Expression of P32 protein of goatpox virus in Pichia pastoris and its potential use as a diagnostic antigen in ELISA

Journal of Virological Methods 162 (2009) 251–257

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Expression of P32 protein of goatpox virus in Pichia pastoris and its potential use as a diagnostic antigen in ELISA V. Bhanot, V. Balamurugan, V. Bhanuprakash, G. Venkatesan, A. Sen, V. Yadav, R. Yogisharadhya, R.K. Singh ∗ Division of Virology, Indian Veterinary Research Institute, Mukteswar, Nainital District, Uttarakhand 263138, India

a b s t r a c t Article history: Received 3 June 2009 Received in revised form 21 August 2009 Accepted 27 August 2009 Available online 3 September 2009 Keywords: GTPV P32 protein Pichia pastoris ELISA Serodiagnosis Capripox infections

The present study was undertaken to express goatpox virus (GTPV) P32 protein in Pichia pastoris and evaluate its potential use as a diagnostic antigen in ELISA. The amplified P32 gene of GTPV was cloned into pPICZ␣A vector and characterized by PCR, restriction enzyme digestion and sequencing. The characterized linear recombinant plasmids were transformed in Pichia host GSII5 strain by electroporation and the zeocin resistant Pichia transformant containing P32 gene was selected and confirmed by PCR. The expression of P32 protein in Pichia was induced with 0.5% methanol at 30 ◦ C. The optimum expression was observed at 72 h post-induction and the yield was 100 mg/L of culture. The expressed protein was precipitated with polyethylene glycol and analyzed by SDS-PAGE and Western blot using GTPV specific serum and GTPV-P32 protein specific monoclonal antibody. Further, the protein precipitated with acetone was evaluated as diagnostic antigen in indirect ELISA in order to replace the whole GTPV. The standardized P32 protein based indirect ELISA had relative specificity and sensitivity of 84.2% and 94.2–100%, respectively when compared with serum neutralization test and whole virus based indirect ELISA. This study showed a potential of the yeast expressed GTPV-P32 protein as safe antigen in ELISA for seroepidemiological study of the capripox infection in sheep and goats, in India as well as capripox enzootic countries. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Goatpox and sheeppox (collectively known as capripox diseases) are the most important World Organization for Animal Health notifiable viral diseases that pose serious socio-economic impact to small ruminant productivity in terms of morbidity, mortality, hide damage, and trade restrictions. Capripox is enzootic in Africa, north of the Equator, the Middle East, Turkey, Iran, Afghanistan, Pakistan, India, Nepal, parts of the People’s Republic of China and Bangladesh (Tulman et al., 2002). Fever, mucopurulent nasal discharge, generalized pocks, and high mortality are the important clinical symptoms of these diseases. The causative agents, goatpox virus (GTPV), and sheeppox virus (SPPV), are enveloped dsDNA viruses; belong to the genus Capripoxvirus of the subfamily Chordopoxvirinae in the family Poxviridae (Van Regenmortel et al., 2000). Genomes of capripoxviruses are approximately 150 kbp and are similar to each other. Viral genome contains a central coding region flanked by

∗ Corresponding author. Present address: National Research Centre on Equines, Sirsa Road, Hisar 125001, Haryana, India. Tel.: +91 01662 275787; fax: +91 01662 276217. E-mail address: rks [email protected] (R.K. Singh). 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.08.020

two identical inverted terminal repeats (ITR), a feature unique to pox viruses. Members of the genus are closely related, with genomic identities ranging from 96% (among species) to 99% (among isolates of the same species) (Tulman et al., 2001, 2002). Capripoxviruses tend to be host specific; however, there are incidences of viruses crossing species barrier between goats and sheep (Bhanuprakash et al., 2006b). Goatpox is an important viral disease of goats, which is enzootic in India for several decades. In a recent study, the losses due to capripox in Maharashtra state alone are estimated INR 100 million with average morbidity and mortality of 63.5% and 49.5%, respectively (Garner et al., 2000). Extrapolating this data, the total estimated annual loss at the national level would be INR 1250 millions. Countries that are free from capripox diseases enjoy a significant advantage in the export market of meat products. Control of the disease is vital to improve the small ruminant productivity in the subcontinent. In India, socio-economic factors preclude application of test and slaughter policy for disease control and the movement of animals is difficult to monitor. Therefore, disease surveillance and vaccination are the best alternatives to control the prevalence of disease and its eradication. The diagnosis of goatpox is based on clinical signs and serological tests such as agar gel immunodiffusion (AGID) (Pandy and Singh, 1972), counter immunoelectrophoresis (CIE) (Sharma et al., 1988a),

