Journal of Virological Methods 199 (2014) 86–94

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Development and characterization of monoclonal antibody against non-structural protein-2 of Chikungunya virus and its application Soma Chattopadhyay a,∗ , Abhishek Kumar a , Prabhudutta Mamidi a , Tapas Kumar Nayak b , Indrani Das a , Jagamohan Chhatai a , Itishree Basantray a , Umarani Bramha a , Prasanta Kumar Maiti c , Sujay Singh c , Amol Ratnakar Suryawanshi a , Subhasis Chattopadhyay b a

Institute of Life Sciences, Nalco Square, Bhubaneswar 751023, India School of Biological Sciences, National Institute of Science Education & Research, Bhubaneswar, India c Imgenex India Pvt. Ltd., Bhubaneswar, India b

a b s t r a c t Article history: Received 9 August 2013 Received in revised form 6 January 2014 Accepted 10 January 2014 Available online 21 January 2014 Keywords: Chikungunya virus nsP2 Monoclonal antibody

The recent epidemics of Chikungunya viruses (CHIKV) with unprecedented magnitude and unusual clinical severity have raised a great public health concern worldwide, especially due to unavailability of vaccine or specific therapy. This emphasizes the need to understand the biological processes of this virus in details. Although CHIKV associated research has been initiated, the availability of CHIKV specific reagents for in-depth investigation of viral infection and replication are scanty. For Alphavirus replication, non-structural protein 2 (nsP2) is known to play a key regulatory role among all other non-structural proteins. The current study describes the development and characterization of nsP2 specific monoclonal antibody (mAb) against a synthetic peptide of CHIKV. Reactivity and efficacy of this mAb have been demonstrated by ELISA, Western blot, Flow cytometry and Immunofluorescence assay. Time kinetic study confirms that this mAb is highly sensitive to CHIKV-nsP2 as this protein has been detected very early during viral replication in infected cells. Homology analysis of the selected epitope sequence reveals that it is conserved among all the CHIKV strains of different genotypes, while analysis with other Alphavirus sequences shows that none of them are 100% identical to the epitope sequence. Moreover, using the mAb, three isoforms of CHIKV-nsP2 have been detected in 2D blot analysis during infection in mammalian cells. Accordingly, it can be suggested that the mAb reported in this study can be a sensitive and specific tool for experimental investigations of CHIKV replication and infection. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Chikungunya fever is an acute mosquito-borne febrile arthritis caused by Chikungunya virus (CHIKV), an Alphavirus belonging to Togaviridae family (Brooks et al., 2004; Griffin, 2007; Strauss and Strauss, 1986). The disease is characterized by abrupt onset of high fever, myalgia, headache, rash (Thaikruea et al., 1997; Diallo et al., 1999; Powers et al., 2000; Pialoux et al., 2007) and polyarthralgia,which is very painful and may persist for several months in some cases (Enserink, 2007). This virus was first isolated in Tanzania, Africa in 1952 (Ross, 1956) and in the last decade, numerous incidences of CHIKV outbreak have been reported globally (AbuBakar et al., 2007; Arankalle et al., 2007; Pialoux et al., 2007; Santhosh et al., 2008; Volk et al., 2010). Phylogenetic analyses based on E1 gene sequences of CHIKV

∗ Corresponding author. Tel.: +91 674 2301676; fax: +91 674 2300728. E-mail address: [email protected] (S. Chattopadhyay). 0166-0934/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2014.01.008

grouped the viruses into three genotypes, Asian, East/Central/South African (ECSA) and West African (Powers et al., 2000; Schuffenecker et al., 2006). However, the recent outbreaks from 2004 onwards were found to be caused by ECSA type (Arankalle et al., 2007; Santhosh et al., 2008). The recent resurgence of CHIKV in India and in several other countries with unprecedented magnitude and unusual clinical manifestation raised a major public health concern worldwide. Chikungunya is a positive-stranded RNA virus with about 11.8 kb long genome which encodes four non-structural proteins (nsP1–4) and three major structural proteins (C, E1 and E2) (Strauss and Strauss, 1986; Brooks et al., 2004; Griffin, 2007). The non-structural proteins are the essential components of the viral replicase and transcriptase complex (Pastorino et al., 2008). The RNA synthesis of Alphaviruses occur in the cytoplasm, where the 5 two-thirds of the genomic RNA is translated into a large polyprotein of ∼2500 amino acids (aa). This polyprotein, termed P1234, is autoproteolytically cleaved to yield the four subunits of the non-structural proteins, nsP1–nsP4 (Kaariainen et al., 1987;

