Journal of Virological Methods 154 (2008) 154–159

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Journal of Virological Methods

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Expression of the capsid protein of Chikungunya virus in a baculovirus forserodiagnosis of Chikungunya disease

Byungki Choa,b, Jungho Kima, Jang-Eun Choc, Bo-Young Jeonc,∗, Sun Parkb

a Standard Diagnostics Inc., Yongin 446-930, Republic of Koreab Department of Microbiology, Ajou University School of Medicine, Suwon 442-721, Republic of Koreac Department of Microbiology and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea

Article history:Received 11 May 2008Received in revised form 24 July 2008Accepted 31 July 2008Available online 1 October 2008

Keywords:Chikungunya virus (CHIKV)

a b s t r a c t

Chikungunya virus (CHIKV) causes endemic or epidemic outbreaks of CHIK fever, which typically mani-fests as a febrile illness. To develop a CHIKV-specific diagnostic test, CHIKV capsid protein was expressedusing a baculovirus expression system. The seroreactvity of the recombinant CHIKV capsid protein wasevaluated by ELISA and immuochromatographic assay (ICA), using 40 anti-CHIKV-positive and 20 anti-CHIKV-negative sera, an additional 20 normal sera samples from healthy Koreans, and 20 anti-Denguevirus sera samples. The sensitivity of the recombinant CHIKV capsid protein was 85% and 87.5% as mea-sured by ELISA and ICA, respectively. The specificity of the recombinant CHIKV capsid protein was 100%both by ELISA and by ICA. No cross-reactivity of the capsid protein was seen with anti-Dengue virus sera

Capsid protein

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samples. There was a significant correlation between the ELISA- and ICA-measured seroreactivities of therecombinant CHIKV capsid protein for anti-CHIKV IgM-positive sera samples. These results suggest that

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. Introduction

Chikungunya virus (CHIKV) is the causative agent of Chikun-unya fever in humans, typified by clinical symptoms that includeever, arthralgias, arthritis, conjunctivitis, and rash (Bodenmannnd Genton, 2006; De Ranitz et al., 1956; Mason and Haddow,957; Pialoux et al., 2007). CHIKV is an alphavirus of the fam-ly Togaviridae (Karabatsos, 1975), and is transmitted by variouspecies of mosquitoes including Aedes (Ae.) africanus, Ae. luteo-ephalus, Ae. albopictus, Ae. furcifer, Ae. taylori, and Ae. aegypti (Diallot al., 1999). The CHIKV genome consists of linear, positive-sense,ingle-stranded RNA of approximately 11.8 kb, and contains struc-ural genes that express a capsid protein and two major envelopeurface glycoproteins E1 and E2 (Konishi and Hotta, 1980; Simizut al., 1984).

CHIKV was isolated originally by R.W. Ross during an outbreak

f dengue-like fever in Tanzania, East Africa in 1952 (Ross, 1956).uring 2005–2006, an epidemic outbreak of CHIK fever occurred on

everal islands in the Indian Ocean and in India, resulting in millionsf clinically suspected cases (Mavalankar et al., 2007; Schuffenecker

∗ Corresponding author at: Department of Microbiology, Yonsei University Col-ege of Medicine, 134 Shinchon-dong Seodaemoon-gu, Seoul 120-752, Republic oforea. Tel.: +82 2 2228 2548; fax: +82 2 392 9310.

E-mail address: bojeon@yuhs.ac (B.-Y. Jeon).

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166-0934/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2008.07.031

protein could be used in a diagnostic test for identifying CHIKV disease.© 2008 Elsevier B.V. All rights reserved.

t al., 2006). In 2006, there was a major outbreak of CHIKV infec-ion in India with 1.39 million reported cases (Lahariya and Pradhan,006). The major clinical symptom resulting from CHIKV infection

s febrile illness, which is similar clinically to symptoms of Dengueirus infection (Karabatsos, 1975). The outbreaks of infection byhese viruses often occur within similar geographical areas andoth diseases are transmitted by same vector, Aedes mosquitoes,rimarily Ae. aegypti (Mourya and Yadav, 2006). Dual infection ofHIKV and dengue virus has also been reported (Myers and Carley,967). However, CHIKV infection, unlike Dengue virus infection, isarely fatal and usually does not require close clinical supervision.herefore, the ability to distinguish CHIKV infection from Dengueirus infection would be extremely beneficial, particularly in areashere Dengue virus infection is endemic or epidemic.

