Accepted Manuscript Title: Purification and immuno-gold labeling of Lily mottle virus from lily leaves Author: Yubao Zhang Yajun Wang Zhongkui Xie Ruoyu Wang Zhihong Guo Wopke van der Werf Wang Le PII: DOI: Reference:

S0166-0934(15)30075-6 http://dx.doi.org/doi:10.1016/j.jviromet.2016.02.001 VIRMET 12952

To appear in:

Journal of Virological Methods

Received date: Revised date: Accepted date:

3-11-2015 3-2-2016 5-2-2016

Please cite this article as: Zhang, Yubao, Wang, Yajun, Xie, Zhongkui, Wang, Ruoyu, Guo, Zhihong, Werf, Wopke van der, Le, Wang, Purification and immunogold labeling of Lily mottle virus from lily leaves.Journal of Virological Methods http://dx.doi.org/10.1016/j.jviromet.2016.02.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Purification and immuno-gold labeling of Lily mottle virus from lily leaves Yubao Zhang a, Yajun Wang a, Zhongkui Xiea*, Ruoyu Wang a, Zhihong Guoa,Wopke van der Werfb, Wang Le a

a

Cold and Arid Regions Environmental and Engineering Research Institute, Chinese

Academy of Sciences, Lanzhou 730000, China b

Wageningen University, Department of Plant Sciences, Centre for Crop Systems

Analysis, Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands

E-mail addresses: [email protected] (Y. Zhang), [email protected](Y. Wang), [email protected](Z. Xie), [email protected] (R.Wang), [email protected] (Z. Guo), [email protected](W. van der Werf), [email protected](L.Wang)

* Corresponding author: Zhongkui Xie Tel. + (86) 931 452-8920; Fax + (86) 931 827-3894 E-mail: [email protected];[email protected] Postal address: Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, No 320 Dong Gang West Road, Lanzhou 730000, China

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Highlights 

An efficient procedure was developed for purification of LMoV from infected leaves.



Viral purity & integrity were confirmed by OD260:280, antibodies, PCR and TEM.



An IGL assay was successfully applied to identify a lily virus for the first time.



The IGL is the label viz. goat anti-rabbit 15 nm colloidal gold.

Abstract Lily mottle virus (LMoV) is prevalent in Lilium species worldwide causing dwarfing, flower breaking, and reduced bulb yield. In this paper, an easy to use and efficient procedure is described for purification of LMoV from lily leaves. The resulting sample is characterized by a 260/280 nm absorbance ratio of 1.20 at a concentration of 1.27mg/mL. The procedure results in high protein purity and particle integrity as shown by UV-spectrophotometry, polyacrylamide gel electrophoresis (PAGE), Western blotting, reverse transcriptase (RT)-PCR and transmission electron microscopy (TEM)in combination with immuno-gold labeling. This is the first time that an immuno-gold labeling (IGL) assay was performed to identify a virus of lily. Purified products can be used as a source of antigen in the preparation of antibodies against LMoV and may assist in the development of a diagnostic test for LMoV and in epidemiological surveys.

Keywords: Lily mottle virus, purification, Transmission electron microscope, immuno-gold labeling assay.

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1. Introduction Viral diseases cause quantitative and qualitative yield reductions of lilies around the world (Wang et al., 2010). More than 10 different viruses have been reported to infect lilies worldwide, and lily mottle virus (LMoV) is one of the most common (Zhang et al., 2014). LMoV is closely related to the tulip breaking virus (TBV; Alper et al., 1982) and is a member of the Potyvirus genus within the Potyviridae family. It is aphid transmitted and only infects plants of the genera Lilium and Tulipa L.(Liliaceae family)(Derks et al., 1994).The particles of LMoV are flexuous, non-enveloped, and rod-shaped; they are 680 to 900 nm long and 11 to 15 nm wide (King et al., 2011). Symptoms of LMoV in lily include leaf mottle, leaf mosaic, reddish-brownish necrotic spots, vein clearing, chlorosis and yellow streaking, and leaf curling and narrowing (Fig.1). Other symptoms included dwarfing, flower breaking and reduced bulb yield. The symptoms may be more severe if plants are simultaneously infected with Cucumber mosaic virus (CMV) and Lily symptomless virus (LSV) (Asjes, 2000; Zhang et al., 2014).Symptoms may also be very mild, or plants maybe symptomless duringearly growth stages. Enzyme-linked immunosorbent assay (ELISA; Sharma et al., 2005; Tong et al., 2010) is commonly used to detect LMoV in lily. However, the method is time consuming, and requires technical expertise and specialized laboratory equipment. Therefore, an immunochromatographic strip (ICS) test was developed to rapidly detect LMoV(Zhang et al., 2015). The ICS assay can be completed within 10 min without the need for special instruments or skilled personnel, and can be used virtually anywhere. Currently, immunochromatographic strips are prepared using two anti-LMoV polyclonal antibodies, to capture and detect LMoV particles (Zhang et al., 2015). Polyclonal antibodies are prone to batch-to-batch variability, which could