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Hwang et al., 1999). Polyprotein processing intermediates have distinct essential functions during the early phase of RNA replication and the synthesis of negative strand RNA (Lemm et al., 1994; Shirako and Strauss, 1994; Wang et al., 1994). In the late phase of infection, the negative strand RNAs are used as stable templates for the synthesis of progeny-positive strands and for the synthesis of sub-genomic mRNAs coding for the structural proteins of the virus. In Alphaviruses, nsP1 is responsible for methylation and capping of viral mRNAs (Laakkonen et al., 1994; Ahola and Kaariainen, 1995). The function of nsP3 is poorly characterized, but it is considered as an essential co-factor of nsP4 (Hahn et al., 1989; Salonen et al., 2003). The nsP4 protein is expressed at a very low level in the infected cells and contains the catalytic subunit of RNA dependent RNA polymerase (Barton et al., 1988). Non-structural protein-2 is the key replication protein with RNA helicase, NTPase and RNA triphosphatase activity and is an auto-protease responsible for cleavage of the non-structural polyprotein (Strauss and Strauss, 1994; Pastorino et al., 2008). In the Semliki forest virus (SFV) and Sindbis virus (SINV) replication, it has been shown that nsP2 is critically involved in shutting off the host transcriptional and translational machinery (Atasheva et al., 2007). Moreover, the highly expressed nsP2 is distributed widely in the cytoplasm as well as in the nuclei of the infected cells and its expression is found to be highly cytotoxic to the host cells (Atasheva et al., 2007; Bourai et al., 2012). Hence, the information regarding Alphavirus nsP2 indicates additional activities of this protein apart from replication and transcription of the sub genomic RNA during infection. This protein has been found to be one of the most important molecules in Alphavirus research; however, our understanding of the molecular mechanisms of its activities is still not very clear. Thus, its structure–function investigation is highly warranted. The most biologically relevant way of investigation of any nonstructural protein is to express it during infection and a specific antibody will serve as an important reagent to explore this in details. The present study describes the development, characterization and extensive evaluation of anti-CHIKV-nsP2 monoclonal antibody (mAb) using different biological techniques during in vitro CHIKV infection in host cell lines and also suggests its further potential usage in experimental investigations associated to the viral replication and infection. 2. Materials and methods 2.1. Materials 2.1.1. Animals BALB/c mice were used for generating the monoclonal antibody. All animal experiments were carried out at the Animal house facility in Imgenex India with prior approval by the Institutional Animal Ethics Committee (IAEC, Imgenex India, Bhubaneswar, India) following the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Govt. of India. 2.1.2. Cells, Viruses and antibodies Vero cells (African green monkey kidney fibroblast cell line), C6/36 (Aedes albopictus mosquito larva cell line), Chikungunya virus prototype African strain S 27 and Indian strain DRDE-06 (isolated in 2006) and anti-CHIKV E2 monoclonal antibody were gifted by Dr. M. M. Parida, DRDE (Defense Research and Development Establishment), Gwalior, India. RAW 267.4 cells (murine macrophage cell line) were obtained from National Centre for Cell Sciences, Pune, India. Mammalian cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; PAN Biotech, Aidenbach, Germany) supplemented with either 5% (for Vero) or 10% (for RAW 267.4)

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fetal bovine serum (FBS; PAN Biotech), Gentamycin and PenicillinStreptomycin (Sigma, New York, USA). C6/36 cells were maintained in Minimum Essential Medium (MEM; Sigma,) with 10% FBS. AntiGAPDH (Clone: IMG13H12) antibody was procured from Imgenex India, Bhubaneswar, India. Anti-Hsp70 mAb was obtained from BD Bioscience (New Jersey, USA). 2.2. Methods 2.2.1. Designing immunogenic epitope of CHIKV nsP2 Chikungunya nsP2 protein sequence was analyzed and the best immunogenic epitope was designed by using Protein Hydroplotter (Protein Lounge, San Diego, USA; www.proteinlounge.com). This tool essentially works on the principles of hydrophilicity and antigenicity (Hopp and Woods, 1981; Kyte and Doolittle, 1982) and determines the best epitope for immunogenic purpose. “ELVRAERTEHEYVYDVDQR” peptide sequence was selected and was synthesized by Imgenex Corporation (San Diego, USA). A cysteine (Cys, C) residue has been incorporated at the N-terminal of the peptide to facilitate the conjugation of the peptide to a carrier protein to make it immunogenic. The identified peptide sequence was subjected to BLAST analysis against the sequences of different CHIKV strains as well as other Alphaviruses deposited in GenBank in order to find out the sequence homology. 2.2.2. Conjugation to carrier protein The peptide was conjugated to keyhole limpet hemocyanin (KLH; Pierce, Rockford, USA) following the method published elsewhere (Bieniarz et al., 1996). In brief, KLH was activated by incubating it with a heterobifunctional crosslinker, sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate, Pierce). The peptide and the KLH-sulfo-SMCC were mixed at 1:1 ratio and were incubated for 2 h at room temperature (RT). The peptide conjugation to KLH was confirmed by Ellman’s reaction (Ellman, 1959). 2.2.3. Immunization of mice Six to seven week old female BALB/c mice were immunized intraperitoneally (i.p.) with 100 ␮g of KLH-conjugated peptide emulsified in complete Freund’s adjuvant (CFA; Sigma). After the first immunization, the mice were boosted (i.p.) twice in the interval of 14 days with 50 ␮g of conjugated peptide emulsified in incomplete Freund’s adjuvant (IFA; Sigma). Blood samples were collected seven days after the last immunization and the sera were subjected to ELISA. The mouse that showed the highest antibody titer by ELISA received another immunization (intravenous, i.v.) of 50 ␮g of conjugated peptide in PBS. 2.2.4. Detection of antibody by ELISA The 96-well microtiter plates were coated with 100 ng of free peptide by incubating overnight at 4 ◦ C. After blocking the plates with (2.5% skimmed milk) mouse serum (1:5000) was added to the wells and the plates were incubated for 1 h at RT. Preimmunization serum from the same mouse was used as negative control. Next, horseradish peroxidase-conjugated goat anti-mouse IgG (1:10,000, Jackson, West Grove, USA) was added and TMB (3,3 ,5,5 -Tetramethylbenzidine, Sigma) was used to detect the color development. The color intensity was measured at 450 nm in an ELISA Plate Reader. An OD value of 1.0 was considered as a positive threshold, while the OD value of pre-immunization control serum was less than 0.2. 2.2.5. Preparation of hybridomas Spleen cells were isolated from the mouse 5 days after the last immunization and were used to prepare hybridomas by the standard procedure as described elsewhere (Kohler and Milstein,

