Journal of Virological Methods 203 (2014) 15–22

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Cloning, expression and evaluation of diagnostic potential of recombinant capsid protein based IgM ELISA for chikungunya virus Raj Priya a , Mohsin Khan a , M. Kameswara Rao b , Manmohan Parida a,∗ a b

Division of Virology, Defence Research & Development Establishment, Gwalior 474002, India Biochemistry Division, Defence Research & Development Establishment, Gwalior 474002, India

a b s t r a c t Article history: Received 2 July 2013 Received in revised form 5 March 2014 Accepted 7 March 2014 Available online 25 March 2014 Keywords: Chikungunya Recombinant capsid protein IgM ELISA

The resurgence of chikungunya virus in the form of unprecedented explosive epidemic with unusual clinical severity after a gap of 32 years is a point of major public health concern. Definitive diagnosis is critical in differentiating the disease, especially in dengue endemic areas. The immunoglobulin M (IgM) enzyme-linked immunosorbent assay (ELISA) is widely used for diagnosis of chikungunya infection. However IgM ELISA based on whole virus antigen is associated with biohazard risk. The present study describes the development and evaluation of recombinant capsid protein based indirect IgM antibody capture micro plate enzyme linked immunosorbent assay (ELISA) for rapid and accurate diagnosis of chikungunya infection. The gene coding for capsid protein was cloned in frame with GST tag in pET41a+ vector and expressed in E. coli followed by purification with affinity chromatography. The comparative evaluation of in-house chikungunya IgM ELISA vis-a-vis commercially available SD ELISA kit with 90 chikungunya suspected acute phase human patient serum samples revealed 97% accordance. The overall sensitivity and specificity of the reported capsid protein based IgM ELISA was 100% and 95% respectively with 96% PPV and 100% NPV. These findings clearly demonstrated the usefulness of the recombinant capsid protein based CHIKV IgM ELISA for reliable clinical diagnosis of CHIKV infection in human patient. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Chikungunya virus (CHIKV) is a mosquito transmitted Alphavirus belonging to the family Togaviridae. CHIKV was first isolated from human in Tanzania, East Africa in 1953. The virus has gained renewed interest because of its resurgence in the form of explosive unprecedented epidemic in India, Sri Lanka, and a number of smaller islands in the Indian Ocean. CHIKV is a positive ssRNA virus with 60–70 nm diameter which comprises of a nucleocapsid enclosed within a phospholipid envelope. The genome is about 11.8 Kb and has 2 open reading frames (ORF), encoding a non-structural polyprotein and a structural polyprotein. The non-structural polypeptide is processed to form 4 non-structural proteins i.e. nsP1, nsP2, nsP3 and nsP4 proteins and the structural polyprotein is cleaved to form 5 structural proteins i.e. Capsid, E3, 6K, E2 and E1 (Strauss and Strauss, 1994). While the non structural proteins have been attributed to play a role in viral replication and

∗ Corresponding author at: Division of Virology, Defence R & D Establishment, Jhansi Road, Gwalior 474002, M.P., India. Tel.: +91 751 2347523; fax: +91 751 2341148. E-mail address: [email protected] (M. Parida). http://dx.doi.org/10.1016/j.jviromet.2014.03.005 0166-0934/© 2014 Elsevier B.V. All rights reserved.

gene expression, the structural proteins have been attributed to play a role in the viral budding and infection. Chikungunya fever is characterized by fever, arthralgia, muscular pain and in some cases maculopapular rash. All these symptoms are self limiting and generally last 1–10 days except arthralgia which is often very debilitating usually persists for months to years (Brighton and Simson, 1984) causing serious economic and social impact on both the individual and the affected communities. In human, the disease starts after 48 h of mosquito bite. Patient shows high viremia load in their blood during 3–4 days of infection which declines during days 5–6 and disappears by days 7–8. Symptoms usually appear 4–7 days post infection. Clinical manifestations of chikungunya are very much similar to those of Dengue fever; hence the ability to distinguish CHIKV infection from dengue fever is a challenge in the areas where both the viruses circulate simultaneously (Carey, 1971). Virus isolation in mosquito cells (C6/36), Vero cells or in neonatal mice (Couderc et al., 2008) is the gold standard for chikungunya diagnosis. The main disadvantages of virus isolation are biohazard due to handling of live virus, being tedious and labor intensive. The chikungunya confirmation can also be done by using gene amplification technologies viz.; RT-PCR (Hasebe et al., 2002; Pfeffer et al., 2002; Pastorino et al., 2005; Edwards et al., 2007; Joseph et al.,