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affect the stability of the ICS test. In contrast, homogeneity of monoclonal antibodies is very high. Once hybridomas are made, a constant and renewable source of antibodies would be available, enabling improved standardization of the ICS assay. Simple and easy to use methods have been published for the purification of LSV and CMV from lily(Hsu et al., 1995; Wang et al., 2007; Liang et al., 2007), and purification of potyviruses from host plants has been described (Rupar et al., 2013).There is no information however, on the purification of LMoV from lily leaves. Purification of viral particles of LMoV is an important step in the production of highly specific antibodies and thus there is a need for more research. In this study, an easy to use and efficient procedure for isolation and purification of LMoV particles was developed. The viral protein purity and morphological intactness of purified viral particles were verified by SDS-PAGE, Western blotting, RT-PCR and transmission electron microscope. In addition, we report the successful application of an immuno-gold labeling (IGL) assay to detect and unequivocally identify LMoV particles. To our knowledge, this is the first time that the IGL technique was applied to the identification of a lily virus.

2. Materials and Method 2.1 Viral material We collected naturally infected plants of oriental hybrid lily(Lilium oriental cv. Sorbonne) showing visual dwarfing, or leaves displaying chlorotic yellow spots, stripes or mosaic symptoms at the Gaolan Research Station(Gaolan County, Lanzhou, Gansu Province, China, 36°13″N 103°47″E)1. Leaves near the flower bud were tested

1Field

Station, Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences in Lanzhou, Gansuprovince, China

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by RT-PCR according to the method of Zhang et al. (2010). Leaves from plants that tested positive for LMoV served as sources of LMoV and were stored at -70°C. 2.2 Purification of LMoV Twenty grams of LMoV positive leaf tissue was crushed into powder in liquid nitrogen with a mortar, homogenized quickly with chilled (4°C)extraction buffer(EB: 0.5M potassium phosphate(pH7.0) containing 10 mM EDTA and 0.1% (v/v) 2-mercaptoethanol)in a 1:4 (w:v) ratio. The resulting crude homogenate was passed through a cloth and centrifuged at 5000 g for 5min at 4°C to remove plant debris. Chloroform was added to the supernatant to a concentration of 50% (v/v) and intensely stirred for 15min and then centrifuged at 5000g for 15 min. First PEG-6000 and then NaCl was slowly added to the supernatant to concentrations of 7.5% and 4% w/v, respectively. The mixture was kept at 4°C and stirred slowly for 30min and then centrifuged at 8000g for 20 min at 4°C. The supernatant was discarded and the pellet was resuspended in 20 ml of resuspension buffer(RB: 0.5M potassium phosphate(pH 7.0) containing 10 mM EDTA, 0.1% (v/v) 2-mercaptoethanol and 2% Triton X-100) and stored overnight. The second day the suspension was centrifuged at 10000g for 20 min. First PEG-6000 and then NaCl were slowly added to the supernatant to concentrations of 6% and 3% w/v, respectively. The mixture was kept at 4°C and stirred slowly for 20min and centrifuged at 8000g for 20 min at 4°C. The supernatant was discarded and the pellet was resuspended in 20 ml of RB. The suspension was kept at 4°C with slow stirring for 20min and centrifuged at 10000g for 20 min at 4°C. Subsequently, the suspension was ultracentrifuged at 25000g for two and a half hours at 4°C.The supernatant was discarded; the pellet was resuspended in 500μl EB and stirred slowly at 4°C for 1 hour and then centrifuged at 5000g for 10 min at 4°C to obtain the final suspension of pure LMoV for further analysis.