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1975; Maiti et al., 2003). In brief, spleen cells were fused with F0 myeloma cells at a ratio of 5:1 in polyethylene glycol (PEG; Sigma). Fused cells were grown in IMDM (Iscove’s Modified Dulbecco’s Media; Invitrogen, Carlsbad, USA) supplemented with HAT (hypoxanthine-aminopterin-thymidine medium; Sigma) and 20% FBS at 37 ◦ C with 5% CO2 . HAT medium was replaced by IMDM supplemented with 20% FBS, 2 weeks after the fusion. Positive hybridomas were cloned by the extensive limiting dilution method. After 8 weeks, positive hybridomas were transferred to IMDM supplemented with 10% FBS. Hybridoma culture supernatants were screened by ELISA for clone selection. Amongst the clones screened, 3F3.2E10 was selected and expanded. Isotyping of this clone was carried out using Rapid ELISA mouse mAb isotyping kit (Pierce) as per the manufacturer’s protocol. 2.2.6. Purification of anti-CHIKVnsP2 monoclonal antibody (mAb) The nsP2 mAb was purified from the hybridoma culture supernatant by protein G affinity chromatography column, Sepharose-protein G (GE Healthcare, Uppsala, Sweden) as per manufacturer’s protocol. The hybridoma supernatant was loaded on the column and after washing with PBS, the bound antibody was eluted with 0.1 M glycine buffer. The fractions were collected and analyzed at 280 nm. The fractions containing proteins were pooled, dialyzed and filter sterilized. 2.2.7. Chikungunya virus infection Vero and RAW 264.7 cells were maintained as mentioned above. Approximately, 0.5 × 106 cells were seeded in 6 well plates. Once 100% confluency was attained, cells were infected with S 27/DRDE06 strain of CHIKV with MOI 1 as described earlier (Dash et al., 2008). In brief, cells were incubated with virus at 37 ◦ C for 90 min with intermittent rocking at every 10 min interval. After three times washing with PBS, maintenance media was added to the cells and infection was allowed to proceed at 37 ◦ C. Samples were collected at different time post infection according to the assay. Serum free media without the virus was used for mock infection. 2.2.8. Western blot Protein expression was examined by Western blot analysis according to the procedure described earlier (Chattopadhyay and Weller, 2006). In brief, cells were harvested at different hours post infection (hpi) and lysed with RIPA buffer containing 300 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% Sodium deoxycholate and 50 mM Tris (pH 8.0). Equal amount of protein (40 ␮g) was separated on 10% SDS-polyacrylamide gel and was transferred onto PVDF membrane. Protein expression was checked with anti-nsP2 mAb and to confirm the equal load of samples, the same membrane was stripped and re-probed with anti-GAPDH antibody (Imgenex India) for Vero and RAW cells and anti-Hsp 70 (BD Bioscience) antibody for C6/36 cells. 2.2.9. Flow cytometry CHIKV infected and mock Vero cells were subjected to Flow cytometric analysis at 18 hpi. Intracellular staining was performed by using the IC-Flow Kit (Imgenex Corp, San Diego, USA) according to the manufacturer’s instructions. Vero cells were incubated with anti-CHIKV-nsP2 mAb followed by Alexa Fluor (AF) 488 conjugated chicken anti-mouse antibody (Invitrogen). Around ten thousand cells were acquired by FACS CaliburTM flow cytometer (BD Biosciences) for each sample and analyzed by CellQuest Pro software (BD Biosciences). Statistical analysis was performed by using Student’s t test. p < 0.05 was considered as statistically significant. 2.2.10. Immunofluorescence assay Vero cells were grown on glass coverslips and infected as described above. At 12 hpi, coverslips were washed twice with cold

