rainfall may have an effect on the larvalhabitat and population size, and finally an in-crease in humidity could favourably affect vectorsurvival (Gubler et al., 2001). The use of plasticcontainers in developing countries, where theyare usually not correctly disposed of and remainin the environment for years, has also been linkedwith the spread of the mosquitoes: acting as rain-water receptacles, and being exposed to sunlight,they can become perfect “incubators” for mos-quito eggs, where the ideal conditions of tem-


perature and humidity are achieved easily andnaturally. Human beings serve as the main CHIKV reser-voir during epidemic periods. In Africa some an-imals (monkeys, rodents, and birds) constitutethe virus reservoir during non-epidemic periods,sustaining virus circulation in the environmentin the absence of human cases. Outbreaks mightoccur in monkeys when herd immunity is low;the animals develop viremia but no pronouncedphysical manifestations (Wolfe et al., 2001; Inoueet al., 2003). An animal reservoir has not beenidentified in Asia, where humans appear to bethe only host.

TREATMENT AND PREVENTION

There are no specific drugs against CHIKV andpatients are symptomatically treated with non-steroidal anti-inflammatory drugs, fluids, andmedicines to relieve symptoms of fever andaching, such as ibuprofen, naproxen, acetamino-phen, or paracetamol. Steroids have occasional-ly been used but their efficacy was not significant(Taubitz et al., 2007). Some time ago chloroquine,a drug useful for prophylaxis and treatment ofmalaria, showed promising results for treatingchronic Chikungunya arthritis (Brighton, 1984),even if a further trial conducted on La RéunionIsland proved that there was no justification forthe use of chloroquine to treat acuteChikungunya disease (de Lamballerie et al.,2008b); overall, the usefulness of chloroquinetreatment remains unclear. Ribavirin (200 mgtwice a day for seven days) given to patients whocontinued to have crippling lower limb pains andarthritis for at least two weeks after a febrileepisode, seems to be effective against CHIKV,leading to faster resolution of joint and soft tissuemanifestations (Ravichandran and Manian,2008). Briolant and colleagues (2004) screenedvarious active antiviral compounds against virus-es of the alphavirus genus in vitro and demon-strated that 6-azauridinet was more effective thanribavirin against CHIKV. Moreover, the combi-nation of interferon (IFN)-α2b and ribavirin hada synergistic antiviral effect on CHIKV (Briolantet al., 2004). Since inhibitors of monocyte chemo-taxis can greatly alleviate alphavirus-inducedarthritides in mice (Rulli et al., 2009) the use of in-

hibitors of chemokine pathways associated withmonocyte/macrophage recruitment may be apromising approach in humans, to be further ex-plored. It is widely recognized that passive vaccination isan appropriate preventive and therapeutic optionfor many viral infections in humans, includingthose spread by viral vertical transmission, espe-cially when no alternative therapy is available(Dessain et al., 2008). CHIKV infection seems toelicit long-lasting protective immunity, and ex-periments performed using animal models haveshown a partial cross-protection among CHIKVand other alphaviruses (Hearn and Rainey, 1963;Edelman et al., 2000). Since human polyvalent im-mune globulins, purified from plasma samples ob-tained from donors in the convalescent phase ofCHIKV infection, exhibited high neutralizing ac-tivity in vitro and a powerful prophylactic and ther-apeutic efficacy against CHIKV infection in in vi-vomouse models (Couderc et al., 2009), it could beused in humans for prevention and treatment, es-pecially in individuals at risk of severe CHIKV dis-ease, such as neonates born to viremic mothersand adults with underlying conditions. Polyclonalimmune globulins present the advantage of abroad reactivity but the therapeutic intervention islimited, due to the short viremia in the acute phaseof CHIKV infection: thus the only benefit thistreatment has to offer would be to help reducingviremia faster (Kam et al., 2009). As an alternativeapproach, more specific human monoclonal an-tibodies (MAbs) could be used. In a recent studytwo unique human MAbs, specific for the CHIKVE1gp, strongly and specifically neutralized CHIKVinfection in vitro (Warter et al., 2011). To date a number of CHIKV vaccines have beendeveloped, but none have been licensed. While anumber of significant questions remain to be ad-dressed related to vaccine validation, such as themost appropriate animal models (species, age,immune status), the dose and route of immu-nization, the potential interference from multi-ple vaccinations against different viruses, andlastly, the practical cost of the vaccine, since mostof the epidemic geographical regions belong tothe developing countries, there is real hope thata vaccine to prevent this disease will not be toolong in arriving.Although no licensed vaccines are currently avail-able for CHIKV, potential vaccine candidates