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2.3Preparationof LMoV antiserum The rabbit anti-LMoV antiserum was obtained following the procedure of Tong et al.(2010). Briefly, the coat protein (CP) gene of LMoV was amplified by RT-PCR from infected plants of oriental hybrid lily (Lilium oriental cv. Sorbonne) and cloned into the prokaryotic pET-28a vector to generate the recombinant plasmid pET-28a-CP.The resulting carboxyterminal His-tagged CP was over-expressed in Escherichia coli BL 21 cells by isopropyl-b-d-thiogalactoside(IPTG) induction and purified over Ni-NTA affinity columns. The purified CP was used to produce polyclonal antiserum in rabbits. Rabbit anti-LMoV CP IgG was purified by ammonium sulfate graded precipitation followed by DE52 anion-exchange chromatography. 2.4Virus purity estimation by Abs 260/280 Concentration of the pure viral suspension was determined by measuring the UV-absorbance at 260 nm and 280 nm with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The ABS260/ABS280 ratios of viral products were calculated to determine the purity. 2.5 SDS-PAGE and Western blotting analysis Purified virus samples were mixed with 2×SDS-PAGE loading buffer, and then boiled for 5 min. SDS-PAGE was carried out on a 12% acrylamide gel. Protein bands were visualized by Coomassie Blue R250 staining. For Western blotting, the proteins were separated by 12% SDS-PAGE under a reducing condition and then electro-blotted onto a nitrocellulose membrane (Millipore, USA) using a Semi-Dry Transfer System (Bio-Rad, USA). Non-specific protein binding was blocked by incubating the membrane overnight at 4°C in TBS containing 0.05%Tween-20 and 5% skim milk. After washing with TBS-Tween 20 (TBST), the membrane was incubated

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with a 1/3000 dilution of rabbit anti-LMoV CP IgG (at 2.1×10−3 mg/mL) at 37°C for 1 h under constant shaking. The membrane was washed five times with TBST and incubated at 37°C with the appropriate Alkaline phosphatase AffiniPure goat anti-rabbit IgG for 1 h, and subsequently washed five times with TBST. Conjugates binding

to

the

membrane

were

nitrobluetetrazolium/5-bromo-4-chloro-3-indolyl-phosphate

visualized (NBT/BCIP)

using as

the

substrate. 2.6 Nucleic acid extraction and RT-PCR analysis The viral RNA of LMoV preparations was extracted from 100μL samples using RNAprep pure Plant Kit (TiangenBiotech, Beijing, China) according to the manufacturer's instructions. Final elution was done with 50μL of RNAse free H2O.The RNA concentration of the sample was measured with a NanoDropND-1000 spectrophotometer (NanoDrop Technologies,Wilmington, DE, USA). Subsequently, a quadruplex RT-PCR assay was used to detect the viral RNA (Zhang et al., 2010; Zhang et al., 2015). Due to its abundance and uniform distribution in plant cells, the 18S rRNA housekeeping gene is usually used as an internal control (Zhang et al., 2015). The first strand of cDNA was synthesized by M-MLV reverse transcriptase with the Oligo(dT)-18 primer (TaKaRa Biotech, Dalian, China). Quadruplex PCR was carried out in 25 μL volumes containing 2μL of cDNA products, 4mM Mg2+, 0.6mM dNTPs, 0.625U of Taq polymerase (TaKaRa EX Taq, TaKaRa Biotech, Dalian, China), and 0.2μM of each quadruplex sense and anti-sense primer. The PCR amplification consisted of 30 cycles at 94°C for 30s, 52.5°C for 45s, and 72°C for 1 min. Following PCR, The reaction products were analyzed by electrophoresis on a 2.0% agarose gel and were of the expected sizes (395, 198, 248, and 303bp, respectively). 2.7Transmission Electron Microscopy and immuno-capture of virus particles