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2008; Panning et al., 2009), Real-time RT-PCR (Carletti et al., 2007; Grivard et al., 2007; Laurent et al., 2007; Santhosh et al., 2007) and RT-LAMP (Parida et al., 2007). In these techniques, results could be available in 3–4 h with the advantages of rapidity, lower contamination rate, higher sensitivity and higher specificity but these techniques are very costly and requires sophisticated instruments and hence unaffordable by most of the laboratories in rural settings or resource poor countries. Presently, serodiagnostic methods for the detection of immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against CHIKV in acute and convalescent sera are used (Grivard et al., 2007). These include enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescent method (Litzba et al., 2008), haemagglutination inhibition, or neutralization techniques. In majority of cases antibodies are being used for diagnosis of CHIKV infection. The IgM antibody is detectable as early as 3–5 days post infection by ELISA and it typically persists for periods ranging from several weeks to 3 months. Instances of persistence of IgM antibodies 18 months after disease onset in some patients have also been described (Grivard et al., 2007). The IgG antibodies can be detected in convalescent samples and persists for years. ELISA based on detection of CHIKV antigen has also been developed (Shukla et al., 2009; Kashyap et al., 2010; Kumar et al., 2012; Khan et al., 2013). However, these assays are effective only for the early phase confirmation i.e. only within 2–6 days of onset of infection when the virus persists in the circulation. CHIKV IgM can be detected as early as 3–5 days after the onset of fever and persists for several months. So the detection of IgM antibody against CHIKV would offer more window period for diagnosis of virus infection. In recent years, few commercial kits were launched in the market for the detection of chikungunya IgM but because of poor sensitivity, their use is limited for the diagnosis of chikungunya infection (Rianthavorn et al., 2010; Blacksell et al., 2011). The use of whole virus preparations as antigen is associated with biohazard. CHIKV IgM ELISA based on recombinant structural protein of virus expressed in baculovirus infected insect cells has been reported (Cho et al., 2008a,b). However it is difficult to isolate proteins from insect expression system in comparison to E. coli expression system. Prokaryotic expression system is more simple and cost effective than eukaryotic system (Yathi et al., 2011; Khan et al., 2013). Thus, there is a need to develop a safer and effective detection assay for chikungunya. Keeping these points in view, in the present study CHIKV capsid gene was cloned and expressed in fusion with GST in order to obtain stable expression and further evaluated for its usefulness in clinical diagnosis of chikungunya infection. The data on the sensitivity, specificity of the recombinant capsid protein based IgM ELISA is discussed and its applicability for clinical diagnosis of chikungunya is evaluated using human patient serum samples.

2.2. Enzymes, chemicals and bacterial strain Enzymes (Taq DNA polymerase, Restriction enzymes Xho I, Nde I, Nco I and T4 DNA ligase), DNA marker, Protein ladder, 2X SDS Loading buffer were purchased from MBI Fermentas, Hanover, USA. The Escherichia coli (E. coli) competent cells (DH5␣ and BL21 (DE3)) were purchased from Novagen, Madison, WI, USA. Luria Bertani (LB) was purchased from Difco laboratories, Detroit, Michigan. Anti-GST (Glutathione-S-Transferase) antibody, Ni-NTA (Nickel-Nitrilotriacetic) super flow resin, viral RNA mini kit was purchased from Qiagen, Hilden, Germany. Glutathione S-Transferase (GST) agarose was purchased from GE Healthcare, USA. ELISA plate was purchased from Nunc, Denmark. Isopropyl ␤-d-1-thiogalactopyranoside (IPTG), Diaminobenzidine (DAB), 3,3 ,5,5 -tetramethylbenzidine (TMB), hydrogen peroxide (30% H2 O2 ), kanamycin, Enhanced Avian HS RT-PCR Kit and secondary HRP Conjugate were purchased from Sigma Chemical, St. Louis, MO, USA. Amicon ultra centrifugal filter device was from Millipore, Bedford, USA. Automated DNA sequencer model ABI310 was from Applied Biosystem, Foster city, CA, USA. 2.3. Viral RNA preparation Viral RNA was isolated from 140 ␮l of CHIKV infected Vero cell supernatant using a QIAamp viral RNA kit (Qiagen, Germany) according to manufacturer’s protocol. The viral RNA was quantified, aliquoted and stored at −80 ◦ C for further experiments. 2.4. Designing of capsid forward and reverse primers According to the complete genome sequences of CHIKV in GenBank (EF-210157), the CHIKV capsid specific primers were designed with the addition of restriction sites at 5 and 3 end. The primer pairs [5 -GCGCATATGGAGTTCATCC-3 (forward) and 5 -CGCCTCGAGACTCCACTCTT-3 (reverse)] were used to amplify 783 bp (7551–8331 bp) long full capsid gene. Bold segment shows 5 overhang added to incorporate Nde I and Xho I site in the forward and reverse primers respectively. 2.5. RT-PCR of capsid gene