Currently, serological tests for the diagnosis of CHIK fever arehe hemagglutination test (HI test), enzyme-linked immunosor-ent assays (ELISA), and indirect immunofluorescence test (Grivardt al., 2007; Karabatsos, 1975; Litzba et al., 2008). Presently, wholeirus antigens have been used as a diagnostic reagent for the diag-osis of CHIK fever. However, a CHIKV-specific antigen that hasigh specificity and low cross-reactivity with other related dis-

ases is needed urgently to diagnose CHIKV disease. Viral proteinn the native form is ideal for the diagnostic purpose, but the proce-ure of protein purification is laborious and time-consuming androduction of native viral protein is limited. Advanced molecularechnology has made it possible to scale up the production of viral

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B. Cho et al. / Journal of Virolo

rotein to process. The purity and activity of recombinant proteinas been shown to be comparable to native viral protein (Michel etl., 2008) and moreover, this method reduces the risk of exposureo biohazardous viruses.

In this study, recombinant CHIKV capsid protein was expressedsing a baculovirus expression system, and the seroreactivity ofhis antigen was validated as a diagnostic reagent for CHIKV fevery IgM indirect ELISA and an immunochromatographic assay (ICA).

. Materials and methods

.1. Performance panel

The evaluation panel for CHIKV was purchased from Labora-oire Marcel Merieux (Lyon, France), and consisted of 40 positivend 20 negative sera samples, based on the presence or absencef anti-CHIKV IgM antibody. As a negative control, 20 normal seraamples were collected from healthy Koreans who have never trav-lled to areas with endemic or epidemic CHIKV or Dengue virus.o check cross-reactivity with Dengue virus infection, 20 Dengueirus-positive sera samples were kindly provided by Truong Uyeninh in Arboviruses Laboratory, National Institute of Hygiene andpidemiology, Hanoi, Vietnam.

.2. Construction of a baculovirus transfer vector containing theHIKV capsid protein gene

To clone the capsid protein gene, CHIKV (strain TSI-GSD-218)as propagated in C6/36 cells. CHIKV genomic total RNA was

xtracted from CHIKV-infected C6/36 cells using an RNeasy miniit (Qiagen Inc., Valencia, CA, USA). The cDNA synthesis and ampli-cation of the capsid protein gene was performed using RT-PreMixBioneer Inc., Seoul, Korea). The sequences of the primers formplification of the CHIKV capsid protein gene are sense primer;′-CCA TGG ATG GAG TTT ATC CCA ACC CAA AC-3′ (NcoI) and anti-ense primer: 5′-CTC GAG CCA CTC TTC GGC CCC CTC AGG-3′ (XhoI).he PCR-amplified CHIKV capsid protein gene containing NcoI andhoI enzyme sites was ligated into the pCR2.1 Topo vector (Invit-

ogen, Carlsbad, CA, USA) and then the CHIKV capsid protein geneas subcloned into the baculovirus vector pFastBac HT (Invitrogen,arlsbad, CA, USA). The resulting plasmid pFast HT-CHIKV-capsidrotein was used to generate recombinant viruses.

.3. Generation of recombinant baculovirus

Recombinant viruses were generated as described previouslyJurutka et al., 2002; Nagesha et al., 1996). Briefly, Sf900 II SFM (Sf9,nvitrogen, Carlsbad, CA, USA) was transfected with the recombi-ant transfer vector pFastBac HT-CHIKV-capsid protein to generateecombinant baculovirus. As a control, pFastBac HT-CAT vectoras transfected into cells. Liposome-mediated gene transfer was

mployed using Cellfectin (Invitrogen, Carlsbad, CA, USA). A fewiral plaques were picked and recombinant viruses were purifiedy plaque assay and confirmed by PCR amplification and Westernlot analysis.