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Formvar-coated nickel grids were deposited on top of the virus suspension samples for 10 min and then negatively stained with 1% phosphotungstic acid solution (PH 6.8) for 2 min. The specimens were examined with a JEM-1230 transmission electron microscope (JEOL, Tokyo, Japan). In the case of the immuno-capture of virus particles, grids were incubated 20 min with a 1/100 dilution of rabbit anti-LMoV CP IgG(at 6.3×10−2mg/mL), washed and then blocked with a solution of 0.2% BSA in PBS for 15 min. Grids were floated on drops of the virus suspension samples for 30 min and then washed five times with PBS. Samples were negatively stained, dried and examined as described above. 2.8 Immuno-gold labeling Immuno-gold labeling was performed according to Asurmendi et al.(2007).Briefly, formvar-coated nickel grids were floated on drops of virus suspension for 20 min. The grids were treated with PBS containing 1% BSA (w/v) for 20 min and the first antibodies(rabbit anti-LMoV CP IgG) were applied and incubated for 1 h (1/300dilution in PBS containing 0.2% BSA).After washing four times with PBS/BSA (0.2% w/v), the second, gold conjugated antibodies were incubated with the grid for 1 h (1/25 dilution of goat anti-rabbit 15 nm colloidal gold, Beijing Biosynthesis Biotech, Beijing, China). The grids were washed five times with PBS/BSA (0.2% w/v) and rinsed twice with deionized water. Finally, the grids were negatively stained and observed as described above. 3. Results 3.1 Viral yield estimation The virus preparation was analyzed specrophotometrically. The ultraviolet spectrum of the purified preparation showed a typical nucleoprotein curve with a peak near 263nm wavelength. The A260/A280 absorbance ratio calculated for this procedure

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was 1.20 and the concentration was 1.27mg/mL. 3.2 Assessment of LMoV purity The purity of LMoV was verified using polyacrylamide gel electrophoresis. The SDS-PAGE profile of purified LMoV showed a single band with a molecular weight of 30 kDa, and the result suggested a single CP of LMoV(Fig.2A).The identity of this band as the LMoV CP was proven by Western blotting using rabbit specific anti-LMoV CP IgG(Fig.2B). 3.3 RNA extraction and RT-PCR analysis Viral RNA of the purified preparations was analyzed by a NanoDropND-1000 spectrophotometer. Only RNA samples with an absorption ratio (A260/A280) of 1.9 to 2.1 were analyzed. Using the viral RNA as templates analyzed by a quadruplex RT-PCR, the expected 398 bp and 303 bp bands of cDNAs were amplified (Fig.3). 3.4 TEM observation and immuno-gold labeling TEM was used to evaluate particle integrity and quantity. A large number of viral particles were found in purified preparations. The great majority were intact (Fig.4 A1-A4). Morphological characteristics of the virus particles in the purified preparations were identical and similar to those described by King et al. (2011). To further demonstrate these virus particles were LMoV, additional observation was done by immuno-capture and immuno-gold labeling.These virus particles were clearly coated with a shell of anti-LMoV CP polyclonal rabbit IgG (Fig.4 B1-B2). In separate tests, the LSV particles did not bind with the anti-LMoV CP polyclonal rabbit IgG (data not shown). Therefore according to the shell of antibody molecules, LMoV and LSV can be distinguished in mixed samples using the immuno-capture assay. Immuno-gold labeling shows that the entire virus rod had antigenic epitopes to which the gold-labeled antibodies could bind (Fig. 4 C1-C3).

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4. Discussion In this study, an efficient procedure for isolation and purification of natural viral particles of LMoV was developed. The use of different methods of analysis (SDS-PAGE, western blotting, RT-PCR and TEM) proved that the procedure described resulted in high protein purity and viral particle integrity. The procedure enabled efficient LMoV antibody production, due to the easily obtainable pure virus suspensions for immunization. In addition, an IGL assay was successfully applied to detect and unequivocally identify LMoV particles. To the best of our knowledge this is the first time that the IGL technique has been used for the identification of a lily virus. Worldwide, lily is used primarily for ornamental flower production (e.g. Lilium oriental cv. Sorbonne), but in China, lilies are also used as a specialty food crop. Lanzhou lily (Lilium davidii var. unicolor) is an important edible bulb crop as well as a traditional medicinal plant with a 150-year cultivation history in the Lanzhou area of northwestern China (Wang et al., 2010). Cultivars of the Lanzhou lily are commonly and seriously affected by the CMV and LSV, which have decrease lily production by 50% in recent years(Zhang et al., 2015).Recently, RT-PCR, sequencing and electron microscopy of leaf samples of Lanzhou lily from the Gaolan research station confirmed that LMoV from ornamental lily can infect Lanzhou lily and damage the structure of the chloroplast(Zhang, Y.B., unpublished data).This finding indicates that lily growers should adopt measures to control LMoV in Lanzhou lily. In addition, it is necessary to further improve the available detection methods such as ELISA and ICS using highly specific monoclonal antibodies. For this, the isolation and purification of LMoV particles is a necessary step. Plant viruses are usually purified with classical methods, as described by Leiser