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spot agglutination (Tiwari et al., 1996), reverse phase haemagglutination (RPHA) (Rao and Negi, 1997), latex agglutination (Rao et al., 1996) and enzyme linked immuno-sorbent assay (ELISA) (Sharma et al., 1988b) and molecular diagnostic techniques such as polymerase chain reaction (PCR) (Balamurugan et al., 2009) and restriction fragment length polymorphism (RFLP) (Hosamani et al., 2004a). However in India, CIE is used commonly for goatpox because of its relative simplicity and rapidity. Laboratory confirmation depends on isolation and identification of GTPV, virus neutralization test, or immunofluorescence. ELISA is employed commonly for seroepidemiological studies, as it is rapid, inexpensive, sensitive and useful for mass screening. Nevertheless, the diagnosis of goatpox by classical virological or serological techniques depends on live virus, which is not always safe as there is a risk in the live virus handling and accidental escape into the environment. Thus a recombinant antigen based diagnostic assay would be a better alternative for disease surveillance. P32 is one of the structural proteins present in all the capripoxviruses and contains major immunogenic determinants (Chand, 1992). Several approaches have been used to develop alternative diagnostic assays using recombinant P32 protein. An indirect ELISA based on recombinant P32 protein has been used successfully for detecting capripox-specific antibodies from sheep (Heine et al., 1999) and bovine (Carn et al., 1994; Ngichabe et al., 1999) serum samples. Production of P32 protein in various heterologous host systems such as prokaryotic (Qiang et al., 2007) and eukaryotic (Chen et al., 2008) systems has been attempted. The yeast system is unique, as it combines the advantages of both prokaryotic (high expression levels, easy to scale-up and inexpensive growth media) and eukaryotic (capacity to carry out most of the post-translational modifications) expression systems. In recent past, the methylotrophic yeast, Pichia pastoris has emerged as powerful heterologous expression system for the production of high level of functionally active recombinant proteins (Cereghino and Cregg, 2000). In this paper, the expression of GTPV-P32 protein in P. pastoris and its potential use as a diagnostic antigen in ELISA for serological diagnosis of goatpox or sheeppox or capripox infections is reported.

2. Materials and methods 2.1. Virus, cells and biologicals Attenuated goatpox vaccine virus (Uttarkashi strain, passage60) available in the Poxvirus Disease laboratory, Division of Virology, Indian Veterinary Research Institute (IVRI), Mukteswar was used as a source for amplification of GTPV-P32 gene. Vero cell line (CCL-81) available in the laboratory was grown in tissue culture flask using Eagles minimum essential medium (EMEM) (Sigma–Aldrich, St. Louis, MO, USA) containing 10% fetal calf serum (FCS) and maintained with 2% FCS. After attainment of 70–80% confluency, the cells were used for propagation of viruses. Goatpox hyper immune serum (HIS) raised in rabbit against the GTPV and GTPV-P32 protein specific monoclonal antibody (MAb) available in the laboratory were used in the assay.

2.2. Host strain and plasmid vector The P. pastoris host strain GS115 and the yeast transfer vectorpPICZ␣A, were procured commercially (Invitrogen Corporation, Carlsbad, CA). The vector contains the P. pastoris AOX1 (alcohol oxidase) promoter and transcription termination sequences separated by multiple cloning sites for insertion of the foreign gene of interest.

2.3. Amplification and cloning of P32 gene The DNA from GTPV was extracted using AuPrep GEN DNA Extraction kit (Life Technologies India Pvt Ltd., New Delhi, India) as per prescribed protocols. The P32 gene was amplified using virus-specific primers (GTPVp32F: 5 CACCGAATTCACCATGGCAGATATCCCATTATATG-3 and GTPVp32R: 5 -GATGGCGGCCGCAACTATATACGTAAATAAC-3 ), designed based on the published sequence of GTPV (accession number AY382869). The amplified PCR product was purified from gel using AuPrepTM GELX kit (Life Technologies India Pvt Ltd., New Delhi, India) and ligated into the pPICZ␣A vector at the Eco RI and Not I sites and transferred into E. coli Top 10F strain by heat shock method by following standard procedures. The recombinant colonies were screened by colony PCR using 5 AOX (5 -GACTGGTTCCAATTGACAAGC-3 ) and 3 AOX (5 GCAAATGGCATTCTGACATCC-3 ) alcohol oxidase primers, followed by restriction enzyme analysis. The P32 gene insert in the vector backbone was sequenced to confirm its orientation in the pPICZ␣A vector. 2.4. Expression of P32 protein The recombinant plasmid was linearized with Sac I and transferred into GS115 strain of Pichia by electroporation for 10 ms with field strength of 7500 V/cm using Bio-Rad Gene pulser as described earlier (Balamurugan et al., 2003). Zeocin resistant Pichia transformant containing P32 gene was selected on Yeast Extract Peptone Dextrose Medium (YPDS)–zeocin plates. Further, Mut+ Pichia clones were selected by replica streaking on Minimal Methanol Medium + Histidine (MMH) and Minimal Dextrose Medium + Histidine (MDH) plates. Yeast chromosomal DNA was extracted from individual clones by spheroplasting with Zymolyase, followed by phenol:chloroform method as described in Invitrogen easy select manual (Invitrogen Corporation, Carlsbad, CA). The presence of the insert in the genome of the positive Pichia colonies was confirmed by PCR using 5 AOX and 3 AOX primers, as well as the vector and insert specific primers. The induction of protein expression was carried out as per Cregg and Higgins (1995). Briefly, 25 mL of each of buffered glycerol complex medium (BMGY) in 250 × 2 mL flasks was inoculated with a colony of Mut+ positive clone and the vector transformant of Pichia. Both flasks were incubated at 28–30 ◦ C in a shaker incubator (250–300 rpm) to reach an A600 of 2–6 (16–18 h). The cells were harvested and resuspended in buffered methanol complex medium (BMMY) to an A600 of 1.0 (≈100–200 mL media) in 1 L flasks to induce expression. The flasks were incubated at 30 ◦ C to continue the growth and the methanol was added to a final concentration of 0.5% every 24 h to sustain the induction for 120 h as described earlier (Surgrue et al., 1997) as well as to find (reach) the optimum level of expression. After 72 h of induction, the entire culture supernatant was harvested, the total protein in the supernatant was precipitated with 6% polyethylene glycol (PEG)-8000 in the presence of 2.3% NaCl2 and stored overnight at 4 ◦ C. The precipitated protein was then pelleted at 15,000 × g for 10 min and the pellet was dissolved in PBS–Phenylmethylsulfonyl fluoride (PMSF—1 mM final concentration) for storage at −70 ◦ C till used. Protein content was estimated by BCA Protein assay kit (Pierce Biotechnologies, Rockford, IL, USA). The expressed protein was analyzed by 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing conditions (Laemmli, 1970) and the specificity of the protein was confirmed by immunoblot assay. A duplicate gel was transblotted onto a nitrocellulose membrane (NCM) for immunodetection as per the method described earlier (Burnette, 1981) with some modifications. The recombinant protein on the blot was detected by incubation with GTPV HIS serum at 1:100 dilution or GTPV-P32