2. Material and methods

CHIKV RNA was used as template to amplify the capsid gene. PCR amplification condition selected for amplification was 48 ◦ C for 45 min, 95 ◦ C for 5 min (95 ◦ C for 1 min, 55 ◦ C for 1 min, 72 ◦ C for 1 min) for total of 35 cycles and a final elongation step of 72 ◦ C for 10 min for the given primer set. The amplification was carried out in 50 ␮l reaction using Enhanced Avian HS RT-PCR kit (Sigma). Amplified product was checked on 1% low melting agarose gel and eluted from the gel using Gel Purification Kit (Qiagen) according to manufacturer’s protocol.

2.1. Virus and cell line

2.6. Cloning of capsid genes in pET41a+ vector

An Indian isolate of CHIKV DRDE-06 (Gen bank accession no: EF-210157) of East Central and South African (ECSA) genotype maintained at Virology Division, DRDE, Gwalior was used in the present study. The virus was passaged in Vero cell line for bulk production and aliquots of supernatant were stored at −80 ◦ C. Vero cell line was obtained from National Centre for Cell Sciences (NCCS), Pune, India and was maintained in Eagles Minimal Essential Medium (MEM) supplemented with 1.1 g sodium bicarbonate/L, 2 mM glutamine and 10% heat inactivated Fetal Bovine Serum (FBS) at 37 ◦ C with 5% CO2 humidified incubator (Galaxy 170R, New Brunswick Scientific, USA).

The purified capsid gene and pET28b+ vector having kanamycin resistance gene was digested with Xho I and Nde I restriction enzymes for overnight at 37 ◦ C. The digested capsid gene was cloned in Nde I and Xho I digested pET28b+ vector and transformed into DH5␣ competent cells. Positive clones were screened by colony lysis PCR, RE digestion and further confirmed by sequencing (data not shown). The recombinant pET28b+ capsid recombinant plasmid was digested with Nco I and Xho I enzymes, subcloned in pET41a+ vector and further transformed in DH5␣ competent cells. Plasmid was isolated from positive clone and was further transformed into BL-21(DE3) competent cells. Similarly pET41a+ vector having GST

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tag (without capsid insert) was transformed in BL-21(DE3) cells by giving heat shock at 42 ◦ C.

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5th blot) diluted in blocking buffer for 1 h. After three washes with PBST, color development was carried with 3,3 -diaminobenzidine as substrate.

