Journal of Virological Methods 265 (2019) 22–25 Contents lists available at ScienceDirect Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet Production of a polyclonal antiserum against recombinant nucleocapsid protein and its application for the detection of fig mosaic virus T Morteza Shahmirzaiea, Mohammad Reza Safarnejadb, Farshad Rakhshandehrooa, ⁎ Hossein Safarpourc, Hodjattallah Rabbanid, Hamid Reza Zamanizadeha, Toufic Elbeainoe, a Department of Plant Protection, College of Agricultural Sciences and Food Industries, Science and Research Branch, Islamic Azad University, Tehran, Iran b Department of Plant Viruses, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran c Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran d Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran e Istituto Agronomico Mediterraneo di Bari, Via Ceglie 9, 70010, Valenzano, BA, Italy ARTICLE INFO ABSTRACT Keywords: Mosaic disease (MD), caused by Fig mosaic emaravirus (FMV), is the most important and devastating virus disease Mosaic disease of fig trees worldwide. The detection of FMV in infected plants is possible only through the use of molecular Fig mosaic emaravirus techniques, i.e. RT-PCR and LAMP, which both offer high sensitivity of detection, but are also considered la- Recombinant protein and detection borious when dealing with a large number of samples. To cope with this restriction, a polyclonal antiserum through the immunization of a rabbit by injecting the recombinant nucleocapsid protein (NP) of FMV was raised and evaluated for its efficacy in Western Blot, Dot immuno-binding and DAS-ELISA. The results obtained showed that the raised antiserum was able to identify the nucleocapsid protein of FMV (p3) which was found to have an estimated molecular weight of ca. 35 KDa. In addition, the antiserum, when used in the three serological assays, was able to detect the p3 of FMV in protein extracts of infected plants with different levels of efficacy. Dot immuno-binding, using denatured plant protein extract, proved to be the most efficient serological assay for detecting FMV in samples collected from different fig orchards. This is the first report on an antiserum raised against FMV that could be used for immunological detection of the virus. 1. Introduction et al., 2012). Two molecular assays (RT-PCR and RT-LAMP) are avail- able for its detection in infected plants (Elbeaino et al., 2009a; Ishikawa Fig mosaic syndrome was first reported in California in the 1930's et al., 2015). However, the inability to transmit FMV to herbaceous (Condit and Horne, 1933) and is now found where figs are grown. The hosts, the difficulty in purifying the virus particles from infected plants disease is characterized by a range of symptoms of varying severity, i.e. and the presence of high amounts of contaminants and inhibitors leaf chlorosis, deformation, mosaic or discoloration patterns (Blodgett (polyphenols, polysaccharides, latex, resins, etc.) in figs are aspects that and Gomec, 1967; Martelli et al., 1993). The year 2009 was a turning restrain the development of biological (mechanical inoculation of FMV point in the identification of the etiology of this disease, when the to herbaceous hosts and\or to indicator plants) and serological tools causal agent was found to be a negative-sense single-stranded RNA needed for its detection and\or further characterization. Although RT- virus that belongs to the genus Emaravirus and family Fimoviridae PCR and RT-LAMP assays are sensitive and specific for detecting FMV in (Elbeaino et al., 2009a, 2018). The virus is transmitted in nature by the infected material, they remain precarious when a large variation of eriophyid mite Aceria ficus and by vegetative propagation through in- sequences exists among FMV isolates and the time needed by both fected cuttings (Blodgett and Gomec, 1967; Elbeaino et al., 2009a). techniques to analyze large numbers of samples. Therefore, the pro- The genome of FMV is composed of six RNA segments which encode duction of recombinant virus proteins through gene amplification and the RNA-dependent RNA polymerase (RdRP, p1), a putative glycopro- expression in a bacterial host, to be used for antibody production for tein (GP, p2), a putative nucleocapsid protein (NP, p3), a putative serological assays, could further assist in the detection of FMV. For this movement protein (MP, p4) and two proteins with unknown functions purpose, the NP of FMV was chosen, similarly to European mountain ash (RNA-5, p5 and RNA-6, p6) (Elbeaino et al., 2009a, b; 2012; Ishikawa ringspot-associated emaravirus (EMARaV) which has a genomic ⁎ Corresponding author. E-mail address: [email protected] (T. Elbeaino). https://doi.org/10.1016/j.jviromet.2018.12.016 Received 8 August 2018; Received in revised form 19 December 2018; Accepted 20 December 2018 Available online 21 December 2018 0166-0934/ © 2018 Elsevier B.V. All rights reserved. M. Shahmirzaie et al. Journal of Virological