have been tested in humans and animals withvarying success. Several vaccine strategies havebeen undertaken:1. whole inactivated virus preparation;2. attenuated live vaccines;3. recombinant proteins or virus like particles;4. DNA vaccination. Due to the ease of preparation, the first developedvaccines were formulations of whole-virus grownin cell cultures and inactivated either by forma-lin or tween-ether (Harrison et al., 1967; Eckels etal., 1970; Harrison et al., 1971; White et al., 1972). Further vaccines focused on attenuated strainsof CHIKV obtained after serial passages in cellcultures (Levitt et al., 1986; Edelman et al., 2000).One of these promising candidates is TSI-GSD-218, a serially passaged and plaque-purified liveCHIKV vaccine, tested for safety and immuno-genicity in human Phase II trials by the US ArmyMedical Research Institute (Edelman et al., 2000).Some chimeric candidate vaccines were devel-oped using either Venezuelan EquineEncephalitis virus (VEEV) attenuated vaccinestrain TC-83, a naturally attenuated strain ofEastern Equine Encephalitis virus, or SINV as abackbone and the structural protein genes ofCHIKV. Vaccinated mice were fully protectedagainst disease and viremia after CHIKV chal-lenge (Wang et al., 2008). Traditional attenuationapproaches, relying on cell culture passages, typ-ically result in attenuation that depends only onsmall numbers of attenuating point mutations.In addition to the risk of reactogenicity, attenua-tion based on small numbers of mutations canalso result in residual alphavirus infectivity formosquito vectors. This risk, underscored by theisolation of the TC-83 VEEV vaccine strain frommosquitoes in Louisiana during an equine vacci-nation campaign designed to control the 1971epidemic (Pedersen et al., 1972), is especially highwhen a vaccine that relies on a small number ofpoint mutations is used in a non-endemic loca-tion that could support a local transmission cycle.In 2012, the United States Army developed andtested a live attenuated strain of CHIKV,CHIKV181/25 for vaccine application.CHIKV181/25 demonstrated an excellent im-munogenic profile, however, transient arthralgiawas observed in about 8% of vaccine recipients.Sharma and colleagues tried to inactivateCHIKV181/25 with 1,5 iodonapthyl azide (INA),