2.7. Expression and localization of recombinant protein Positive clone containing recombinant plasmid was grown in Luria–Bertani (LB) broth at 37 ◦ C. When the OD600 (optical density) reached the value of 0.7, culture was induced with 1 mM isopropyl␤-d-thiogalacto-pyranoside (IPTG) and further grown for 4 h at 37 ◦ C. The cells were centrifuged at 4000 × g for 20 min, the pellet was lysed in sample loading buffer and analyzed on 10% SDS PAGE. In order to check the localization of protein cell pellet was diluted in distilled water and sonicated for 2 min on ice. The lysate was centrifuged at 10,000 × g for 10 min and the supernatant (soluble fraction) as well as pellet (insoluble fraction) was analyzed separately on 10% SDS-PAGE and stained with Coomassie brilliant blue R-250. 2.8. Purification of recombinant Capsid protein under native condition BL-21(DE3) cells containing the recombinant plasmid were grown at 37 ◦ C in LB media containing kanamycin until the OD600 reached to a level of 0.7. Cells were induced with 1 mM IPTG for next 4 h. The cells were collected by centrifugation at 9000 × g for 10 min at 4 ◦ C and suspended in 10 ml of PBS (pH-7.4) containing protease inhibitor cocktail and 1% Triton-X 100 and incubated for 30 min on ice. The cell suspension was sonicated for 10 min on ice (with 9 sec pulse on and 9 s pulse off) using microprobe set at 40% frequency of sonicator (Sonics, USA). The supernatant was collected after centrifugation at 9000 × g for 30 min and filtered through 0.45 ␮m filter. The pellet was discarded. Clear cell suspension was loaded on 200 ␮l GST agarose column equilibrated with PBS (pH7.4) and incubated at room temperature for efficient binding in rotatory shaker for 30 min. Column was washed with 10 bed volume of 1X PBS buffer (pH-7.4) for 3 times and the protein was eluted with 10 ml of elution buffer containing 50 mM Tris HCl and 10 mM reduced glutathione (pH-8). Eluted protein was concentrated using an Amicon Ultra centrifugal filter device with a 30-kDa cutoff membrane. Concentrated protein was dialyzed overnight against PBS to remove salts. Protein was analyzed on 10% SDS PAGE followed by Coomassie Brilliant Blue staining. The concentration of purified protein was determined by Bradford assay (Thermo Scientific, USA) and stored at −80 ◦ C. Similarly BL-21(DE3) cells with GST vector without capsid insert were grown at 37 ◦ C in LB media containing Kanamycin until the OD600 reached value of 0.7. Cells were induced with 1 mM IPTG for next 4 h. The cells were collected by centrifugation at 10,000 × g for 10 min at 4 ◦ C and the GST protein was purified similarly as described above. 2.9. Western blot analysis of purified GST-capsid recombinant protein The purified protein was checked on 10% SDS-PAGE gel and then transferred to nitrocellulose membranes. The membranes were blocked by 3% BSA for 1 h at room temperature, washed with PBS and incubated with different antibody viz. CHIKV positive patient sera (1:500), CHIKV negative patient sera (1:500), GST monoclonal antibody (1:3000), Rabbit hyper immune sera generated after CHIKV immunization (1:4000), Rabbit hyperimmune sera generated before CHIKV immunization (1:4000), diluted in blocking buffer for 1 h at room temperature. The membranes were washed 3 times with PBST and incubated with their respective HRP conjugate (anti human HRP conjugate in 1:2000 dilution for 1st and 2nd blot, anti Rabbit HRP conjugate in 1:4000 for 3rd, 4th and

2.10. MALDI-TOF/MS analysis of recombinant GST-capsid protein The GST-capsid protein purified from E. coli cells was further confirmed using MALDI-TOF/MS analysis. The Coomassie stained 1D SDS Gel band of recombinant GST-capsid protein was excised from the gel and washed thrice with 200 ␮l of destaining solvent containing 50% (v/v) acetonitrile (ACN) in 50 mM NH4 HCO3 (1:1) with constant vortexing of 15 min. The gel pieces were dehydrated with 200 ␮l of 100% ACN and dried in speed vac centrifuge (Thermo, USA) for 20 min. The protein in gel slices were subjected to digestion using 25 ␮l of trypsin (20 ␮g/100 ␮l) in 50 mM NH4 HCO3 at 37 ◦ C for overnight in a shaker incubator. The peptides were extracted twice from the gel using 200 ␮l of extraction solvent (50% ACN, 5% TFA) and lyophilized. The peptides obtained after lyophilization were reconstituted in 8 ␮l milli Q water and was subsequently eluted using Mini Tip C18 micro tips (Sigma, USA). Finally, 1 ␮l of the purified peptide along with 1 ␮l of HCCA matrix was spotted on the MALDI plate. Mass spectrometric analysis was performed using MALDI–TOF instrument (Bruker Microflex LRF20, Flex Control Workstation, Bermen, Germany) equipped with delayed extraction (150 ns) and a UV ionization laser (N2, 337 nm) with a 3-ns pulse width. The spectra were evaluated using Flex Analysis Software (Bruker Daltonics). The MS spectra obtained was submitted to MASCOT data base search via Bio tools versions 3.1. 2.11. Recombinant GST-capsid protein based IgM ELISA Recombinant GST-capsid protein was tested for its antigenicity in terms of recognition of antibody against CHIKV present in human serum samples. For this purpose 96 wells of microtitre plate was coated with 300 ng/well of recombinant GST-capsid protein in carbonate-bicarbonate buffer (pH-9.6) and incubated overnight at 4 ◦ C. The plate was washed 3 times with PBST (PBS containing 0.05% of Tween 20) and blocked with 5% BSA for overnight at 4 ◦ C. Human patient sera in the dilution of 1:100 in PBS was added to each well and incubated for 30 min at 37 ◦ C. After 5 times washing with PBST, the plate was incubated with 1:5000 dilution of antihuman IgM HRP conjugate in 5% BSA for 30 min at 37 ◦ C. Wells were again washed 5 times with PBST and the reaction was developed by adding 100 ␮l of TMB in each well. After 15 min of incubation at room temperature, the reaction was stopped by 100 ␮l of 2 N H2 SO4 and the OD was recorded at 450 nm using ELISA plate reader. 2.12. Evaluation of IgM ELISA for clinical diagnosis of chikungunya Ninety chikungunya suspected human patient serum samples collected between 3 and 10 days of fever (Dash et al., 2007; Santhosh et al., 2008) were first tested with in-house recombinant GST-capsid protein based IgM ELISA. The same samples were then evaluated with commercially available SD (Standard Diagnostics, South Korea) chikungunya IgM ELISA kit according to manufacturer’s instructions. Further specificity of in-house GSTcapsid protein based IgM ELISA was established through cross reactivity analysis with a panel of 10 apparently healthy serum samples and 10 dengue confirmed patient serum samples collected during 2004 outbreak (Dash et al., 2006). The values for accordance, specificity, sensitivity, PPV (Positive predictive value) and NPV (Negative predictive value) were determined as described earlier (Shukla et al., 2009).