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protein specific MAb (1:5 dilution) followed by addition of antigoat HRPO or anti-mouse HRPO conjugate (Sigma–Aldrich, St. Louis, MO, USA) as the case may be with dimethyl amino benzidine as an in-soluble substrate. 2.5. Evaluation of expressed protein 2.5.1. Preparation of recombinant GTPV-P32 protein After 72 h of induction with 0.5% methanol, culture supernatant was collected and precipitated with equal amount of pre-chilled 100% acetone. The precipitated protein at 50 ␮L/well (<5 ␮g) was used as a coating antigen in ELISA for standardization. The protein from the fresh or stored culture supernatant of yeast cells having GTPV-P32 gene were precipitated separately with equal volume of chilled acetone and pelleted at 15,000 × g for 10 min. The pellet was resuspended in PBS (pH 7.2) containing PMSF (1 mM final concentration) and stored at −70 ◦ C till used. 2.5.2. Goatpox virus antigen GTPV antigen was prepared by following the method described by Bhanuprakash et al. (2006a). Vero cells were infected with GTPV at 0.01 multiplicity of infection. When 80–90% cytopathic effects were observed, the cells were harvested using cell scrapers. Following centrifugation at 800 × g for 20 min, the cell pellet was sonicated and the clarified supernatant was ultracentrifuged over 36% sucrose cushion at 85,000 × g for 2 h. The pellet and the upper band containing virus and soluble proteins, respectively were collected and used in the assay as virus antigen (Bhanuprakash et al., 2006a). 2.5.3. Serum samples Goat HIS against GTPV and goatpox negative serum were available in the laboratory and were used as positive and negative controls, respectively. For assessing the specificity of the assay, HIS against orf (ORFV), buffalopox (BPXV), camelpox (CMLV), bluetongue (BTV) and Peste des petits ruminants (PPRV) viruses available in the laboratory were also used. The serum samples (n = 30) obtained from apparently healthy goats free of goatpox antibodies with no history of either vaccination or natural exposure to GTPV were considered as negative control. The serum samples (n = 30) obtained after 28 days post-vaccination from goats vaccinated experimentally with live attenuated goatpox vaccine developed at IVRI (Hosamani et al., 2004b) were considered as positive controls. Serum samples (n = 203) collected from sheep vaccinated with 1 and 50 doses of three different sheeppox vaccines namely SPPV Srinagar, Rumanian Fanar (RF) and Ranipet available in the laboratory were included for sero-monitoring. For each dose of different vaccines, six animals were immunized (unpublished data). These samples were collected at 0, 7, 14, 21, 28 days post-vaccination, 10 and 135 days post-challenge. The serum neutralization test results for these sheep serum samples were available in the laboratory and were used to compare the sensitivity and specificity of the standardized GTPV-P32 protein based indirect ELISA. Serum samples (n = 387; goats n = 357; sheep n = 30) of unknown antibody status available in the Poxvirus Disease laboratory, Division of Virology, IVRI, Mukteswar were included in the investigation for serological surveillance. 2.5.4. ELISA standardization ELISA was carried out according to Bhanuprakash et al. (2006a) and Balamurugan et al. (2007) with some modifications. Briefly, expressed GTPV-P32 protein and purified GTPV antigen (1:50) were coated in flat bottomed 96 well plate (Nalgene Nunc Int., Hamburg, Germany) and the left over sites were saturated with blocking buffer (5% skimmed milk powder and 3% lactalbumin hydrolysate in PBS-1% Tween 20). The test serum samples from the animals were diluted 1:50 in blocking buffer. The antigen–antibody reaction was

