Serological Detection of Grapevine leafroll virus 2 Using an Antiserum Developed against the Recombinant Coat Protein

Grapevine leafroll associated virus 2 (GLRaV 2) is one of the important components in the leafroll disease complex. The coat protein gene of GLRaV 2 was cloned into a protein expression vector pMAL-c2x and the recombinant protein, consisting of the maltose binding protein (MBP) and GLRaV 2 coat protein (CP), was expressed in Escherichia coli. The recombinant MBP-CP was used to raise a high quality antiserum. When used in Western blot analysis, the anti-MBP-CP antiserum produced specific reaction to the recombinant protein as well as to the viral coat protein of GLRaV 2. In Immunosorbent electron microscopy study, the anti-MBP-CP antibodies strongly decorated the GLRaV 2 virions. Using the newly developed antiserum, an indirect plate-trapped antigen enzyme-linked immunosorbent assay method was developed and successfully implemented for virus detection. A field survey was conducted to evaluate the virus infection status by GLRaV 2 and GLRaV 3 using antibodies developed against their respective recombinant coat proteins. Introduction Grapevine leafroll is one of the most important viral diseases of grapevine. This disease is widespread and has been reported in almost all major grapevine growing areas in the world. Study of leafroll disease is complicated by the fact that at least nine serologically distinct viruses, Grapevine leafroll associated viruses (GLRaV 1–9) in the family Closteroviridae, are associated with the disease (Alkowni et al., 2004; Fauquet et al., 2005). Of the nine GLRaVs, only GLRaV 2 is mechanically transmissible to an herbaceous host (Nicotiana benthamiana). GLRaVs occur in relatively low concentration in infected grape tissue and are difficult to purify. The presence of mixtures of viruses in a diseased vine is common in the field. Therefore, production of high quality antibodies suitable for large scale detection of these viruses is not an easy task. To date polyclonal antibodies produced using partially purified viruses have generated poor results (Gugerli et al., 1984; Boscia et al., 1995). Furthermore purified virus preparation may still contain host proteins that produce a non-specific reaction and require an extensive pre-absorption with healthy plant extract in order to function effectively in enzyme-linked immunosorbent assay (ELISA). Alternative approaches are needed to produce antibodies that are effective for use in a large scale disease diagnosis. Recombinant protein technology has been applied to produce a number of polyclonal antibodies against viral coat proteins (Nikolaeva et al., 1995; Vaira et al., 1996; Jelkmann and Keim-Konrad, 1997; Rubinson et al., 1997; Ling et al., 2000; Cerovska et al., 2003). However, the usefulness of these antibodies for virus detection in ELISA needs further improvement. Previously, we reported the partial sequence of GLRaV 2 and showed that the genome organization of this virus is typical of the genus Closterovirus (Zhu et al., 1998). Meng et al. (2005) completed the sequence of the genome of GLRaV 2. With the availability of the genome sequence of GLRaV 2, a full length coat protein gene (ORF6) was amplified by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and engineered into a protein expression vector pMAL-C2. Identification of ORF6 as the coat protein had been confirmed by Western blot (Zhu et al., 1998). The objectives of the The use of trade, firm or corporation names in this article does not imply the endorsement or approval by the USDA-ARS of any product to the exclusion of others that may be suitable. www.blackwell-synergy.com J. Phytopathology 155, 65–69 (2007) 2007 The Authors Journal compilation 2007 Blackwell Verlag, Berlin present study were to generate a polyclonal antiserum against the recombinant coat protein of GLRaV 2, to characterize its serological properties, and to evaluate its application for virus detection of field samples using ELISA. We report here that the antiserum produced against the recombinant coat protein was evaluated using Western blot, immunosorbent electron microscopy (ISEM) and ELISA. A plate-trapped antigen ELISA (PTA-ELISA) was successfully implemented for the detection of GLRaV 2 in field-collected samples. Materials and Methods Virus source and dsRNA isolation GLRaV-2 infected Vitis vinifera, cv. Pinot Noir, the PN isolate (Zhu et al., 1998; Meng et al., 2005) was used as the virus source for the coat protein (CP) gene amplification as well as for ELISA development and validation. The