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(Fig. 2B). The optimal yield of protein was 8 mg/l of shake flask culture. The GST protein was also localized in soluble form so it was purified under native condition. SDS PAGE (Fig. 3B) analysis showed presence of 29 kDa GST protein and yield was 12 mg/l of shake flask culture. 3.3. Western blot analysis Western blot analysis (Fig. 4A) with anti-GST monoclonal antibody, Rabbit antibody against CHIKV antigen and CHIKV positive patient sera detected 60 kDa protein band representing recombinant GST-capsid protein hence proving its antigenicity. Pre immune sera of rabbit and healthy patient sera did not produce any signal (data not shown). Similarly 29 kDa of GST protein was confirmed by GST monoclonal antibody (Fig. 4B). 3.4. MALDI-TOF/MS analysis of recombinant GST-capsid protein Fig. 1. The chikungunya capsid PCR product was examined on 1% agarose gel, stained with ethidum bromide. Lane M: Prestained protein marker; lane 2: 783 bp chikungunya capsid PCR product.

3. Results 3.1. Cloning and expression of capsid gene in pET41a Vector

The E. coli expressed recombinant GST-capsid protein was further confirmed by MALDI-TOF/MS analysis. The 60 kDa protein band corresponding to recombinant GST-capsid was enzymatically digested using trypsin. Twenty-three peptides in case of GST and nine peptides in case of capsid were obtained by MASCOT database search which identified GST-capsid protein with sequence coverage of 58% of GST and 42% of chikungunya capsid protein (Table 1).

A 783 bp DNA fragment encoding CHIKV capsid protein was amplified by RT-PCR using specific primers (Fig. 1). The amplified product was cloned in pET 28b+ vector. The product was digested by Nco I and Xho I sites and subcloned in pET 41a+ vector in frame with GST tag. The recombinant plasmid was transformed in E. coli BL-21 (DE3) cells and expression of recombinant protein was confirmed by SDS PAGE. The presence of capsid protein in the BL-21 (DE3) cells before and after induction with IPTG was analyzed. Protein band of 60 kDa (Fig. 2A) was observed in resultant supernatant after centrifugation of the sonicated cell suspension indicating GST-capsid protein was soluble. A 29 kDa GST protein band was observed in induced BL-21(DE3) cells but not in uninduced cells (Fig. 3A).

3.5. Titration of recombinant GST-capsid protein for IgM ELISA

3.2. Purification of recombinant GST-capsid protein

3.6. Specificity of recombinant GST-capsid protein based IgM ELISA

The recombinant GST-capsid protein was found in soluble form. So its purification was performed under native condition thus maintaining its functional conformation. The protein was allowed to bind with Glutathione beads for 30 min at room temperature and further eluted with reduced glutathione. SDS-PAGE gel analysis confirmed the presence of 60 kDa GST-capsid recombinant protein

To determine the optimal concentration of bacterially expressed recombinant GST-capsid protein for detection of CHIKV IgM by indirect ELISA, the protein was serially diluted from 10 ␮g/ml to 1 ␮g/ml. Confirmed positive and negative patient serum samples were then tested against different dilutions of recombinant GSTcapsid antigen. The highest ratio of absorbance value for positive to negative serum was obtained using 3 ␮g/ml of recombinant GSTcapsid protein (Fig. 5). This concentration of protein was therefore used in further experiments to evaluate the diagnostic potential for detection of CHIKV specific IgM antibody.