Methods 265 (2019) 22–25 characteristic similar to that of FMV (Mielke et al., 2008), to develop a 300 mM, Imidazole 10 mM; pH 8) containing 1 mg/ml of lysozyme. The serological detection tool. Furthermore, the advantage from choosing suspension was sonicated six times in short pulses of 30 s (150 W with the NP resides in its high sequence conservation among FMV isolates (El 30 s interval). The purification of expressed fusion protein was carried Air et al., 2016) and immunogenic response to express a stable poly- out under native conditions through immobilized metal ion affinity clonal antiserum for immunodiagnostic tests [Double-Antibody Sand- chromatography (IMAC), following the manufacturer’s protocol wich Enzyme-Linked Immuno-Sorbent Assay (DAS-ELISA), Dot im- (Qiagen, Netherlands). The quality of purified recombinant FMV-NP muno-binding assay (DIBA) and Western Blot (WB)] for which the was determined by coomassie-stained discontinuous SDS-PAGE system results are reported hereinafter. (Laemmli, 1970), as well as by immuno-blotting with anti- 6×His tag monoclonal antibody (Ausubel et al., 1992). 2. Materials and methods 2.3. Immunization, IgG purification and conjugation 2.1. Source of plant material Immunization was performed using a New Zealand White female An Iranian fig accession (ES3) infected with FMV and showing 97% rabbit with five subcutaneous injections, at intervals of two weeks. One of identity at the amino acid level (sequence deposited in the GenBank hundred μg of purified recombinant FMV-NP were emulsified with an under the accession number MG880755) with the FMV Italian isolate identical volume of Montanide complete adjuvant preparation and in- (accession number FM991954) was used as a source material to pro- jected into the rabbit. Test bleeds were taken one week after the last duce a recombinant NP. Forty-seven samples, which consist of leaves injection and IgG purification was performed by affinity chromato- taken from mosaic-diseased and apparently healthy fig plants, were graphy column prepared by coupling the immunogenic antigen to collected from commercial orchards in five main fig-growing provinces CNBr-activated sepharose 4B (GE Healthcare, Uppsala, Sweden). The of Iran [Gilan and Mazandaran (North), Tehran and Markazi (Centre), bound protein was eluted with 0.1 M Glycine/HCl pH 2.5 and dialyzed Fars (South)], in summer 2016. These samples were firstly screened by against Phosphate-buffered saline solution (1X PBS) overnight. The RT-PCR to identify FMV-infected and healthy fig accessions and used purified anti-FMV-NP antibodies were labeled with alkaline phospha- afterward as negative and positive controls to evaluate the im- tase (AP) from calf intestinal mucosa using 1% glutaraldehyde solution munodiagnostic efficacy of the antiserum produced. as homobifunctional reagent (Sigma-Aldrich, USA), as described by Harlow and Lane (1988). The preparation was kept for 4 h at room 2.2. Cloning of FMV-NP fragment and construction of pET-28a(+)-FMV- temperature, and dialyzed for three times against 1X PBS to eliminate NP expression plasmid glutaraldehyde residues. Blocking of reaction was performed by adding 5 mg/ml of bovine serum albumin (BSA) and the labeled antiserum was Total nucleic acids (TNA) were extracted from leaf vein tissues using stored at 4 °C. Total HiYield™ mini RNA extraction kit (Real-Biotech, Taiwan) ac- cording to the manufacturer’s instructions. Complementary DNA 2.4. WB, DIBA and DAS-ELISA (cDNA) was synthesized using 0.5 μg of RNA extract mixed with 0.1 μg of random primers, in the presence of 200 units of Moloney murine The immunodiagnostic efficacy of the produced polyclonal anti- leukaemia virus (M-MLV) reverse transcriptase enzyme, according to the serum was evaluated by WB, DIBA and DAS-ELISA. manufacturer’s instructions (ThermoFisher Scientific, USA). RT-PCR (i) WB was conducted according to Towbin et al. (1979); thus 0.2 g was carried out using 15 pmol of sense (FMV-NP-s: 5′-CCATGGCACCT of leaf vein tissues from healthy and infected fig plants were homo- AAGAGTAAGACTAC-3′) and antisense primers (FMV-NP-a: 5′-CTCGA genized in 5 vol of extraction buffer (Tris-HCl 0.5 mM, pH 8.8, 4% SDS, GAACATGAGCACTTGCAATC-3′), designed to amplify the complete 40% glucose and 4% of 2-mercapthoethanol) for the extraction of coding region of RNA-3 of FMV (947 nt). Two stretches of nucleotide proteins from plant sap. Extracts were heated for 5 min. at 100 °C, sequences (underlined), cleavable by NcoI and XhoI endonucleases, centrifuged at 6000 g for 3 min. and 30 μl were loaded onto 10% SDS- were introduced at sense and antisense primers, respectively to facil- PAGE. Proteins were electro-blotted onto Millipore polyvinylidene di- itate the directional cloning of the PCR product. RT-PCR consisted of an fluoride (PVDF) membrane and further blocked with 5% non fat dry initial denaturation at 94 °C for 4 min followed by 35 cycles at 94 °C for milk and incubated overnight at 4 °C.