PBS. The cells were fixed with freshly prepared 4% paraformaldehyde in PBS (pH 7.4), for 30 min at RT. The cover slips were washed three times with PBS and then permeabilized with 0.5% Triton X100 in PBS for 5 min. After three washes, cells were blocked with 3% Bovine serum albumin (BSA; Sigma) with 10 mM glycine (Blocking solution, Sigma) in PBS overnight at RT. Cells were incubated with anti-nsP2 mAb for 1 h at RT. After washing, cells were incubated with AF 488 conjugated anti-mouse antibody (1:15,000) for 45 min. Then, coverslips were mounted with antifade (Invitrogen) to reduce photobleaching. Fluorescence microscopic images were acquired using Leica TCS SP5 confocal microscope (Leica Microsystems, Heidelberg, Germany) using 20× objectives and images were captured and analyzed using Leica Application Suite Advanced Fluorescence (LASAF) V.1.8.1 software. 2.2.11. 2D-Western blot 2D electrophoresis and Western blot analyses were performed using the protocol described earlier (Suryawanshi et al., 2011) with minor modification. In brief, mock and infected Vero cells were harvested at 18 hpi and lysed overnight at 4 ◦ C using 2D lysis buffer. The protein samples were quantitated using 2D Quant Kit (GE Healthcare). In 2D electrophoresis, 3-11NL, 18 cm immobilized pH gradient (IPG) strips were passively rehydrated with 250 ␮g protein samples and then subjected to active rehydration at 50 V for 10 h followed by prefocusing at 250 V for 1 h, 500 V for 1 h, 1000 V for 1 h, then 2500 V for 1 h and finally isoelectric focusing at gradient 8000 V for 45,000 V h and 8000 V for 30 min using Ettan IPGphor3 IEF system (GE Healthcare). The proteins on the strips were reduced using 65 mmol/L DTT in 10 ml equilibration buffer [50 mmol/L Tris–HCl pH 8.8, 6 mol/L urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.005% (w/v) bromophenol blue] for 20 min and subsequently alkylated in 135 mmol/l iodoacetamide in 10 ml equilibration buffer for 30 min to prevent reoxidation of the proteins. These strips were subjected to seconddimension 10% SDS-PAGE. Gels were either stained using the silver PlusOne staining kit (GE Healthcare) or transferred on to PVDF membrane. Triplicate gels were run for each sample to confirm reproducibility. Western blot was performed as described above. Further, these blot images were analyzed to get the approximate molecular weight (MW) and isoelectric point (pI) of the identified protein spots. 3. Results 3.1. Generation and evaluation of anti-CHIKV nsP2 mAb 3.1.1. Epitope designing The 19-mer long amino acid (aa) sequence, “ELVRAERTEHEYVYDVDQR” was selected for raising mAb as the hydrophilicity, hydrophobicity and antigenicity indexes are 0.557, −1.129 and 1.078, respectively. Fig. 1A is the schematic representation showing the position of the peptide from 656 to 674 aa of the non-structural polyprotein of CHIKV and Table 1 shows the homology of this peptide sequence with several other CHIKV nsP2 sequences randomly chosen from the GenBank database representing different genotypes of this virus isolated worldwide. The result shows that the peptide is fully conserved among all the genotypes of CHIKV strains as all strains have 100% sequence identity (Table 1). In order to find out the specificity of this epitope, a similar analysis was carried out with the non-structural polyprotein sequences of representative Alphaviruses. It was observed that none of the other Alphavirus sequences is 100% identical and maximum identity, i.e. 84.21% is observed with Igbo-Ora, and O’nyong-nyong viruses (Table 2). However, sequence identities with other Alphaviruses range from 68.42% to 31.58% (Table 2). This indicates that the mAb

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Table 1 Homology analysis of the peptide “ELVRAERTEHEYVYDVDQR” with non-structural polyprotein sequences of various CHIKV strains from all over the world isolated at different period of time. Sl. no.

Accession no.

Chikungunya virus strain

Place of isolation

Year of isolation

Genotype of CHIKV strain

Maximum identity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 36 37 38 39

ADG95931.1 AAN05101.1 ADG95929.1 ADG95935.1 ADG95927.1 ADG95884.1 ABN04201.1 ADG95937.1 ADG95933.1 AAU43880.1 ADG95905.1 ABN04197.1 CAJ90474.1 ABD95937.2 ABJ98543.1 ABN04187.1 ABN04189.1 ABN04191.1 ABN04193.1 ABN04195.1 ABP88821.1 ABU93704.1 ACD93567.1 ACD93607.1 ACD93609.1 ACY25939.1 ACY66840.1 BAH97930.1 ACY25937.1 ACY25941.1 ADG95912.1 ABX38963.1 ACY25943.1 ACY25945.1 ACY66844.1 ACY66846.1 ADJ19189.1 ADJ19191.1

Tanzania Ross low-psg 1953 S27 (African prototype) Thailand TH35 1958 India Gibbs263 1963 Congo LSFS 1960 Nigeria IbH35 1964 India MH5 1973 Thailand 1455 1975 Uganda Ag4155 1982 Senegal 37997 1983 Indonesia RSU1 1985 IND-00-MH4 (Yawat) La Reunion 06-021 2005 LR2006 OPY1 (Re-Union) D570/06 (Mauritius) IND-06-AP3 IND-06-KA15 IND-06-MH2 IND-06-RJ1 India TN1 2006 DRDE-06 Wuerzburg (Mauritius) MY002IMR/06/BP TM25 (Mauritius) CHIK31 (Rajasthan) KL06 Singapore 0611aTw 2006 Japan SL11131 2006 KL06, RGCB03 KL07 Sri-lanka CK1 2007 ITA07-RA1(Long Varient) KL07 KL08 Bangladesh 0810aTw 2008 Malaysia 0810bTw 2008 Thailand CU10 2008 Thailand CU683 2009