a photoactive hydrophobic azide molecule thatthey used in a previous study (Sharma et al.,2007) to completely inactivate VEEV, in additionto UV irradiation. The INA-inactivatedCHIKV181/25 formulation may address the issueof residual virulence associated with live attenu-ated CHIKV181/25, but the INA-inactivation re-sults in a relatively weaker binding capacity ofCHIKV181/25 to the neutralizing polyclonal an-ti-CHIKV E2 glycoprotein (E2gp) so that furtherinvestigations are necessary (Sharma et al., 2012).Alternative genetic strategies such as viralchimeras offer the promise of more stable atten-uation (Kennedy et al., 2011). For instance, a re-cent study showed that chimeric alphaviruses,encoding CHIKV-specific structural genes (butno structural or nonstructural proteins capableof interfering with development of cellular an-tiviral response), induce protective immune re-sponse against subsequent CHIKV challenge(Wang et al., 2011). A novel CHIKV vaccine candidate, CHIKV/IRES(internal ribosome entry site), was generated bymanipulation of the structural protein expressionof a wt-CHIKV strain via the encephalomyocardi-tis virus IRES, and exhibited a high degree ofmurine attenuation that was not dependent on anintact IFN type I response, highly attenuated andefficacious after a single dose (Plante et al., 2011).Another approach, recently undertaken by Akataand colleagues (2010), was the use of virus-likeparticles (VLPs) expressing CHIKV structural pro-teins that resemble replication-competent al-phaviruses (Akahata et al., 2010). Immunizationof monkeys with these VLPs elicited neutralizingantibodies against envelope proteins from differentCHIKV strains that could confer passive protec-tion against lethal CHIKV challenge into new mice. The last frontier in the approach of CHIKV vac-cine design is the DNA vaccine strategy. An adap-tive constant-current electroporation techniquewas used to immunize mice (Muthumani et al.,2008) and rhesus macaques (Mallilankaraman etal., 2011) with an intramuscular injection of plas-mid coding for the CHIKV-capsid, E1 and E2.Vaccination induced robust antigen-specific cellu-lar and humoral immune responses in both cases. Kumar and colleagues (2012) aimed to developcandidate vaccines following two different strate-gies: one based on recombinant E2gp; the otherbased on chemically inactivated whole virus, both


with promising results (Kumar et al., 2012). Sincea vaccine is not currently available, protectionagainst mosquito bites and vector control are themain preventive measures. Individual protectionrelies on the use of mosquito repellents and meas-ures to limit skin exposure to mosquitoes.Bednets should be used during the night in hos-pitals and day-care facilities but Aedes mosqui-toes are active all-day long. Control of both adultand larval mosquito populations uses the samemodel as for dengue and has been relatively ef-fective in many countries and settings. Breedingsites must be removed, destroyed, frequentlyemptied, and cleaned or treated with insecticides.Control of A. aegypti has rarely been achieved andnever sustained (Reiter et al., 2006). Recent datashow the different degrees of insecticide resist-ance in A. albopictus and A. aegypti (Cui et al.,2006). Large-scale prevention campaigns usingdichlorodiphenyltrichloroethane have been ef-fective against A. aegypti but not A. albopictus.However, vector control is an endless, costly, andlabor-intensive measure and is not always wellaccepted by local populations, whose coopera-tion is crucial. Control of CHIKV infection, oth-er than use of drugs for treatment of disease, de-velopment of vaccines, individual protection frommosquitoes and vector control programs, also in-volves surveillance that is fundamental for earlyidentification of cases and quarantine measure-ment. A model used in investigation of the trans-mission potential of CHIKV in Italy has provenuseful to provide insight into the possible impactof future outbreaks in temperate climate regionsand the effectiveness of the interventions per-formed during the outbreak (Poletti et al., 2011).

CLINICAL MANIFESTATIONS

After infection with CHIKV, there is a silent in-cubation period lasting about 2-4 days (range 1-12days) (Lam et al., 2001). Clinical onset is abrupt,with high fever, headache, back pain, myalgia,and arthralgia; the latter can be intense, affectingmainly the extremities (ankles, wrists, phalanges)but also the large joints (Robinson, 1955; Lam etal., 2001; Hochedez et al., 2006; Quatresous, 2006;Saxena et al., 2006). Skin involvement is presentin about 40-50% of cases, and consists of a pru-riginous maculopapular rash predominating on