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followed by incubation with anti goat or sheep HRPO conjugate (1:8000 dilution) and detected by colour development with the chromogen, ortho phenyl diamine (OPD) and H2 O2 as a substrate. For optimization of working dilution of antigen and antibodies, a chequerboard titration was performed as per standard procedures. The specific dilution of recombinant GTPV-P32 protein or GTPV antigen and standard positive serum that induced approximately 75% absorbance (A492 ) of the plateau was arbitrarily selected. To arrive at it, the antigen, the reference serum samples from animals of various immune status (pre-vaccinated) and (post-vaccinated) was tested in two-fold dilutions. The antigen and serum dilutions that gave maximum difference in absorbance at 492 nm between positive and negative (P–N) were selected. After optimization of the assay, samples with known status of GTPV antibody were employed for deciding cut-off value. A mean optical density (OD) value of the negative serum samples with double the standard deviation (S.D.) was decided for specificity of the assay. 3. Results 3.1. Cloning of GTPV-P32 gene in to pPICZ˛A transfer vector The GTPV-P32 gene was amplified from viral DNA extracted from attenuated goatpox vaccine virus (Uttarkashi strain, passage60) by PCR using virus-specific primers, which resulted in specific product of 983 bp. The amplified products were purified and digested with Eco RI and Not I to facilitate cloning into pPICZ␣A vector at the Eco RI and Not I directional sites. Following ligation of PCR product and pPICZ␣A with T4 DNA ligase at 22 ◦ C for 4 h, it was transformed into E. coli TOP 10F strain. Transformation efficiency was moderate, i.e., 50%. Out of 6 colonies screened, three colonies were found recombinant. The recombinant plasmids were designated as pPICZ␣A/GTPV-P32 and were used for expression in P. pastoris. 3.2. Expression of GTPV-P32 protein in P. pastoris GSII5 strain The recombinant pPICZ␣A/GTPV-P32 plasmid was linearized with Sac I and used for transforming yeast cells by electroporation. Pichia integrants obtained were screened for the presence of P32 gene insert by PCR using vector specific primers. Intactness of AOX1 gene (Mut+ or Muts ) was tested by assessing their methanol utilizing efficiency and their growth rates on replica plates. Out of 20 colonies screened by replica plating on MMH and MDH plates, 10 were found Mut+ indicating that AOX1 gene was intact. The genomic DNA was isolated from six colonies and screened by PCR for integrations (Fig. 1). Two colonies were found to carry vector with the insert P32 and these two recombinants were utilized for protein production. The PCR positive Pichia clones of each were grown separately and the expression was induced with 0.5% methanol. Methanol was added every 24 h interval to sustain induction up to 120 h. After the induction, the proteins in the culture supernatants were collected at various intervals and the proteins secreted out were analyzed by 15% SDS-PAGE along with supernatant from the control vector transformants. The expressed protein was concentrated by either acetone or 6% PEG-8000. The PEG precipitated protein was used for characterization in SDS-PAGE, while, acetone precipitated protein was used in ELISA. After optimization, expression was scaled up by increasing the culture volume using large baffled flasks. Methanol (0.5%) was added at every 24 h interval until 72 h as it was optimized for protein expression. Protein secreted was precipitated with 6% PEG after 72 h post-induction and were analyzed by SDS-PAGE (Fig. 2A). The specificity of the expressed protein was confirmed in immunoblotting. The protein was transferred on to nitrocellulose membrane and

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Fig. 1. Agarose Gel Electrophoresis of PCR products from Pichia integrants. Genomic DNA extracted from Mut+ transformants was subjected to PCR amplification using vector and gene specific primer. Specific amplicons size obtained with Lane 1: 5 AOX/3 AOX (1515 bp). Lane 2: ␣-factor/GTPVp32 R (1047 bp). Lane 3: GTPVp32 F/GTPVp32 R (983 bp). Lane M: 100 bp plus DNA ladder (MBI, Fermentas, MD, USA). Lane 4: 5 AOX/3 AOX (2200 bp) (Pichia GSII5 strain control). Lane 5: 5 AOX/3 AOX (588 bp) (pPICZ␣A vector control). Lane 6: Non-template control.

immunodetected with GTPV HIS and GTPV-P32 specific MAb. An intensive colour reaction was observed with the protein band corresponding to 40 kDa P32 protein of GTPV with HIS (Fig. 2B(i)) and with GTPV-P32 specific MAb (Fig. 2B(ii)). There was no colour reaction with the proteins from induced culture of parent strain GS115 and vector-transformed Pichia clone. The secreted protein was concentrated and the amount was estimated by BCA Protein assay kit along with BSA standard. The estimated recombinant GTPV-P32 protein concentration was 100 mg/L of culture supernatant. The culture supernatant was saved after 72 h post-induction, chilled at 4 ◦ C, and concentrated with equal volume of pre-chilled acetone for further use in ELISA.