Tanzania Tanzania Thailand India Congo Nigeria India Thailand Uganda Senegal Indonesia India Reunion Island Reunion Island Mauritius India India India India India India Germany Malaysia Mauritius India India Singapore Japan India India Sri Lanka Italy India India Bangladesh Malaysia Thailand Thailand

1953 1954 1958 1963 1960 1964 1973 1975 1982 1983 1985 2000 2005 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2007 2007 2007 2007 2007 2008 2008 2008 2009

ECSA ECSA Asian Asian ECSA West African Asian Asian ECSA West African Asian ECSA ECSA ECSA ECSA ECSA ECSA ECSA ECSA ECSA ECSA ECSA Asian ECSA ECSA ECSA ECSA ECSA ECSA ECSA ECSA ECSA ECSA ECSA ECSA ECSA ECSA ECSA

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

Table 2 Homology analysis of different Alphavirus non-structural polyproteins (nsPs) with the CHIKV-nsP2-mAb peptide sequence “ELVRAERTEHEYVYDVDQR”. Dot (.) represents the identical amino acid.

Position of Amino Acid

mAb peptide sequence Accession no. and name of the Alphavirus member AAC97206.1|Igbo-Ora virus AAC97204.1|O’nyong-nyong virus AAM64226.1Semliki Forest Virus CAB62256.1|Semliki Forest Virus AAB40701.1|Barmah Forest Virus AAA86133.1|Sindbis-like virus AAA96972.1|Ockelbo virus ACT68008.1|Fort Morgan virus ACT32134.1|Highlands J virus CAA52868.2|WEEV AAM10974.1|Sindbis virus AFJ15604.1|Salmonid alphavirus subtype 3 CAC87660.1|Sleeping disease virus AAC53734.1|EEEV ABB45867.1|EEEV ABB45865.1|EEEV AAU89533.1|VEEV

6 8 5 E

6 8 6 L

6 8 7 V

. . . . . . . K. . K. . K. KVT KVT KVI KV. KT . KI T K. . K. . KV. KVL KVL KT .

6 8 8 R

. . . . P K K . K K K D D K . . K

6 8 9 A

V V . . V . . S S T . . . S S S P

6 9 0 E

. . . . . . . S H Q A A A T G S S

6 9 1 R

K K . . . L L E E D L K K E E E E

6 9 2 T

. . . . A A A A A . A A A . A A H

6 9 3 E

6 9 4 H

. . . . DA DA . S . T . T . S . S DS . T KD KD DS . S DS DG

6 9 5 E

6 9 6 Y

. . . . . . . . D. . . . . D. . . . . . F . . . . . . . . . . . .

6 6 9 9 7 8 VY

. . . . . . . . . . . . . . . . L

. . F F F F F F F F F . . F F F .

67 90 90 DV

. . . . . . . . . . . . . . . I . I . I . . EL EL . I . I . I . I

77 00 12 DQ

. . . . . . . . . . . S S . . . .

. . K K . K K . A A K S S A A A R

7 0 3 R

K K K K K K K . . . . K K K K . K

Percent Identity 84.21 84.21 68.42 68.42 57.90 47.37 47.37 42.10 42.10 42.10 42.10 36.90 36.90 36.90 36.90 36.90 31.58

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Fig. 1. Generation and evaluation of anti-CHIKV nsP2 mAb. (A) Schematic representation of the non-structural polyprotein of the Chikungunya virus. All four gradient bars indicate different non-structural proteins nsP1 (1–535), nsP2 (536–1333), nsP3 (1334–1863), and nsP4 (1864–2474). Black bar (from amino acid 656-674, 19 mer) in the N-terminal part of the nsP2 protein indicates the peptide sequence that was used for monoclonal antibody generation. (B) Isotyping of 3F3.2E10 clones by using the Pierce Rapid ELISA mouse mAb isotyping kit. (C) Detection of the reactivity of anti-nsP2 mAb clone 3F3.2E10 hybridoma cell with the peptide by ELISA. Data are representative of three independent experiments. (D) Detection of nsP2 in CHIKV S 27 strain infected Vero cell lysate in Western blot using anti-nsP2. GAPDH was used as a loading control.

may not cross-react with other Alphaviruses which needs experimental support. 3.1.2. mAb generation and characterization The anti-CHIKV-nsP2 mAb was developed by conventional hybridoma technology. The hybridoma clones were screened using ELISA against the peptide, subcloned and finally 3F3.2E10 clone was selected based on its consistent higher ELISA titer. The isotype of heavy and light chains of the selected antibody was found to be IgG2b and , respectively (Fig. 1B). This clone was expanded and the resulting supernatant was purified for further use. Fig. 1C shows the reactivity of the mAb (OD value 2.204 ± 0.04) against the immunizing peptide in ELISA. This experiment was repeated independently more than three times and the result suggests that the purified mAb binds very strongly with the CHIKV peptide as compared to control.

found that 7.75 ± 1.89% CHIKV infected Vero cells showed the presence of the nsP2 protein as compared to mock (0.57 ± 0.15, p < 0.05) (Fig. 2A and B). Moreover, in terms of mean fluorescence intensity (MFI) of nsP2 expression, a positive shift was observed in