.4. Expression analysis and immunoblotting

Sf900 II cells were infected with recombinant baculoviruses at

multiplicity of infection of 5:1 PFU:cell and incubated at 27 ◦C.

hree days post-infection, cells were harvested and whole-cellysates were analysed by a discontinuous sodium dodecyl sulfateolyacrylamide gel electrophoresis (SDS-PAGE) system and the gelsere stained with Coomassie blue. Proteins separated by SDS-PAGE

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ethods 154 (2008) 154–159 155

ere transferred electrophoretically onto a nitrocellulose mem-rane for Western blot analysis. The membrane was incubated inBS containing 2% skim milk and then incubated in a 1:100 dilu-ion of CHIKV IgM positive serum, which was prepared by pooling0 CHIKV IgM positive sera. After 1 h, the membrane was washedeveral times and subsequently treated with horseradish perox-dase (HRP)-conjugated goat anti-human IgM antibodies (Santaruz Biotechnology, Santa Cruz, CA, USA) at a 1:2000 dilution forh at room temperature. Protein binding was detected using themersham Biosciences ImmunoBlot System (Amersham Pharma-ia Biotech, Stockholm, Sweden).

.5. Affinity purification of the recombinant CHIKV capsid protein

A high-yield, homogenous preparation of CHIKV capsid pro-ein was obtained using nickel-nitrilotriacetic acid (Ni-NTA) resin,ccording to the standard procedures described by the manufac-urer (Clontech, Mountain View, CA, USA). Briefly, recombinantaculovirus-infected Sf9 cell lysates were pelleted, and the super-atants were added to the equilibrated Ni-NTA agarose in a 1:10olume ratio. The bead slurry was then washed with 10 volumes of0 mM sodium phosphate, 300 mM NaCl, 10% glycerol, and 20 mM

midazole (pH 8.0). The CHIKV capsid protein was then eluted with00 or 500 mM imidazole in 50 mM sodium phosphate, 300 mMaCl, and 10% glycerol (pH 6.0).

.6. Anti-CHIKV IgM indirect ELISA

Enzyme-linked immunosorbent assays (ELISA) were performedo confirm the reactivity of recombinant CHIKV capsid protein tonti-CHIKV IgM antibodies as described before (Cho et al., 2001;hein et al., 1992). Ninety-six-well enzyme immunoassay (EIA)lates (Costar, Cambridge, MA, USA) were coated with serial dilu-ions of recombinant CHIKV capsid protein (1, 2, 4 and 8 �g/ml) inolycarbonate buffer (pH 9.2) overnight at 4 ◦C. Plates were thenashed and blocked with PBS (pH 7.4) containing 1% BSA for 1 h at

7 ◦C. Sera samples were diluted in PBS containing 1% BSA (1:100)nd were then added to the wells and incubated for 1 h at 37 ◦C.he bound antibodies were detected with HRP-conjugated mousenti-human IgM antibody (1:3000 dilution, Standard Diagnostics,ongin, Korea). The plates were washed minimally four times withBS containing 0.05% Tween 20 between each step. An enzyme sub-trate, TMB (3,3′,5,5′-Tetramethylbenzidine, Sigma Chemical Co.,t. Louis, MO, USA), was added to the wells and incubated for0 min. The reaction was stopped by adding 1.5N H2SO4, and thebsorbance was read at 450 nm using an automatic ELISA plateeader (Molecular Devices, Biotek Instruments, Hyland Park, VA,SA). The absorbance ratio of anti-CHIKV-positive serum to neg-tive serum for the recombinant capsid protein (P/N ratio) wasalculated as follows: A450 nm of positive serum/A450 nm of neg-tive serum.

.7. Immunochromatographic assay (ICA)

An ICA was developed for the detection of anti-CHIKV capsidrotein IgM using the method described previously (Chiao et al.,004; Kim et al., 2007; Shyu et al., 2002). In brief, colloidal gold40 nm in diameter, British Biocell International, UK) was used forhe conjugation of anti-human IgM. Colloidal gold solution (1%,/v) was adjusted to a pH of 8.5, and 0.2 ml anti-human IgM

1 mg/ml) was added to 10 ml pH-adjusted colloidal gold solution.he mixture was incubated overnight at 4 ◦C, and then centrifugedt 6000 × g for 30 min at 4 ◦C. After centrifugation, the gold pelletsere suspended in 10 ml of storage buffer (2 mM sodium borate

ontaining 0.1% BSA and 0.1% sodium azide, pH 7.2). This anti-

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uman IgM-coated colloidal gold probe was stored at 4 ◦C whileot in use. Recombinant CHIKV capsid protein was coated at theest line position (1 �g in 1 mg/ml) of the nitrocellulose membraneMillipore Co., Bedford, IN, USA). Five microlitres of gold conjugateas applied to the glass fiber. Fifty microlitres of serum was mixedith 50 �l of assay diluent (20 mM Tris buffer (pH 7.2) containing

.1% Triton X-100 and 0.5% BSA) and then applied to the samplead. The colour intensity was observed visually in 10 min and cate-orised as negative (−), or as positive; weak (+), strong (++), or verytrong (+++).