Fig. 2. Characterization of GTPV-P32 protein expressed in Pichia (A) SDS-PAGE (B) Western blot (i) with GTPV hyperimmune serum (ii) with GTPV-P32 specific monoclonal antibody. Recombinant protein from GSII5 pPICZ␣A/GTPV-P32 Pichia clone: culture supernatant was precipitated with 6% PEG and analyzed in 15% gel. Lanes 1 and 2: 72 h sample of Pichia host GSII5 carrying pPICZ␣A vector and parent strain GSII5, respectively. Lanes 3, 4, 5, 6, 7: 0, 24, 48, 72, 96 h sample of GSII5 pPICZ␣A/GTPV-P32, respectively. Lane M: Prestained protein marker (MBI, Fermentas, MD, USA). Lanes A, B, C: 72 h sample of GSII5 carrying pPICZ␣A/GTPV-P32, pPICZ␣A vector, and parent strain GSII5, respectively.

0.113 (mean + 3S.D.) for deciding the status of serum samples in GTPV-P32 protein based indirect ELISA. The mean ± S.D. optical density values of conjugate, positive, negative and blank goat serum samples were 0.077 ± 0.018, 0.340 ± 0.023, 0.062 ± 0.017 and 0.067 ± 0.023, respectively. In order to rule out the crossreactivity of GTPV-P32 protein with parapox virus, HIS specific to different related viruses were tested in P32 protein based indirect ELISA and there was no reactivity with HIS of ORFV. The reactivity of GTPV-P32 protein was specific for capripox viruses (Fig. 3). 3.4. Relative specificity and sensitivity of the assay The performance of recombinant P32 protein based indirect ELISA in terms of relative sensitivity and specificity was compared

3.3. Evaluation of recombinant GTPV-P32 protein as a diagnostic antigen The expressed GTPV-P32 protein was tested for its suitability as a diagnostic antigen in indirect ELISA instead of whole virus being used as coating antigen. Acetone precipitated GTPV-P32 protein was used in indirect ELISA along with the GTPV antigen to compare the reactivity. The mean reactivity of the positive serum with recombinant P32 protein was 0.340 at OD 492 nm. The expressed P32 protein also reacted well with GTPV-P32 specific MAb in ELISA with a mean reactivity of 0.551 at OD 492 nm. After optimization of the ELISA, samples with known GTPV antibody were employed for deciding cut-off value. Thirty GTPV negative serum samples from goat were screened, which revealed a mean OD value of 0.062 with standard deviation (S.D.) of 0.017. Therefore, the cut-off value was set as 0.096 (mean + 2S.D.) or

Fig. 3. Specificity of the GTPV-P32 protein based indirect ELISA. Reactivity of the hyperimmune serum of various viruses in recombinant GTPV-P32 protein based indirect ELISA. SPPV: Sheeppox virus; GTPV: goatpox virus; CMLV: camelpox virus; BPXV: buffalopox virus; ORFV: orf virus; PPRV: Peste des petits ruminants virus and BTV: bluetongue virus.

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Table 1 Relative specificity and sensitivity of GTPV-P32 protein based indirect ELISA with serum neutralization test (SNT) and whole virus based indirect ELISA based on 203 sheep serum samples. SNT

Whole virus based indirect ELISA Positive

Negative

(A) Serum neutralization test versus whole virus based indirect ELISA Positive 139 0 Negative 8 56 Total 147 56 Relative specificity of whole virus based ELISA (56/64) = 87.5% Relative sensitivity of whole virus based ELISA (139/139) = 100% SNT

Total 139 64 203

P32 protein based indirect ELISA Positive

Negative

Total

(B) Serum neutralization test versus GTPV-P32 protein based indirect ELISA Positive 130 8 138 Negative 9 56 65 Total 139 64 203 Relative specificity of P32 based indirect ELISA (56/65) = 86.2% Relative sensitivity of P32 based indirect ELISA (130/138) = 94.2% Whole virus based ELISA

P32 protein based ELISA Positive

Negative

Total

(C) Whole virus based ELISA versus GTPV-P32 protein based indirect ELISA Positive 138 0 138 Negative 9 56 65 Total 147 56 203 Relative specificity of P32 based indirect ELISA (56/65) = 86.2% Relative sensitivity of P32 based indirect ELISA (138/138) = 100%