To check whether GST tag interfered with GST-capsid ELISA the plates were either coated with purified GST antigen or GST-capsid antigen and OD value was measured on treatment with 10 positive and 10 negative (Fig. 6) serum samples. The OD value of positive and negative sera was significantly different when tested on GST-capsid

Fig. 2. A. SDS PAGE analysis of GST-capsid fusion protein. Lane M: Prestained protein marker; Lane 1: uninduced E. coli BL-21(DE3) cells; lane 2: supernatant after sonication of E. coli cells. Lane 3: protein present in pellet. B. Lane M: Prestained protein marker; Lane 1: Purified GST-capsid protein.

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Fig. 3. (A) SDS PAGE analysis of GST protein. Lane M: Prestained Protein marker; Lane 1: uninduced BL-21(DE3) cells; Lane 2: supernatant after sonication of cells. (B) Lane M: Prestained marker; Lane 1: Purified GST protein.

Fig. 4. (A) Confirmation of purified GST-capsid protein by western blotting. Lane M: Prestained protein Marker; Reactivity of GST-capsid protein with Lane1: GST monoclonal antibody; Lane2: chikungunya positive patient sera; Lane 3: Chikungunya virus immunized rabbit sera. (B) Confirmation of purified GST protein with GST monoclonal antibody. Lane M: Prestained protein Marker; Lane1: Reactivity of GST protein with GST monoclonal antibody. Table 1 Tryptic peptides obtained from recombinant GST-capsid of chikungunya virus identified by MALDI-TOF/MS analysis in reflectron mode using HCCA as matrix. S.no.

Position

Measured mass

Theoretical mass

Mass error

Miss cleave

Peptide sequence

G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15 G16 G17 G18 G19 G20 G21 C1 C2 C3 C4 C5 C6 C7 C8 C9

65–73 65–73 1–9 1–9 28–35 104–113 19–27 109–119 1–11 126–136 126–136 120–131 126–136 126–136 114–125 90–103 90–103 74–87 88–103 88–103 198–218 1–12 13–36 51–62 55–62 123–133 142–151 178–200 253–261 238–247

1032.5024 1048.4710 1094.4869 1110.4439 1138.4537 1143.4557 1149.5007 1314.5775 1335.6977 1408.4989 1424.5773 1435.5624 1440.5700 1440.6215 1441.6178 1516.5722 1532.5517 1546.5646 1801.6230 1817.6137 2326.5995 1546.5992 2952.2993 1440.6610 923.4585 1125.4742 1017.5132 2720.4361 1031.4640 1118.5404

1031.5797 1047.5746 1093.5630 1109.5579 1137.5090 1142.6084 1148.6328 1313.6867 1334.7420 1407.6890 1423.6839 1434.7792 1439.6788 1439.6788 1440.7500 1515.7966 1531.7916 1545.7432 1800.9403 1816.9353 2325.1331 1545.7286 2951.6583 1439.8031 922.5348 11124.5536 1016.5138 2719.2316 1030.4607 1117.6132

−82 −106 −76 −109 −55 −140 −121 −88 −38 −140 −80 −156 −80 −44 −96 −153 −161 −120 −180 −181 197 −88 −124 −104 −90 −77 −7 72 −3 −71

0 0 0 0 0 1 0 1 1 1 1 1 1 1 1 0 0 1 1 1 0 0 1 1 0 0 0 0 0 0

K.LTQSMAIIR.Y K.LTQSMAIIR.Y .MSPILGYWK.I .MSPILGYWK K.YEEHLYER.D R.YGVSRIAYSK.D R.LLLEYLEEK.Y R.IAYSKDFETLK.V .MSPILGYWKIK.G K.LPEMLKMFEDR.L K.LPEMLKMFEDR.L K.VDFLSKLPEMLK.M K.LPEMLKMFEDR.L K.LPEMLKMFEDR.L K.DFETLKVDFLSK.L R.AEISMLEGAVLDIR.Y R.AEISMLEGAVLDIR.Y R.YIADKHNMLGGCPK.E K.ERAEISMLEGAVLDIR.Y K.ERAEISMLEGAVLDIR.Y K.YIAWPLQGWQATFGGGDHPPK .MEFIPTQTFYNR.R R.RYQPRPWTPRPTIQVIGPRPRPQR.Q K.LTMRAVPQQKPR.R R.AVPQQKPR.R K.VTGYACLVGDK.V K.GTIDNADLAK.L K.FTHEKPEGYYNWHHGAVQYSGGR.F K.ITPEGAEEW. R.TALSVVTWNK.D