the thorax. The clinical presentation may also in-volve facial oedema and, in children, a bullousrash with pronounced sloughing, localised pe-techiae and gingivorrhagia (Fourie and Morrison,1979; Brighton et al., 1983). Radiological findingsare normal, and biological markers of inflamma-tion (erythrocyte sedimentation rate and C-reac-tive protein) are normal or moderately elevated(Fourie and Morrison, 1979; Kennedy et al., 1980).Iridocyclitis and retinitis are the most commonocular manifestations associated with CHIKF; lessfrequent ocular lesions include episcleritis. All oc-ular manifestations have a benign course withcomplete resolution and preservation of vision.Retinitis shows gradual resolution over a period of6 to 8 weeks (Mahendradas et al., 2008). Erratic,relapsing, and incapacitating arthralgia is the hall-mark of Chikungunya, although it rarely affectschildren. These manifestations are normally mi-gratory and involve the small joints of hands,wrists, ankles, and feet with pain on movement. The symptoms generally resolve within 7-10 days,except for joint stiffness and pain: up to 12% ofpatients still have chronic arthralgia three yearsafter onset of the illness. Arthralgia experiencedby CHIKV patients closely resembles thesymptoms induced by other viruses like RRV andBFV (et al., 2002; Jacups et al., 2008).Neurological complications such as meningo-en-cephalitis were reported in a few patients duringthe first Indian outbreak in 1973, and during the2006 Indian outbreak (Chatterjee et al., 1965 a,b; Ravi, 2006). Moreover, during the 2006 Indian-Ocean outbreak, rare cases of Guillain-Barrésyndrome associated with CHIKV infection havebeen described (Wielanek et al., 2007; Lebrun etal., 2009). The possible mechanisms underlyingthese processes remain unknown, even if it wasfound that mouse CNS tissues such as the cho-roid plexi could also be targets of CHIKV, len-ding more credence to the fact that CHIKV in-fections do affect CNS cells and tissues (Coudercet al., 2008). Other rare complications describedafter CHIKV infection are mild hemorrhage,myocarditis, and hepatitis (Lemant et al., 2008). CHIKV is not generally considered a life-threat-ening disease. Usually the clinical course is fair-ly mild, but fatal cases directly or indirectly linkedto infection with CHIKV were observed duringthe Indian-Ocean outbreak (Josseran et al., 2006).The main evidence of a mortality linked to


CHIKF epidemics was obtained in La Réunion,Mauritius, and India, by comparing expected andobserved mortality data. In all cases, during themonths when the epidemics were raging, the ob-served mortality significantly exceeded the ex-pected rate. In particular, in La Réunion themonthly crude death rates in February andMarch 2006 were respectively 34.4% and 25.2%higher than expected. This corresponded to 260excess deaths (an increase of 18.4%) with a roughestimate of the case-fatality rate for CHIKF of≈1/1,000 cases. The case-fatality rate calculatedon increased crude death rates in Mauritius andAhmedabad, India, is substantially higher thanthat calculated in La Réunion: approximately4.5% (15,760 confirmed or suspected cases and743 excess deaths) and 4.9% (60,777 confirmed orsuspected cases and 2,944 excess deaths), re-spectively (Beesoon et al., 2008; Mavalankar et al.,2008). These differences may be attributed tomany factors (greater disease severity, preexistingpatient conditions, different patient management,or coincident excess deaths from other causes)but may also be due to a different efficacy of thesurveillance systems for CHIKF, that probablyworked poorly in Mauritius and India, leading tounderestimation of the total number of cases(Fusco et al., 2010). The possible link betweenCHIKV infection and multiorgan failure is stillunder investigation.

VIRUS DISSEMINATION AND TARGET ORGANS

Following intradermal inoculation by infectedmosquitoes, CHIKV directly enters the subcuta-neous capillaries where its replication starts im-mediately (Figure 3) with some viruses infectingsusceptible cells in the skin, such as macrophagesor fibroblasts and endothelial cells. Local viralreplication seems to be minor and limited in time,with the locally produced virus probably beingtransported to secondary lymphoid organs closeto the site of inoculation, where infected migra-tory cells produce new viruses which can, in turn,infect susceptible resident cells. Even if the hostis mounting a response to control the virus in theskin dermis, the virus disseminates quite rapidlyto the blood circulatory system. Viruses producedin the draining lymph nodes are released into the