3.1.3. Evaluation of the mAb The specificity of the mAb was determined by Western blot using S 27 virus infected Vero cell lysate (18 hpi). The purified mAb showed strong reactivity only with the CHIKV infected cell lysate and the reacted band was found to be around 90 kDa which is the predicted size of nsP2 of CHIKV (Fig. 1D). Only one band was observed in the infected sample even though the whole cell lysate had all host proteins as well as viral proteins. Moreover, the mock cell lysate did not show any reactivity as shown in Fig. 1D. GAPDH was used as loading control. Thus, the result confirms that the newly developed mAb using synthetic peptide is able to recognize only the CHIKV-nsP2 protein in virus infected cell lysate and does not show any cross-reactivity. 3.2. Application of anti-CHIKV nsP2 in flow cytometry and immunofluorescence assay The specific reactivity of the mAb was demonstrated by Flow cytometric analysis. The uninfected mock and CHIKV infected Vero cells were acquired and analyzed in terms of dot-plot analysis for percent positive cells of nsP2 at 16–18 hpi (Fig. 2). It was

Fig. 2. Detection of nsP2 in CHIKV (S 27) infected Vero cell line by flow cytometry. (A) Dot–plot analysis showing percent positive cells for nsP2 at 16–18 hpi. Right panel of the dot-plot shows nsP2 positive cells during CHIKV infection and left panel shows the mock infected cells. (B) Graphical representation of flow cytometric analysis of nsP2 percent positive cells (n = 3). Experimental data are presented as mean ± SEM. * p < 0.05. (C) Mean fluorescence intensity (MFI) of nsP2 positive cells with respect to mock. Filled and open histogram shows nsP2 expression for mock and CHIKV infected Vero cells respectively. Data are representative of three independent experiments.

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Fig. 3. Detection of nsP2 in CHIKV infected Vero cells by immunofluorescence assay. Mock and CHIKV infected cells are stained with CHIKV nsP2 monoclonal (A1, A2, A3, B1, B2, B3), CHIKV-E2 monoclonal (C1, C2, C3, D1, D2, D3) followed by staining with secondary antibody anti-mouse Alexa Fluor 488. Mock cells stained with nsP2 or E2 mAb were used as negative control.

CHIKV infected cells (3.38 ± 0.46) as compared to mock (2.21 ± 0.13, p < 0.05). Together, it shows that the anti-CHIKV nsP2 mAb can be used to determine CHIKV nsP2 expression and its frequency level, i.e. % positive cells during CHIKV replication. The non-structural protein nsP2 is synthesized during replication of the virus life cycle inside the host cell. Hence, the result indicates that viruses in only

10% healthy cells are in replication phase at 18 hpi, while the majority of the viruses inside the cells are probably in packaging or egress phase. Similarly, the virus infected Vero cells were subjected to immunofluorescence assay for the detection of the CHIKV-nsP2 protein using the newly generated mAb. It was observed that infected

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were found to be 90 kDa while the pI values of these spots were 9, 9.2, and 9.6 approximately, indicating the existence of 3 isoforms of the nsP2 protein in S 27 infected Vero cells. This result also confirms the specificity of the mAb to CHIKV-nsP2 only and no cross-reactivity to other proteins.

4. Discussion

Fig. 4. Sensitivity of CHIKV-nsP2 monoclonal antibody. (A) Detection of CHIKV nsP2 in S 27 virus infected different cell lines (Vero, C6/36 and RAW cells). Cells were infected with CHIKV virus with MOI 1 and harvested at 8 hpi for Vero and 24 hpi for C6/36 and RAW cells. Equal amount of protein was separated in 10% SDS-PAGE. After transfer, PVDF membranes were processed with CHIKV-nsP2 mAb. GAPDH/Hsp70 was used as loading control. (B) Detection of CHIKV nsP2 in Vero cell lines infected with S 27 and DRDE-06 strain of virus with MOI 2. Cells were harvested at 0, 1, 2, 3, 4, 5, 6 and 7 hpi and analyzed by Western blot analysis using nsP2 mAb.