.8. Statistical analysis

Differences between experimental groups were analysed usinghe GraphPad Prism (Version 4) program. Differences were consid-red significant when P was <0.05.

. Results and discussion

.1. Construction and generation of recombinant baculovirusontaining the CHIKV capsid protein gene

The CHIKV capsid protein gene was amplified with primers con-aining NcoI/XhoI enzyme sites (Fig. 1) and was ligated into the pCR.1 Topo vector. The sequence of CHIKV capsid protein gene wasonfirmed by sequencing (data not shown). The CHIKV capsid pro-ein gene was then subcloned into the baculovirus vector pFastBacT using the NcoI/XhoI sites of the baculovirus vector. The resultinglasmid pFastBac HT-CHIKV-capsid protein was used to generateecombinant viruses.

.2. Expression of CHIKV capsid protein in Sf9 cell cultures

The CHIKV capsid protein gene constructs were expressed in Sf9ells. To validate the expression of CHIKV capsid protein, infectedells were harvested 24, 48, and 72 h post-infection, whole-cell

ysates were analysed by SDS-PAGE, and the gels were stained

ith Coomassie blue (Fig. 2A). The identity of the recombinantHIKV capsid protein was confirmed by Western blot analysis usingooled anti-CHIKV-positive serum (Fig. 2B). The expression levelf recombinant CHIKV capsid protein peaked at 72 h post-infection

ig. 1. PCR-amplification of CHIKV capsid protein gene. CHIKV capsid protein geneas amplified and electrophoresed in a 1% agarose gel. M: 1 kb plus DNA ladder,

ane 1: PCR product of capsid protein gene (783 bp).

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data not shown). A protein band with an approximate molecularass of 27 kDa was observed in the purified recombinant CHIKV-

apsid protein, and a corresponding protein band was seen in theell lysates infected with recombinant baculovirus supplying theHIKV-capsid protein constructs. The corresponding protein wasot present in mock-infected cells.

These results demonstrate that CHIKV capsid protein was suc-essfully expressed and purified via the baculovirus expressionystem and by affinity purification.

.3. Titration of recombinant CHIKV capsid protein fornti-CHIKV antibody detection by indirect ELISA

Recombinant baculovirus-expressed CHIKV capsid protein wasitrated to detect anti-CHIKV IgM antibodies using indirect ELISA. Toetermine the optimal concentration of recombinant CHIKV capsidrotein, recombinant capsid protein was serially diluted from 0.5 to.0 �g/ml (from 50 to 800 ng/well, respectively), and CHIKV capsidrotein-specific IgM antibodies were detected using pooled anti-HIKV-positive or negative serum. As shown in Fig. 3, as little as0 ng/well (0.5 �g/ml) of recombinant capsid protein produced a7-fold stronger ELISA signal for anti-CHIKV-positive serum thanor negative serum. The highest ratio (21.4-fold) of absorbancealue for positive to negative serum (P/N ratio) was obtained using00 ng/well (1.0 �g/ml) of recombinant capsid protein. This amountf protein was considered the optimal concentration of recombi-ant capsid protein and the following experiments, therefore, usedhis concentration of recombinant protein.

.4. Evaluation of the recombinant CHIKV capsid protein as aiagnostic reagent using ELISA

The recombinant CHIKV capsid protein was evaluated as a diag-ostic reagent for the detection of anti-CHIKV IgM antibodies usingLISA. Sixty anti-CHIKV sera samples (40 positive and 20 negative)ere used to evaluate the recombinant CHIKV capsid protein. To

heck the cross-reactivity of the CHIKV capsid protein with Dengueirus infection, twenty anti-Dengue virus-positive sera samplesere included. As a negative control of CHIKV and Dengue virus,0 normal sera from healthy Koreans were also used.