with that of purified whole virus indirect ELISA and serum neutralization test using a two-sided contingency table. Serum samples collected periodically from 28 sheep vaccinated with experimental sheeppox vaccines of different strain were tested in GTPV-P32 protein based indirect ELISA for sero-monitoring study. Out of 203 sheep serum samples tested, 138 samples were positive and P32 protein based indirect ELISA showed a specificity of 84.2% and a sensitivity of 94.2–100%, when compared with serum neutralization test and whole virus based indirect ELISA (Table 1A–C). Employing GTPV-P32 protein based indirect ELISA, a steady increase in the antibody titre was observed after the second week in all sheep, which exceeded the cut-off value (0.113). These serum samples were also tested by serum neutralization test. Overall, an increased antibody response was observed after the second week postvaccination (Fig. 4A and B). Further, for sero-surveillance, the serum samples received from areas endemic for goatpox were also tested, which could differentiate clearly between infected and uninfected population. In this preliminary study, out of 387 serum samples tested, 159 samples were found positive by GTPV-P32 protein based ELISA and 154 samples were found positive by GTPV antigen based indirect ELISA. 4. Discussion The control of the disease is vital to improve the small ruminant productivity in the subcontinent. Diagnosis of the capripox is made initially on clinical grounds followed by laboratory confirmation. Serology is limited in its application due to low antibody response following capripox infections (Kitching and Hammond, 1991) and the difficulties encountered with conventional, tissue culture dependant techniques (Carn et al., 1994). Several approaches have been developed as alternative diagnostic assays using recombinant GTPV-P32 protein. There are no reports of expression of GTPVP32 antigen in yeast systems except some Chinese studies showing expression in bacterial and baculovirus systems. Yeast system was selected in this study as it is unique and combines the advantages

Fig. 4. Sero-monitoring of sheep vaccinated with different sheeppox vaccine strains by using GTPV-P32 protein based indirect ELISA. (A) Sheep received a single dose of three different vaccines (average OD values of the animals). (B) Sheep received a 50 dose of three different vaccines (average OD values of the animals). Line shows negative to positive cut-off. DPV: Days post-vaccination; DPC: days post-challenge.

of both prokaryotic and eukaryotic systems and the P32 protein of GTPV was expressed in P. pastoris and evaluated its potential use as a diagnostic antigen in ELISA for serological diagnosis of capripox infections. The GTPV-P32 gene was amplified by PCR, cloned into pPICZ␣A vector and characterized. Basis for selection of pPICZ␣A yeast vector is reasoned due to its strong and highly inducible PAOX1 promoter for the expression of P32 protein. pPICZ␣A is a small size expression vector as it secretes protein in supernatant and contains zeocin resistance gene for selection in E. coli and Pichia. The ␣-factor secretory signal sequences present in vector get the expressed protein secreted out into the culture medium. In order to express GTPV-P32 protein, Pichia host strain GSII5 was used for transformation of the characterized recombinant plasmid DNA. Electroporation method was preferred as it is more efficient than other chemical methods as described earlier (Scorer et al., 1994). It is easier and more convenient than other chemical methods since it does not require spheroplasting of yeast cells. Spheroplasting procedure requires the removal of cell wall by treating the yeast cells with ˇ-glucanase enzyme (Zymolyase) which hydrolyses the ˇ1–3 linkage in the cell wall and form spheroplasts (Hinnen et al., 1978), otherwise the cell wall prevents the uptake of DNA. Electroporation yielded 2 × 102 transformants per ␮g DNA which was in agreement with the recommended range of 102 –104 /␮g DNA described earlier (Cregg et al., 1985). However, transformation efficiency in Pichia was reported to be less than Sacchromyces (Hinnen et al., 1978; Cregg et al., 1985). Transformation of the DNA linearised with Sac I can generate stable transformants of Pichia cells homologous recombination between the transforming DNA and the region of homology within the yeast genome (Cregg et al., 1985, 1989). These integrants show high stability in the absence of selective pressure even when present in multiple copies. It is presumed that the gene insertion events at AOX1 loci arise from a single cross over event between the loci and any of the 3AOX1 regions of vector such as AOX1 promoter, the AOX1 transcription termination (TT) sequences further downstream the AOX1 gene. The phenotype of such transformants is Mut+ His+ or Muts His+ (Cregg et al., 1989).