20

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In house IgM ELISA

3.0

SD ELISA

OD value at 450 nm

2.5 2.0 1.5 1.0 0.5 0.0

Fig. 5. Optimization of GST-capsid protein concentration for In-house IgM ELISA. ELISA plate was coated with different concentration of antigens and evaluated for their reactivity with confirmed chikungunya positive or negative samples. Absorbance was read at 450 nm. The P/N ratio is OD value of positive sera/OD value of negative sera at 450 nm.

coated plate than that tested on GST coated plate. However, the OD of positive and negative sera was not significantly different when tested by GST antigen. Steric interference by fused GST tag was not observed, since the IgM ELISA by using purified native virus did not affect the result (data not shown). To check the cross reactivity, 10 serum samples from positive cases of dengue and 10 healthy sera were tested. None of these sera samples reacted with GSTcapsid antigen (Fig. 7), hence proving the specificity of recombinant GST-capsid protein in picking chikungunya specific IgM antibodies only. 3.7. Evaluation of in house ELISA using GST-capsid protein A total of ninety clinically suspected acute phase (3–10 days of fever) human patient serum samples from different outbreaks were tested with in house IgM ELISA based on recombinant GSTcapsid antigen. The cut-off value was set at twice the average value of the negative controls. The samples showing OD value ≥0.6 was considered as positive. Out of 90 samples, 50 samples were positive and 40 samples were negative for CHIKV IgM antibody (Fig. 7). The OD value for positive samples at 450 nm was in the range of 2.6–0.8. All the 90 sera samples mentioned above were further assessed with SD ELISA kit. While 48 samples were detected CHIKV IgM positive, 42 samples were detected CHIKV IgM negative (Fig. 7). In house GST-capsid based ELISA showed 100% sensitivity, 95% specificity, 96% PPV and 100% NPV with commercial SD kit. 4. Discussion

GST-CAPSIID

2

GST

1.5 1 0.5 0 1

2

3 4 5 6 7 8 9 Chikungunya positive samples

10

CHIK -

HEALTHY DENGUE CHIK+

CHIK -

Fig. 7. Detection of anti-CHIKV IgM antibodies in 90 suspected chikungunya patient sera by In house GST-capsid ELISA and by SD ELISA commercial kit. To check the cross reactivity, 10 healthy and 10 dengue positive sera were also tested by In house GST-capsid ELISA. Absorbance was read at 450 nm.

infected about 1.3 millions people across 13 states (Arankalle et al., 2007) causing the concern among public health and administrative authorities for the management of situation (Dash et al., 2007). As there is no antiviral therapy or vaccine available for CHIKV, a specific detection system is the need of hour. Several techniques for CHIKV detection e.g. IFT (Immunofluorescence technology), Plaque assay and virus isolation have been reported but the complexity of these techniques has replaced them by more convenient methods e.g. RT-PCR and ELISA. RT-PCR is highly specific and sensitive test for CHIKV (Edwards et al., 2007) but the reagents and equipment are too costly for widespread use. Serum IgM detection of CHIKV suspected patient is a useful tool for diagnostic as it can be detected as early as 4–5 days after the onset of fever and generally persists for several months. Earlier whole virus was being used as an antigen for detection of serum IgM but it was associated with biohazard risk. Handling of live virus requires BSL3 laboratory and expertise. Hence, these days specific protein produced by recombinant DNA technology (RDT) is being used for CHIKV diagnostic purpose instead of whole live virus. The utility of baculovirus expressed capsid protein and envelope proteins (E1 and E2) as an antigen for serodiagnosis of CHIKV have already been investigated (Cho et al., 2008a,b). However, there are certain drawbacks with the baculovirus system, especially the complicated procedure of protein purification, requirement of expensive culture media by insect cells and the fact that baculovirus kills the cells in few days, which make continuous production of the recombinant protein difficult. Hence this approach is expensive and tedious. In spite of presence of different expression systems, E. coli is most attractive choice for recombinant protein production due to low cost of production, well characterized genetics and cultivation conditions (Babaeipour et al., 2010; Huang et al., 2012; Nausch et al., Optical density at 450 nm

Optical density at 450 nm

Chikungunya virus is a mosquito born Alphavirus which has caused many outbreaks in different parts of world including Asia. Recently it has evolved to a new lineage that rapidly spreads to many parts of Africa, Asia and Europe. In India this outbreak

CHIK+

2

GST-CAPSID

GST

1.5 1 0.5 0 1

2 3 4 5 6 7 8 9 10 Chikungunya negative samples

Fig. 6. ELISA using GST as reference for evaluation of non specific binding. Twenty serum samples including 10 positive and 10 negative sera were used. Each serum sample was run in duplicate, both on GST-capsid antigen and GST antigen wells to check whether GST tag reacts with patient sera to produce false positive results.