mature canes were harvested in the Fall of 1997 and stored at 4 C until used. Phloem tissue was scraped from GLRaV 2 infected grapevines and used for dsRNA isolation. Double stranded RNA was extracted with phenol/chloroform followed by CF11 cellulose column chromatography essentially as described by Hu et al. (1990). Purification of maltose binding protein-coat protein fusion protein and production of antibody The construction of the protein expression vector containing the coat protein gene of GLRaV 2, designated as pMAL-C2xGLRaV2CP, was described in detail by Zhu et al. (1998). To purify the maltose binding protein-coat protein (MBP-CP) fusion protein, Escherichia coli K12 TB1 containing the recombinant plasmid was cultured at 37 C in Luria-Bertani broth containing 0.2% glucose and 100 lg/ml of ampicillin. Induction of the recombinant protein was achieved with incubation for 3 h after the addition of 0.03 mm isopropylb-Dthiogalactopyranoside (IPTG) to an active growing cell culture (OD600 : 0.5). Bacterial cells were harvested by centrifugation, resuspended in the column buffer (20 mm Tris-Cl, 200 mm NaCl, 1.0 mm EDTA and 10 mm 2-mercaptoethanol) and disrupted by sonication. Supernatant was then passed through an amylose column according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). After a thorough washing, the MBP-CP fusion protein was eluted from the column with the column buffer containing 10 mm maltose. The eluted MBP-CP fusion protein was then concentrated with ultrafree centrifugal filters (MilliPore Corporation, Bedford, MA, USA). The concentration of the purified MBPCP fusion protein was estimated using the Bio-Rad Bradford assay and spectrophotometer at 280 nm. The purity of this purified MBP-CP fusion protein was evaluated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) as described in the following section. The concentrated MBP-CP fusion protein, resuspended in the column buffer at 1 mg/ml, was used for injection. A New Zealand white rabbit was immunized subcutaneously with one milligram of the purified MBP-CP fusion protein (1 mg/ml) emulsified with Freund’s complete adjuvant. This was followed by a second injection 1 week later with incomplete adjuvant, and a booster shot 2 months after the initial immunization. One week after the second injection, antiserum was collected weekly and its reaction titre to GLRaV2 was evaluated by Western blot analysis. The final collection of antiserum was carried out 1 week after the booster injection. This antibody was designated as MBP-CP antiserum. SDS-PAGE and Western blot The pulverized grapevine phloem tissue or N. benthamiana leaf tissue was ground in Bradley buffer (50 mm Tris, pH7.5, 10 mm KCl, 20% glycerol, 0.4 m sucrose, 5.0 mm MgCl2, 10 mm b-mercaptoethanol) containing 20 lg/ml pefabloc and chymostatin protease inhibitor (Roche Applied Science, Indianapolis, IN, USA). An aliquot of tissue extract was separated on a 12% SDSPAGE gel. After electrophoresis, the gel was either stained with Coomassie Brilliant Blue or subjected to immunodetection. For immunodetection in Western blot, SDS-PAGE separated proteins were electro-blotted onto an Immobilon transfer membrane (Millipore Corporation, Bedford, MA, USA), which was then subjected to immunodetection using either an anti-MBP-CP antiserum developed in the present study or an antiGLRaV 2 antiserum against purified virions kindly provided by Dr Zimmerman (Zimmerman et al., 1990). The target proteins were detected with SuperSignal WestPico Rabbit IgG detection kit according to the manufacturer’s instructions (Pierce Biotechnology, Rockford, IL, USA). The immuno-reaction was visualized by exposure of the treated membrane to a Kodak X-ray film which was then developed accordingly. Immunosorbent electron microscopy The general procedure, described by Milne and Luisoni (1977), was applied in immunosorbent electron microscopy (ISEM) study. For immuno-decoration, comparative studies were conducted to evaluate the strength of virus decoration with the MBP-CP antiserum and the anti-GLRaV 2 antiserum (Zimmerman et al., 1990). The Formvar grids were placed first on a diluted antiserum (1 : 10) for 5 min. The antibody-coated grids were then used to trap virus particles by floating on crude plant sap for 15 min. The trapped virus particles were finally decorated by an additional incubation for 15 min with the respective antiserum, diluted 1 : 50 in 0.1 m phosphate buffered saline (PBS), pH 7.0. After washing, the grids were stained with 1.5% uranyl acetate and observed using an electron microscope (JEOL Ltd., Tokyo).