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After the characterization of the positive Pichia clones (Fig. 1), expression of recombinant GTPV-P32 protein was induced using 0.5% methanol at 30 ◦ C. Since the inserted gene was in the down stream of the secretary signal sequences, the expressed gene product was expected to be secreted out in to the medium. Even though the Mut+ /Muts phenotypes would not make any difference in the expression level as reported earlier (Clare et al., 1991), we selected Mut+ for expression studies since they grew faster than Muts in the presence of methanol. Methanol was added every 24 h interval to sustain induction up to 120 h. Samples collected at various time intervals were analyzed by SDS-PAGE. Upon comparison of protein profiles of the recombinant clones with that of the control (pPICZ␣A vector and Pichia GSII5 strain cells), MW of 40 kDa expressed protein was observed in case of recombinant clones. In un-induced and control cultures, such a specific band was not observed even after prolonged incubation. Therefore, 40 kDa band corresponded to cloned GTPV-P32 gene product (Fig. 2). There was no increase in the quantity of secreted protein with increased frequency of methanol addition and increased duration of induction. As reported in the earlier studies (Balamurugan et al., 2003), 72 h culture showed intense band, while no proteins could be detected before 24 h in case of induced cultures (Fig. 2A). The optimum time of harvest was 72 h post-induction and thereafter, there was a decrease in the protein concentration in the supernatant (Fig. 2A). This decrease may be due to degradation of secreted protein by the proteases released by the lysed cells. But, experimental evidences for this presumption are not available. As reported by Clare et al. (1991), though the level of expression is largely independent of site of chromosomal integration of the gene (AOX1 or his4), the type of integrant (integration or replacement) and methanol utilization of phenotype of host strain (Mut+ His+ ), may vary with the number of fragments of target gene integrated with host genome. However, in this study amount of protein expression was in agreement with the reported range. Varying levels of protein secretion (ranging from 6.3 mg/L to 12 g/L of culture) were observed by various groups depending on the nature of the protein (Romanos et al., 1991; Vozza et al., 1996). The proteins resolved in SDS-PAGE were transferred on to a NCM and were detected by using a GTPV-P32 protein specific MAb (Fig. 2). On analysis, specific band of 40 kDa molecular weight (MW) size was observed. This confirmed that the expressed recombinant protein was specific to GTPV and no other non-specific bands were noticed. The calculated size as per aa composition along with part of ␣secretory signal cleavage sequence from vector and as observed by the mobility in SDS-PAGE, was in agreement with the reported size indicating that the 40 kDa protein was the product from the cloned gene. This MW corresponds with the reported size of P32 protein expressed in E. coli using plasmid vector pGEX-2T (Carn et al., 1994; Heine et al., 1999). Western blot analysis with MAb against the GTPV-P32 protein indicated that the band observed in SDS-PAGE was virus specific. Further, to evaluate recombinant GTPV-P32 protein for its potential use as antigen in diagnosis, ELISA was performed. Though other immuno-assays are available, ELISA is one of the most sensitive methods for evaluating expressed proteins and which has been extensively applied (Carn et al., 1994; Heine et al., 1999). P32 is one of the structural proteins present in all the capripoxviruses, contains major immunogenic determinants (Chand, 1992). There is a report of expression of recombinant P32 antigen in prokaryotic system and its suitability as antigen in ELISA (Carn et al., 1994). A recombinant protein was used in ELISA along with the GTPV antigen to compare the reactivity. The results indicated that the expressed P32 protein reacted well with GTPV HIS in ELISA making it evident that the epitopes present in the expressed protein are well recognized by antibody. The reactivity of either precipitated or concentrated P32 protein in ELISA with positive serum

indicated that possibility of using the recombinant P32 protein as antigen in place of whole virus in indirect ELISA. Precipitating the supernatant with acetone avoided a tedious method of protein purification for down stream process of the expressed protein. This would imply that method of concentration of protein is economical and used easily in ELISA. The reactivity of expressed GTPV-P32 protein with specific MAb in ELISA ensures the retention of conformational epitopes of expressed protein and its antigenicity or immunogenicity. The reactivity of the GTPV-P32 protein and GTPV antigen with MAb indicated that the recombinant GTPV-P32 protein can replace the whole virus as coating antigen in indirect ELISA. After optimization of the ELISA, samples with known negative GTPV antibody were employed for deciding cut-off value. The cutoff value was set as 0.096 (mean + 2S.D.) or 0.113 (mean + 3S.D.) for deciding the status of serum samples in GTPV-P32 protein based indirect ELISA. In this, we opted for a slightly higher cut-off value (two times standard negative serum samples mean + 3S.D.) to increase the specificity of the assay without greatly compromising the sensitivity. The assay included standard controls (conjugate, positive, negative and antigen blank) to assess the quality, accuracy and repeatability as per standard procedure (Jacobson, 1998). The OD values of all the standards used in this assay did not show much variation between the plates, indicating repeatability of the assay. Capripox viruses share common antigen with parapoxviruses (Roy et al., 2008). Hyperimmune serum specific to different related viruses were tested in P32 based indirect ELISA to rule out the crossreactivity of the expressed GTPV-P32 protein, that showed the P32 protein was specific only for capripox viruses (Fig. 3). However, further evaluation for assessing the specificity of the assay with lumpy skin disease serum samples is required. In the present study, using a two-sided contingency table, the performance of recombinant GTPV-P32 based indirect ELISA in terms of relative sensitivity and specificity was compared with that of purified whole virus indirect ELISA and serum neutralization test. In the sero-monitoring study, the GTPV-P32 protein based indirect ELISA showed a specificity of 84.2% and a sensitivity of 94.2–100%, when compared with serum neutralization test and whole virus based indirect ELISA (Table 1). Carn et al. (1994) also reported 100% sensitivity of recombinant P32 based ELISA when compared to the serum neutralization test while testing lumpy skin disease serum samples obtained from bovines. Recently, Babiuk et al. (2009) reported that detection of antibodies against capripoxviruses using an inactivated sheeppox virus ELISA using serum samples obtained experimentally from animals infected with virulent sheeppox or goatpox virus isolates. The sensitivity of the ELISA was 96% with a specificity of 95%, where the sensitivity of the virus neutralization assay was 96% with a specificity of 100%. Non-virulent capripoxvirus isolates, in contrast, did not elicit antibody responses (Babiuk et al., 2009). In this study, the result of one dose correlated very well with 50 doses of vaccine, both showing increased antibody response after the second week of vaccination (Fig. 4). A decrease in antibody response was observed 10 days post-challenge, which may be due to binding of some amount of neutralizing antibodies to antigen in challenge virus. However, the antibody response was maintained above the cut-off value when the serum samples were tested at 135 days post-challenge. Subsequent monitoring at different days of postvaccination or post-challenge to determine the antibody kinetics is required. In this preliminary sero-surveillance study, the relative sensitivity and specificity of the GTPV-P32 protein based indirect ELISA assay was not compared with that of serum neutralization test, while testing the samples of unknown status of antibody with respect to GTPV from endemic area. This is for the reason that the limited volume of field serum samples available for the test