cells were detected by the mAb and nsP2 protein was localized in the cytoplasm as well as in the nucleus of the infected cells (Fig. 3, B1–B3). However, the mock cells did not show any staining. The data indicate strong reactivity and specificity of 3F3.2E10 clone against the CHIKV-nsP2 protein. In parallel, cells were stained with CHIKV-E2 mAb for the confirmation of viral infection (Fig. 3, D1–D3) and this showed cytoplasmic localization of the E2 protein (Kumar et al., 2012). Taken together, the result suggests that the newly developed anti-nsP2 mAb can be a useful tool to study CHIKV-nsP2 in various immunofluorescence assays. 3.3. Specificity and sensitivity of the nsP2 mAb Further to check the specificity of this mAb in different CHIKV strains and different cell lines, another CHIKV strain, DRDE-06 was included in this study. In addition to Vero cells, mouse macrophage cell lines (RAW 267.4) and mosquito larva cell lines (C6/36) were infected with S 27 strain of CHIKV. Infected cell lysates were subjected to Western blot analysis using this mAb. Fig. 4A shows that the nsP2 mAb detected only the 90 kDa nsP2 protein of CHIKV virus in different cell lines, while no reactivity was seen in mock cells. As a loading control, GAPDH was used for Vero and RAW and Hsp70 was used for C6/36 cell lines (Fig. 4A). To investigate the sensitivity of the mAb, Vero cells were infected with MOI 2 of S 27 and DRDE-06 separately, harvested at every hour up to 7 hpi and cell lysates were used for Western blot analysis using the CHIKV-nsP2 mAb. Fig. 4B shows that nsP2 was detected by this anti-CHIKV-nsP2 mAb as early as 0 hpi (for DRDE-06) and from 1 hpi for S 27 and nsP2 expression level increased gradually in case of both the viral strains. GAPDH was used as loading control. Hence, the result shows that the mAb is very specific and sensitive; and this can be applied to nsP2 study in a virus infection model using different strains of CHIKV in different cell lines. 3.4. Detection of multiple nsP2 isoforms during CHIKV infection In order to determine the molecular weight (MW) and isoelectric pH (pI) of nsP2 protein, 2D Western blot was performed using virus infected and mock cell lysates. 2D blot showed 3 protein spots in CHIKV infected samples as shown in Fig. 5B. No reactivity was noted in mock sample (Fig. 5A). The MWs of all three protein spots

In the present study, the monoclonal antibody against the nsP2 protein of CHIKV has been developed, characterized and evaluated as a tool for experimental study using different techniques like Western blot, Flow cytometry and Immunofluorescence assay. In addition, a time kinetic study with virus infected cell lysate and homology analyses with CHIKV and other Alphavirus sequences suggest that this mAb may be a sensitive and specific tool for CHIKVnsP2. Moreover, using this mAb, three isoforms of CHIKV-nsP2 has been detected in 2D blot analysis during viral infection in mammalian cells. The resurgence of CHIKV in the form of an epidemic with unusual clinical symptoms and high fatality rates in India and in different parts of the world has created an immense public health concern globally. The unavailability of the vaccine or any antiviral drug for this virus is one of the reasons for the severity of the recent explosive epidemics which urge a better understanding of the whole viral replication process in details so that proper control strategies can be developed in future. The CHIKV associated experimental and clinical research has got momentum in recent years after several outbreaks in the last decade, however, the availability of CHIKV specific reagents for in-depth investigation of viral infection and replication are scanty. In the field of Alphavirus research, very few reagents are available to study the nsP2 protein in details. In one of the studies, it has been reported that co-injection of the CHIKV-pnsP2 adjuvant with pEnv qualitatively and quantitatively increased antigen specific neutralizing antibody responses in comparison to pEnv alone (Bao et al., 2013). The His antibody was used in this study to detect nsP2 expression in transfected cells. In another study, Atasheva et al., used a recombinant Sindbis virus that could express enhanced green fluorescence protein (EGFP)-tagged nsP2 and identified the host cell factors co-isolated with nsP2 from the virus infected cells (Atasheva et al., 2007). Similar reagent was used for understanding the role of nsP2 in down regulating cellular transcriptional machinery (Garmashova et al., 2006; Frolov et al., 2009). However, a polyclonal antibody against nsP2 of Semliki Forest virus was used for studying the importance of the nuclear localization signal and it was observed that mutations in this region influenced RNA synthesis, protein expression and cytotoxicity of this virus (Tamm et al., 2008). Accordingly, it appears that till date the anti-CHIKVnsP2-mAb is not available to investigate the molecular mechanism related to multiple roles of this highly important viral protein during viral replication and infection processes. The most biologically relevant way of studying this protein is to express it in the context of replicating virus during infection. For detection of nsP2, a short or long sequence can be tagged in nsP2 clone, but this may make the protein less functional or non-functional. In addition, this approach is not suitable for nsP2 study in biologically relevant infection model and also limits the use of natural virus isolates collected from patients containing different mutations. Hence, lack of appropriate reagent is a major constrain for investigating the role of nsP2 in CHIKV infection. In the present study, the mAb has been developed against a linear epitope in the N-terminal part of CHIKV-nsP2. This epitope comprises of the 19 amino acids, “ELVRAERTEHEYVYDVDQR” corresponding to the position 656–674 of CHIKV non-structural polyprotein. This epitope is shared by all CHIKV strains of different

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Fig. 5. Detection of multiple isoforms of CHIKV nsP2 during infection in Vero cells. 2D Western blot analysis was performed after harvesting the mock and S 27 virus infected Vero cells at 16 hpi. In 2D electrophoresis, 3-11NL, 18 cm IPG strips were used to separate 250 ␮g protein samples. Protein samples were analyzed in 10% SDS-PAGE followed by Western blot. (A) Mock Vero cell proteome. (B) S 27 infected Vero cell proteome. Three spots reacted with CHIKV-nsP2 mAb are marked as 1, 2 and 3.