lymph circulation and then to the blood throughthe thoracic duct. Once in the blood, the viruswill have access to various parts of the body, in-cluding the liver, muscle, joints and brain. Inthese tissues, the infection is associated with amarked infiltration of mononuclear cells, in-cluding macrophages, that can be consideredTrojan horses for virus spread to sanctuary bodysites. The pathological events associated with tis-sue infection are mostly subclinical in the liver(hepatocyte apoptosis) and lymphoid organs(adenopathy), whereas mononuclear cell infil-tration and viral replication in the muscles andjoints are associated with very strong pain, withsome patients presenting arthritis (Dupuis-Maguiraga et al., 2012).During the first week of CHIKV infection, viremiacan reach very high levels (viral loads of 3.3×109

copies/ml) (Parola et al., 2006). Thus, it remainsunclear if the virus detected in the blood is re-leased from virus-infected peripheral bloodmononuclear cells, or is spilled out from otherreplication sites.

IMMUNOPATHOGENESIS

The innate immune response is the first barrieragainst viruses, being able to inhibit viral repli-cation through cytolytic and non-cytolytic mech-anisms. The IFN system plays an important rolein limiting virus spread at an early stage of in-fection. In vitro growth of all alphaviruses can begreatly suppressed by the antiviral effects of IFN-α/β when it is added to cells prior to infection(Sourisseau et al., 2007; Courderc et al., 2008;Schilte et al., 2010). The finding that aberrantType I interferon signalling in mice led to severeforms of CHIKF (Couderc et al., 2008) furtherhighlighted the important roles cytokines play inthe pathology of CHIKV infection. Moreover, in a recent study Wauquier and col-leagues (2011) demonstrated that CHIKV infec-tion in humans elicits strong innate immunity in-volving the production of numerous proinflam-matory mediators. Interestingly, high levels ofInterferon IFN-α were consistently found.Production of interleukin (IL), IL-4, IL-10, andIFN-γ suggested the engagement of the adaptiveimmunity. This was confirmed by flow cytometryof circulating T lymphocytes that showed a CD8+


T lymphocyte response in the early stages of thedisease and a CD4+ T lymphocyte-mediated re-sponse in the later stages (Wauquier et al., 2011).CHIKV interactions with monocytes and with oth-er blood leukocytes induced a robust and rapid in-nate immune response with the production of spe-cific chemokines and cytokines, including IFN-α. The involvement of monocytes during the earlyphase of CHIKV infection in vivo is massive, andinfected monocyte/macrophages migrate in thesynovial tissues of chronically CHIKV-infected pa-tients, where they contribute to the inflammationprocess. This may explain the persistence of jointsymptoms despite the short duration of viremia(Her et al., 2010). Infected monocyte/macrophages may be the main cells responsible

for viral dissemination in other sanctuary bodysites, such as the nervous system, and, in turn, maycontribute to the development of clinical manifes-tations mediated by excess immune response. Usually, CHIKF is a self-limiting disease, with adefined duration of clinical course (7-10 days).Recovery is associated with a vigorous immuneresponse, that may confer protection from re-in-fection. However, in some cases, chronic disease(arthralgia) may be established. Chronic symp-toms may persist even after clearance of the virusfrom the blood, but it is possible that an activeviral reservoir persists locally in the joints. Fivestudies have tried to identify the factors associ-ated with chronic Chikungunya disease in groupsof patients in Singapore (Chow et al., 2011), La


FIGURE 3 - Schematic representation of CHIKV dissemination to different tissues and organs.