As shown in Fig. 4, the mean absorbance value of the recom-inant CHIKV capsid protein was approximately 0.9 for thenti-CHIKV-positive sera samples, and was about 0.05 for the anti-HIKV-negative sera samples. Based on these numbers, the cut-offalue was determined (A450, 0.16 = 0.05 ± 3 × 0.037 (S.D.)). Theensitivity of recombinant capsid protein for anti-CHIKV-positiveera samples was 85% (based on the cut-off values, A450 = 0.16,able 1). No cross-reactivity of the recombinant CHIKV capsidrotein was seen with any of the 20 Dengue virus sera samplesA450 < 0.15). Additionally, reactivity of the recombinant CHIKVapsid protein was not seen with the 20 normal control sera sam-les derived from healthy Koreans (A450 < 0.15). The specificity ofhe recombinant CHIKV capsid protein was 100% when used withnti-CHIKV-negative sera samples and normal control sera samplesf health Korean (Table 1). These results indicate that the recombi-ant CHIKV capsid protein is strongly reactive towards anti-CHIKV

gM antibodies, and had no cross-reactivity to anti-Dengue virusgM antibodies.

.5. Evaluation of the recombinant CHIKV capsid protein using

CA

To evaluate the recombinant CHIKV capsid protein as a rapidiagnostic reagent, an ICA assay using recombinant CHIKV cap-id protein was developed. Gold conjugates react with antibodies

B. Cho et al. / Journal of Virological Methods 154 (2008) 154–159 157

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ig. 2. Analysis of expression of CHIKV capsid protein in Sf9 cells. Sf9 cell lysates wy Western blot with anti-CHIKV-positive serum (B). M; Prestained protein markeontaining CHIKV capsid protein, lane 3; purified recombinant CHIKV capsid protei

n the serum, and the antibody–gold conjugate complex theninds specifically with CHIKV capsid protein. The colour appearsccording to the concentration of CHIKV capsid protein-specificntibodies present in the serum, and the colour change can be

isually observed immediately.

Thirty-five of 40 anti-CHIKV-positive sera samples were reactiveo the recombinant CHIKV capsid protein in the ICA (Fig. 5). Theesults of colour intensity using ICA were significantly correlated

ig. 3. Detection of recombinant CHIKV capsid protein by capsid protein-basedLISA. Various concentrations of recombinant CHIKV capsid protein were coatedn each well of a microtiter plate and evaluated for their reactivity with poolednti-CHIKV-positive or negative serum. Absorbance was read at 450 nm. The P/Natio is A450 nm of positive serum/A450 nm of negative serum.

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alysed by SDS-PAGE, and the gel was stained with Coomassie blue (A) or analysede 1; lysate of Sf9 cells, lane 2; lysate of Sf9 cells infected with recombinant virus

ith the absorbance values attained using ELISA with the 40 anti-HIKV-positive sera samples (R2 = 0.257, p < 0.001). Additionally,here was no positive reaction for any of the 20 anti-CHIKV-negativeera samples or for the 20 normal sera samples derived from healthyoreans (data not shown). No cross-reactivity was seen in any of the

wenty anti-Dengue virus sera samples. The sensitivity and speci-city of the ICA for anti-CHIKV capsid protein IgM antibodies were7.5% and 100%, respectively (Table 1).

While both CHIKV and Dengue virus cause febrile disease withinhe same geographical area, the prognosis of Dengue virus infection

ig. 4. The immunoreactivity of the recombinant CHIKV capsid protein using ELISA.orty anti-CHIKV-positive sera samples, 20 anti-CHIKV-negative sera samples, 20ormal sera samples of healthy Koreans, and 20 anti-Dengue virus sera samplesere evaluated. The absorbance was read at 450 nm.

158 B. Cho et al. / Journal of Virological M

Table 1Relative sensitivity and specificity of anti-CHIKV IgM indirect ELISA and ICA usingrecombinant CHIKV capsid protein

IgM capture ELISA (LMM)

Positivea Negativea

Anti-CHIKV IgM ELISA(CHIKV capsid protein)

Positiveb 34 0

Negativeb 6 20

Anti-CHIKV IgM ICA(CHIKV capsid protein)

Positive 35 0

Negative 5 20

Sensitivity [%] of ELISA 85.0%Sensitivity [%] of ICA 87.5%Specificity [%] of ELISA 100%Specificity [%] of ICA 100%

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a Based on the cut-off value, A450 nm = 0.15 (Laboratorie Marcel Merieux (Lyon,rance)).