genotypes indicating that this epitope is well conserved among CHIKV strains. However, homology analyses with other Alphaviruses revealed that it does not show complete identity with any other Alphavirus sequences, however this mAb may cross-react with Igbo-Ora and O’nyong-nyong viruses as the epitope is showing 84.21% identity. The epitope sequence is quite different as compared to other Alphaviruses which may predict that the CHIKV-nsP2 mAb may be a specific reagent for this virus. In addition, nsP2 was detected in virus infected cells as early as 0 hpi (just after 1.5 h adsorption) in case of DRDE-06 and from 1 hpi in case of S 27, used in this study, suggesting that the nsP2 mAb may work as a sensitive and universal detection tool for nsP2 in different strains of CHIKV. Further, it has been demonstrated that the nsP2 mAb, although developed against synthetic peptide, recognizes CHIKV-nsP2 in denatured form in Western blot, as well as in native form in Flow cytometric analysis and Immunofluorescence assay. During Western blot analysis only one band was recognised by this mAb in the whole cell lysate containing entire host as well as viral proteins. Further, this protein band was excised from silver stained gel and subjected to Mass spectrometry analysis which confirms the identity of this band as CHIKV-nsP2 (data not shown). This indicates that the mAb does not cross-react with any other host and viral proteins. 2D blot analysis showed the presence of three isoforms of CHIKV-nsP2 during infection in mammalian cells which indicates that nsP2 may undergo post translational modifications during infection in mammalian host cells. However, further investigation is required to decipher their identity or modifications and understand their functional significance in CHIKV replication. Since CHIKV-nsP2 can be detected in host cells during viral replication, the anti-CHIKV-nsP2 mAb can be useful in understanding the stages of viral replication or infection, correlating morphological changes of the infected cells with viral protein expression and sub-cellular localization of nsP2 in different compartments in host cells. This can also be used in comparing the replication efficiency or infection patterns of different CHIKV strains containing different mutations. In addition, this mAb can be used to investigate viral replication and infection associated with altered host cell responses across the species. Moreover, its efficacy towards neutralizing CHIKV-nsP2 during replication in hosts and/or to induce host protective immunity, if any, may be explored in future. In conclusion, our results suggest that the mAb developed in this study will be a sensitive and specific tool for detection of the nsP2

protein during CHIKV infection, which further ensures its potential usage for experimental investigations of CHIKV research associated to viral replication and infection. Acknowledgements We thank Dr. M.M. Parida for kindly providing the virus strains (S 27 and DRDE-06), CHIKV polyclonal antibody and Vero cell line. We also wish to thank Mr. B.S. Sahoo for his technical assistance in confocal imaging using LASAF software and Ms. S. Muduli for her technical assistance in carrying out confocal experiment. This work was supported by the Department of Biotechnology, Ministry of Science and Technology, Govt. of India vide grant No. BT/PR13118/GBD/27/186/2009 and BT/PR15173/GBD/27/356/2011 and by Council of Scientific and Industrial Research (CSIR), Ministry of Science and Technology, Govt. of India vide Project No. 37 (1542)/12/EMR-II). References AbuBakar, S., Sam, I.C., Wong, P.F., MatRahim, N., Hooi, P.S., Roslan, N., 2007. Reemergence of endemic Chikungunya, Malaysia. Emerg. Infect. Dis. 13, 147–149. Ahola, T., Kaariainen, L., 1995. Reaction in alphavirus mRNA capping: formation of a covalent complex of nonstructural protein nsP1 with 7-methyl-GMP. Proc Natl. Acad. Sci. U.S.A. 92, 507–511. 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. 88, 1967–1976. Atasheva, S., Gorchakov, R., English, R., Frolov, I., Frolova, E., 2007. Development of Sindbis viruses encoding nsP2/GFP chimeric proteins and their application for studying nsP2 functioning. J. Virol. 81, 5046–5057. Bao, H., Ramanathan, A.A., Kawalakar, O., Sundaram, S.G., Tingey, C., Bian, C.B., Muruganandam, N., Vijayachari, P., Sardesai, N.Y., Weiner, D.B., Ugen, K.E., Muthumani, K., 2013. Nonstructural protein 2 (nsP2) of Chikungunya virus (CHIKV) enhances protective immunity mediated by a CHIKV envelope protein expressing DNA vaccine. Viral Immunol. 26, 75–83. Barton, D.J., Sawicki, S.G., Sawicki, D.L., 1988. Demonstration in vitro of temperaturesensitive elongation of RNA in Sindbis virus mutant ts6. J. Virol. 62, 3597–3602. Bieniarz, C., Husain, M., Barnes, G., King, C.A., Welch, C.J., 1996. Extended length heterobifunctional coupling agents for protein conjugations. Bioconjug. Chem. 7, 88–95. Bourai, M., Lucas-Hourani, M., Gad, H.H., Drosten, C., Jacob, Y., Tafforeau, L., Cassonnet, P., Jones, L.M., Judith, D., Couderc, T., Lecuit, M., Andre, P., Kummerer, B.M., Lotteau, V., Despres, P., Tangy, F., Vidalain, P.O., 2012. Mapping of Chikungunya virus interactions with host proteins identified nsP2 as a highly connected viral component. J. Virol. 86, 3121–3134. Brooks, G.F., Butel, J.S., Morse, S.A., 2004. Human arbovirus infections. In: Jawetz, M., Adelberg (Eds.), Medical Microbiology. Mc Graw Hill, Singapore, pp. 514–524.

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