Réunion (Hoarau et al., 2010), Dakshina Kannada(India) (Manimunda et al., 2010; Chaaitanya etal., 2011), and Emilia Romagna (Italy) (Kelvin etal., 2011). Regulatory mechanisms silencing the vigorous(even localized) inflammatory response seem tobe required to prevent the establishment ofchronic disease weeks or even months after viralclearance from the blood. The absence of suchmechanisms leads to chronic arthralgia. In fact,in patients from the La Réunion study, variousmarkers of inflammation (IFN-α, IL-6, monocytechemotactic protein-1/CCL-2, IL-8, and matrixmetalloproteinase 2) were detected in the syn-ovial fluid of a patient suffering from chronicpain, but not in patients who fully recovered(Hoarau et al., 2010). The persistence of a localreservoir of CHIKV in joints may therefore becharacteristic of chronic disease, consistentlywith findings in the macaque model, in whichCHIKV was detected after up to 90 days espe-cially in joint tissues, leading to chronic local in-flammation (Labadie et al., 2010). Moreover,Hoarau et al. (2010) reported high plasma con-centrations of IL-12 and IFN-α mRNA in bloodmononuclear cells after the convalescent phasein patients with chronic disease between 6months and 1 year after infection. In patientsfrom Singapore, the concentrations of these twocytokines, measured by alternative techniques,peaked in the acute phase and returned to normallevels at 2-3 months, even in patients who stillhad clinical symptoms. According to these find-ings, Chaaithanya and colleagues (2011) andKelvin and colleagues (2011) reported high lev-els of Th1-type cytokines in the blood of patientswith chronic disease (Chaaitanya et al., 2011;Kelvin et al., 2011). Thus, despite certain dis-crepancies, the available studies suggest thatchronic disease is associated with a de-regulationof inflammation during the acute and convales-cence phases. This lack of regulation results in adeleterious inflammatory process that persists for≥ 1 year after the first clinical signs (Dupuis-Maguiraga et al., 2012).Concerning the possible implication of viral fac-tors in the pathogenesis, attention has focusedon the A226V mutation, that has been associatedwith enhanced replication and fitness of CHIKVin A. albopictus vector, and has also been shownto modulate the cholesterol requirement for in-

fection of insect cells (Tsetsarkin et al., 2007). Arecent study by our group investigated the possi-ble involvement of A226V mutation in enhanc-ing human pathogenesis by testing the replica-tion competence in primate cell cultures of twoisolates, differing for the presence or absence ofthis mutation (Bordi et al., 2011). We observedthat the presence of A226V mutation did not in-fluence the replication kinetics on primate cells.Moreover, the two isolates displayed very similartime course of cytopathic effect onset, numberand extent of CHIKV antigen-positive cells, aswell as the t-shape of the virus-positive multicel-lular foci, thus suggesting a similar mechanism ofspread of the virus in the infected cell cultures. In addition, we considered the possibility that theA226V mutation could be associated with partialresistance to the antiviral activity of recombinantIFN-α in classical experiments of virus replica-tion inhibition. Surprisingly, the A226V-carryingstrain was more susceptible than the wt virus tothe antiviral action of IFN-α. Overall, our result did not support the conceptthat A226V mutation confers a replicative ad-vantage in primate cell cultures, nor did it supportthe possibility that partial resistance to the in-hibitory action of IFN-α could account for the ex-plosive spread of the mutated strain in the hu-man population in the countries where this mu-tation had occurred. However, the possibility thatthe interplay between the virus and the innate de-fence system may act at different levels of thevirus/host interaction is to be taken into consid-eration, by exploring, for instance, other steps ofthe IFN response activation.At the moment, understanding CHIKV immuno-biology is still in its infancy and there is a longway to go before answers related to the interac-tion between virus and host immunity are ob-tained. These will certainly be important in de-signing novel antiviral control strategies againstthe spread of CHIKV infection.

DIAGNOSIS

Chikungunya infection is diagnosed on the basisof clinical, epidemiological and laboratory criteria.An acute onset of fever and severe arthralgia orarthritis that is not explained by other medicaldisorders is considered a possible CHIKV case.