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to perform as well as limited number of samples analyzed in this seroepidemiological study. However, to explore the potential of this assay, it is desirable to evaluate it by screening more number of serum samples from sheep, goat and even from bovine for its suitability as serological diagnosis of capripox infections before field application. For an effective control program of any infectious disease, detailed epidemiology must be studied by extensive clinical and serological surveillance before the launch of control program. As such there is no gold standard test for detection of capripox antibodies. Therefore, the performance of both serum neutralization test and ELISA were compared. The ELISA assay differentiates infected from uninfected population and compared very well with serum neutralization test. After analysis, GTPV-P32 protein based indirect ELISA was found efficient for detection of capripox antibodies as like whole virus based ELISA (Bhanuprakash et al., 2006a). The recombinant GTPV-P32 protein based indirect ELISA described above is an ideal alternative to purified whole virus based indirect ELISA for sero-surveillance and sero-monitoring of capripox virus antibodies in sheep and goats. Acknowledgements The authors thank the Director, Indian Veterinary Research Institute (IVRI) for providing necessary facilities to carry out this work and the staff of Poxvirus Disease laboratory, Rinderpest and Allied Diseases Laboratory, IVRI, Mukteswar, for their valuable and timely help in carrying out this work. Further, V. Bhanot acknowledges IVRI for financial support in terms of Senior Research Fellowship to carry out the Ph.D. program. The financial support provided by the Indian Council of Agricultural Research (ICAR), New Delhi, India under Niche Area of Excellence: Production and Quality control of Veterinary Immunodiagnostics and immunoprophylactics is also acknowledged. References Babiuk, S., Wallace, D.B., Smith, S.J., Bowden, T.R., Dalman, B., Parkyn, G., Copps, J., Boyle, D.B., 2009. Detection of antibodies against capripoxviruses using an inactivated sheeppox virus ELISA. Transbound. Emerg. Dis. 56, 132– 141. Balamurugan, V., Danappa Jayappa, K., Hosamani, M., Bhanuprakash, V., Venkatesan, G., Singh, R.K., 2009. Comparative efficacy of conventional and TaqMan polymerase chain reaction assays in the detection of capripoxviruses from clinical samples. J. Vet. Diagn. Invest. 21, 225– 231. Balamurugan, V., Renji, R., Saha, S.N., Reddy, G.R., Gopalakrishna, S., Suryanarayana, V.V.S., 2003. Protective immune response of the capsid precursor polypeptide (P1) of foot and mouth disease virus type ‘O’ produced in Pichia pastoris. Virus Res. 92, 141–149. Balamurugan, V., Singh, R.P., Saravanan, P., Sen, A., Sarkar, J., Sahay, B., Rasool, T.J., Singh, R.K., 2007. Development of an indirect ELISA for the detection of antibodies against Peste-des-petits-ruminants virus in small ruminants. Vet. Res. Commun. 31, 355–364. Bhanuprakash, V., Hosamani, M., Juneja, S., Kumar, N., Singh, R.K., 2006a. Detection of goat pox antibodies: comparative efficacy of indirect ELISA and counterimmunoelectrophoresis. J. Appl. Anim. Res. 30, 177–180. Bhanuprakash, V., Indrani, B.K., Hosamani, M., Singh, R.K., 2006b. The current status of sheep pox disease. Comp. Immunol. Microbiol. Infect. Dis. 29, 27–60. Burnette, W.N., 1981. ‘Western Blotting’ electrophoretic transfer of proteins from sodium dodecyl sulphate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195–203. Carn, V.M., Kitching, R.P., Hammond, J.M., Chand, P., Anderson, J., lack, D.N., 1994. Use of a recombinant antigen in an indirect ELISA for detecting bovine antibody to capripoxvirus. J. Virol. Methods 49, 285–294. Cereghino, J.L., Cregg, J.M., 2000. Heterologous protein expression in methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24, 45–66. Chand, P., 1992. Molecular and immunological characterization of a major envelope protein of capripoxvirus. Ph.D. Thesis. University of Surrey, Surrey, UK.

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