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2013). However many proteins precipitate in insoluble inclusion bodies inside E. coli cytoplasm. Although refolding is one of the choices to obtain correctly folded protein but refolding cannot be applied for all the proteins and it also decreases the yield of protein. There are certain vectors e.g. pGEX and pET-41 that allow expression of recombinant protein in soluble form without interfering its biological activity. These vectors contain GST tag which acts as a chaperone to facilitate protein folding and allows easy purification of recombinant protein. This approach has been successfully used in the purification of diagnostically useful antigen of Dengue virus (Rodrıguez et al., 2012) and human cytomegalovirus (Vornhagen et al., 1994). So in an attempt to improve the solubility and simplify the means of production of capsid antigen this gene was cloned in frame with a GST (Glutathione S-transferase) tag in pET41a+ vector. The recombinant capsid protein was expressed with a GST tag at the C-terminal end in E. coli. The GST tag not only enhanced the stability of protein but also efficiently increased the solubility of protein. The expressed protein was present in the soluble phase hence it was purified under native condition in one step affinity chromatography on immobilized reduced glutathione. SDS PAGE analysis showed the protein was 90% pure and its concentration was about 8 mg/l of bacterial culture. Western blot assay showed that the recombinant GST-capsid protein reacted with chikungunya positive serum sample and CHIKV immunized rabbit sera and hence demonstrating its biological activity and also suggesting possible use in diagnostic assay. No reactivity was observed in case of healthy patient sera and CHIKV pre immunized rabbit sera. An in-house indirect ELISA using GST-capsid fusion protein as an antigen showed 97% accordance with the commercially available SD ELISA kit. No cross reactivity was observed in case of healthy sera and dengue patient sera. GST tag also did not cross react with patient sera to produce false positive result. Thus it is concluded that the present study for detection of chikungunya IgM antibody by indirect ELISA using capsid protein as an antigen is specific, rapid and easy to perform and therefore can be a useful tool for rapid surveillance of CHIKV suspected samples. The strategy adapted for production and purification of GST-capsid antigen is easy, less time consuming and can be easily adapted for other viruses. The most important advantage of this assay is that it can differentiate chikungunya infection from Dengue fever. Acknowledgements The authors are thankful to Prof (Dr). M. P. Kaushik, Director, DRDE for his keen interest in this study. Ms. Raj Priya also acknowledges the fellowship and the contingency grant received from CSIR, India. References Arankalle, V.A., Shrivastava, S., Cherian, S., Gunjikar, R.S., Walimbe, A.M., Jadhav, S.M., Sudeep, A.B., Mishra, A.C., 2007. Genetic divergence of Chikungunya viruses in India (1963–2006) with special reference to the 2005–2006 explosive epidemic. J. Gen. Virol. 7, 1967–1976. Babaeipour, V., Shojaosadati, S.A., Khalilzadeh, R., Maghsoudi, N., Farnoud, A.M., 2010. Enhancement of human gamma-interferon production in recombinant E. coli using batch cultivation. Appl. Biochem. Biotechnol. 160, 2366–2376. Blacksell, S.D., Tanganuchitcharnchai, A., Jarman, R.G., Gibbons, R.V., Paris, D.H., Bailey, M.S., Day, N.P., Premaratna, R., Lalloo, D.G., de Silva, H.J., 2011. Poor diagnostic accuracy of commercial antibody-based assays for the acute diagnosis of chikungunya infection. Clin. Vaccine Immunol. 18, 1773–1775. Brighton, S.W., Simson, I.W., 1984. A destructive arthropathy following Chikungunya virus arthritis – a possible association. Clin. Rheumatol. 3, 253–258. Carey, D.E., 1971. Chikungunya and dengue: a case of mistaken identity? J. Hist. Med. Allied Sci. 26, 243–262. Carletti, F., Bordi, L., Chiappini, R., Ippolito, G., Sciarrone, M.R., Capobianchi, M.R., Di Caro, A., Castillett, C., 2007. Rapid detection and quantification of Chikungunya virus by a one-step reverse transcription polymerase chain reaction real-time assay. Am. J. Trop. Med. Hyg. 77, 521–524.

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