The case becomes probable if the patient has livedin or visited epidemic areas in a time frame con-sistent with the incubation period (WHOGuidelines for prevention and control ofChikungunya fever http://www.searo.who.int/LinkFiles/Publication_SEA-CD-182.pdf (accessedAug 01, 2011).However, laboratory confirmation is crucial, be-cause the case should be distinguished from var-ious disorders with similar clinical manifesta-tions, such as dengue fever, other alphavirusesand arthritic diseases and also endemic malaria.The interpretation of laboratory findings is de-pendent on knowledge of the kinetics of viremiaand antibody response in human beings. The de-tection of viral nucleic acid or of infectious virusin serum samples is useful during the initialviremic phase, at the onset of symptoms and nor-mally for the following 5-10 days, when CHIKVRNA reaches very high levels (viral loads of3.3x109 copies/ml) and can be easily detected.Afterwards, the diagnosis is based mainly on thedetection of specific immune response by sero-logical methods.Molecular assays constitutes a rapid and sensi-tive technique for diagnosis of CHIKV infectionduring the early stages of illness before an anti-body response is evident. Conventional RT-PCR(Hasebe et al., 2002; Pfeffer et al., 2002) are avail-able, together with real time loop-mediated RT-PCR (Parida et al., 2007) and real time TaqManRT-PCR assay targeting the envelope E1 gene(Pastorino et al., 2005) or the non-structural thensP1 gene (Carletti et al., 2007). Moreover a one-step SYBR green-based real time assay targetingthe non-structural nsp2 gene was described morerecently (Ho et al., 2010).Viral isolation can be performed from serum ofinfected patients on insect or mammalian celllines (i.e. C6/36 or Vero E6) or by intracerebral in-oculation of 1-day-old mice during the earlyphase of the disease when the viral load is veryhigh and the immune response is still not de-tectable. In fact, the presence of early antibodyseems to prevent isolation of the virus, hencevirus isolation has been shown to be successfullargely in antibody-negative samples obtained onor before day 2 of illness (Panning et al., 2006).Moreover, viral isolation is useful for epidemiol-ogy or pathogenesis studies or for thorough mo-lecular characterization (Fusco et al., 2010).

The detection of CHIKV-specific immune re-sponse is based on serological methods such asenzyme-linked assays (ELISA), indirect im-munofluorescence assays (IFA), hemoagglutina-tion inhibition (HI) and micro-neutralization(MNt).IFA and ELISA are rapid and sensitive techniquesfor detection of CHIKV-specific antibodies, andcan distinguish between IgG and IgM. IgM aredetectable 2-3 days after the onset of symptomsand persist for several weeks, up to 3 months(Sam and AbuBakar, 2006; Litzba et et al., 2008).Rarely, IgM can be detected for longer periods, upto 1 year. CHIKV-specific IgG appear soon afterIgM antibodies (2-3 days) and persist for years.Various in-house ELISA techniques using wholeantigen or recombinant capsid or envelope anti-gens have been described (Cho et al., 2008).Commercial serological assays are available andresults obtained from a comparison of the assayssuggested that the sensitivity for detection of anearly antibody response before day 5 is depend-ent on the strain of the virus used for the assay orthe source of the antigen; assays based on re-combinant antigens might be too specific withregard to mutations (Cho et al., 2008; Litzba etal., 2008; Yap et al., 2010).Testing of a couple of sera collected in the acuteand convalescent phases of the disease is manda-tory for the identification of recent infection us-ing serological methods that cannot distinguishIgG Ab from IgM Ab (i.e. HI and MNt). It is alsovery useful to confirm results obtained with oth-er methods, especially taking into account thepossibility of rare persistence of IgM antibodies. Moreover, rapid bedside tests are commerciallyavailable, but their sensitivity and specificity arepoorly established, and the possibility of false-positive reactions resulting from cross-reactivitywith other arthropod-borne alphaviruses has tobe considered (Blackburn et al., 1995). In fact,CHIKV is a member of the SFV antigenic com-plex, and is most closely related to ONNV. In thisrespect, diagnosis based exclusively on CHIKV-specific serological testing is useful only for trav-ellers returning from a geographic area affectedby epidemic CHIKV diffusion (Pile et al., 1999),while in other cases differential diagnosis is nec-essary, taking into account the most commonviruses circulating in the region where the infec-tion has presumably been acquired.


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