b Based on the cut-off value, A450 nm = 0.16.

s much more severe (Queyriaux et al., 2008). Thus, laboratory con-

rmation of suspected cases is essential to launch control measureshen an epidemic or endemic outbreak occurs. Current laboratoryiagnosis of CHIK infection is based on isolation of the virus anderological and molecular methods. Reverse transcriptase PCR (RT-

ig. 5. ICA for the detection of anti-CHIKV capsid protein IgM antibodies. Sera sam-le was applied to the sample pad and the result was visually observed in 10 min.esults with no addition (A) and with anti-CHIKV-positive serum (B) and with anti-HIKV-negative serum (C) are shown.

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CR) is a confirmatory method used to identify CHIKV (Carlettit al., 2007; Edwards et al., 2007), and while this test exhibitsigh specificity, it requires expensive equipment and skilled sci-ntists to perform the test. Serological diagnostic methods suchs the hemagglutination inhibition test (HI test) and ELISA havelso been used to diagnose CHIKV disease. Although the HI tests a simple and rapid test, the results can be difficult to interpretue to cross-reactivity with other viruses (Chhabra et al., 2008;rivard et al., 2007; Karabatsos, 1975). ELISA is another popularethod to detect viral antigen-specific antibodies due to its high

ensitivity and specificity, and ELISA makes it possible to screenlarge number of small volume samples (Kitagawa et al., 1983;

hein et al., 1992). However, these serological methods have lim-tations because whole-virus antigens are used as the diagnosticeagent, which can result in cross-reaction with antigens of relatediruses.

In this study, recombinant CHIKV capsid protein was expressedsing a baculovirus expression system and the seroreactivity of thisapsid protein was evaluated using anti-CHIKV-positive and neg-tive sera samples as well as Dengue virus-positive sera samplesoth by ELISA and ICA. ICA is a very simple and rapid test, and yetas high specificity and sensitivity similar to that of ELISA (Biaginit al., 2006; Kim et al., 2007).

The sensitivity of the recombinant CHIKV capsid protein to anti-HIKV-positive sera samples, as determined by ELISA was 85%based on the cut-off value, A450 < 0.16). ICA using recombinantHIKV capsid protein showed a similar sensitivity (87.5%) to theLISA method using the recombinant CHIKV capsid protein. Theolour intensity in the ICA was significantly correlated with thebsorbance value as measured by ELISA (R2 = 0.257, p < 0.001). Noalse positives were detected in any of the anti-CHIKV-negative seraamples or the normal control sera samples of healthy Koreans inither the ELISA or ICA. Moreover, cross-reactivity of the recom-inant CHIKV capsid protein with anti-Dengue virus sera samplesas absent both in ELISA and in the ICA. Generally, an IgM cap-

ure ELISA is widely used for serodiagnosis of disease because lowevels of specific IgM antibodies can be detected without interfer-nce of IgG antibodies (Porter et al., 1999). In our study, indirectgM ELISA was used to evaluate the seroreactivity of CHIKV cap-id protein and we observed that the sensitivity and specificity ofndirect ELISA were high, consistent with the findings of a previ-us report (Petraityte et al., 2008). However, indirect IgM ELISAould not detect 6 of 40 anti-CHIKV-positive sera as positive evenhough those sera samples did show very low seroreactivity (datarom Laborotoire Marcel Merieux (Lyon, France). These false neg-tives could result from the evaluation method used as well ashe target antigen, the CHIKV capsid protein. The capsid proteins buried inside the virus particle, possibly impeding exposuref the protein to the host immune system (Sharp et al., 2006).xposed CHIKV E1 and E2 glycoproteins might give higher sen-itivity for detection of CHIKV disease. These results suggest thathe expressed recombinant CHIKV capsid protein might be a goodiagnostic reagent for CHIK virus infection as it is able to distinguishHIKV infection from those of related diseases such as Dengue virus

nfection.In conclusion, recombinant CHIKV capsid protein, expressed

sing a baculovirus expression system, was highly specific to anti-HIKV IgM antibodies, and the cross-reactivity of this protein withengue virus was very low.

cknowledgements

We thank Dr. Truong Uyen Nihn for providing the anti-Dengueirus sera samples. This work was supported by the Standard Diag-ostics Inc., Yongin, Korea.

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