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and Richer (1978), which involves differential centrifugation, sometimes after clarification with organic solvents, and generally culminating in a density gradient centrifugation step. Labile viruses may be adversely affected by the latter, and all viruses must generally be free of density gradient material for intact and stable viral properties (Barton, 1977). These methods are complex, time consuming and often inefficient. In this study an efficient LMoV purification method based on differential centrifugation in combination with PEG6000 was developed. Purification showed high protein purity and particle integrity; moreover, it reduced the purification time from four working days required for classic purification to a day and a half. This is the first study where the LMoV was purified. The advantages of this improved purification procedure make it an attractive serological diagnostic tool, although it requires purified virus for the immunization step. In addition, optimization of purification conditions is crucial for the harvest of high quality natural viral particles. In the LMoV purification process, several extraction buffers of different composition and pH were compared. The buffers tested included the 50 mM Tris/HCl buffer, pH 7.4

containing

100mM

NaCl,

10

mM

EDTA,

10

mM

MgSO4,

0.1%

(w/v)polyvinylpolypyrrolidone and 0.1% (v/v) 2-mercaptoethanol, as well as the 500mM sodium citrate buffer, pH 6.5 containing 10 mM EDTA and 0.05 % (v/v) 2-mercaptoethanol and the 500mM potassium phosphate buffer, pH7.0 containing 10 mM EDTA and 0.1% (v/v) 2-mercaptoethanol. From UV spectrum and TEM analyses, the highest viral yield of LMoV was achieved using 500mM potassium phosphate buffer (pH7.0) and the concentration of purified virus preparation was 1.27mg/mL. In contrast, LMoV could not be purified using sodium citrate buffer. The results indicate that LMoV purification is sensitive to the pH of the buffer system. Furthermore, a high concentration of extraction buffer results in better purification than a low

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concentration. Thus, the 500mM potassium phosphate buffer (pH7.0) was shown to be optimum for the purification of LMoV. Buoyant density is one of the non-destructive measurements of viruses, nucleic acids etc. which help in characterizing these entities in terms of their chemical composition and physical conformation. Besides, The MW and other properties of a purified virus are also important and helpful for characterization and research. As for the additional data on MW and buoyant density of the LMoV as well as other parameters, this will be further analyzed using related methods in our future research. The IGL technique is widely used to study human viruses such as hepatitis A, influenza A and B, and others (Kjeldsberg, 1986; Patterson and Oxford, 1986). This technique facilitated detailed morphological studies of these viruses and also proved valuable in diagnostic virology. In addition, the technique has facilitated the identification of different virus serotypes and has also been used to localize specific antigens on virus structures (Patterson and Verduin, 1987). Viruses are commonly labeled after adsorption onto a coated grid. This method is relatively quick and gives a consistently high level of specific labeling and a low background. The IGL assay was originally applied to plant viruses in 1982. Giunchedi and Langenberg (1982) used a lectin to precipitate barley stripe mosaic virus (BSMV), which they then labeled with rabbit anti-lectin and protein-A gold. Pares and Whitecross (1982) labeled five strains of the tobamovirus group with primary antisera and protein-A gold. They called this technique gold-labeled antibody decoration and used it to distinguish between serologically related viruses on the basis of number of gold particles per virus rod. Asurmendi et al. (2007) labeled the tobacco mosaic virus (TMV) with an anti-TMV monoclonal antibody (Mab#16) and gold conjugated antibody.

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To our knowledge, there are few reports of IGL as it relates to lily viruses; mixed infections of LMoV and LSV is very common in lilies, and in fact the morphological characteristics of these two viruses are very similar. Detection of LMoV and LSV in lilies is essential in the production and certification of virus-free lily bulbs. Thus, the IGL technique offers great potential to identify particles of LMoV and LSV with specific antibodies. Furthermore, in our labeling study, 15 nm of goat anti-rabbit antibodies colloidal gold conjugate was used as secondary antibodies to label the particles of LMoV. The labeling results were demonstrated to give a high level of specific conjugation and a low level of background detection. The proper concentration of colloidal gold conjugate antibody is required for the labeling assay or it will lose its sensitivity and increase the background level of detection. Therefore, to obtain the best results, the amount of antibodies colloidal gold conjugate should be optimized in the IGL assay.

Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant No. 31201651), by the “West Light” project of the Chinese Academy of Sciences in 2014, by the China Postdoctoral Science Foundation funded project (Grant No. 2015M580894), by the Chinese Academy of Sciences (CAS) President’s International Fellowship Initiative (PIFI) project (Grant No. 2010T1Z31), and by the Ningxia Agricultural Comprehensive Development Office (NTKJ-2015-05-01). Thank you to Dr. H. R. Kutcher for review of the manuscript.

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References Alper, M.K., Lesemann, D.E., Loebenstein, G., 1982. Mechanical transmission of a strain of tulip breaking virus from Liliumlongiflorum to Chenopodium spp. Phytoparasitica 10, 193-199. Asjes, C.J., 2000. Control of aphid-borne lily symptomless virus and lily mottle virus in Lilium in the Netherlands. Virus Res.71, 23-32. Asurmendi, S., Berg, R.H., Smith, T.J., Bendahmane, M., Beachy, R.N., 2007. Aggregation of TMV CP plays a role in CP functions and in coat -protein-medicated resistance. Virology 366, 98-106. Barton, R.J., 1977. An examination of permeation chromatography on columns of controlled pore glass for routine purification of plant viruses. J. Gen. Virol. 35,77-87. Derks, A.F.L.M., Lemmers, M.E.C., van Gemen, B.A., 1994. Lily mottle virus in lilies characterization, strains and its differentiation from tulip breaking virus in tulips. Acta Hort. (ISHS), 377, 281-288. Giunchedi, L., Langenberg, W.G.,1982. Efficacy of colloidal-gold-labelled antibody asmeasured in a barley stripe mosaic virus lectin-antilectin system. Phytopathology72, 645-647. Hsu, H.T., Kim, J.Y., Lawson, R.H., 1995. Purification of lily symptomlesscarlavirus and detection of the virus in lilies. Plant Dis. 79, 912-916. King, A.M.Q., Lefkowitz, E., Adams, M.J., Carstens, E.B., 2011. Virus Taxonomy:Classification and Nomenclature of Viruses. Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, San Diego, pp.1075. Kjeldsberg, E., 1986. Use of gold IgG complexes and human antisera for

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electronmicroscopic identification of hepatitis Avirus and polioviruses. J. Virol. Methods 13, 207-214. Leiser, R. M., Richter, J., 1978. Purification and some characteristicsof potato virus Y. Arch. Phytopathol. Pflanzenschutz 14, 337-350. Liang Q.L., Wei L.X., Xu B.L., 2009. Serological detection methods of ornamental lily infectedwith cucumber mosaic virus. J. Gansu Agric. Univ. 44(3), 97-101 (in Chinese with English summary). Pares, R.D., Whitecross, M.I., 1982. Gold-labelled antibody decoration (GLAD) in the diagnosis of plant viruses by immuno-electron microscopy. J.Immunol. Methods 51(1), 23-28. Patterson, S., Oxford, J.S.,1986. Analysis of antigenic determinants on internal andexternal proteins of influenza virus and identification of antigenic subpopulationsof virions in recent field isolates using monoclonal antibodies and immuno-goldlabelling. Arch.Virol. 88, 189-202. Patterson, S., Verduin, B.J.M., 1987. Application of immuno-gold labeling in animal and plant virology. Arch.Virol. 97, 1-26. Rupar

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Fig.1. Lily leaves exhibiting mottle symptoms

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Fig.2.(A)SDS-PAGE analysis of purified LMoV preparations(lanes 2- 4) and control (lane1). Samples were separated by electrophoresis on a 12% polyacrylamide gel. (B) Western blot analysis of purified LMoV preparations (lanes 2- 4) and control (lane1), which were separated by 12% SDS-PAGE, blotted to Polyvinylidenedifluoride (PVDF) membranes and probed with polyclonal rabbit anti-LMoV CP IgG.

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Fig.3.RT-PCR products of purified LMoV coat protein gene. Agarose gel electrophoresis of quadruplex RT-PCR products show the 393 and 303 bp bands (Inner control: 18 S rRNA gene) fragments amplified, respectively. Lane M: marker; lane 1: control; lanes 2-4: RT-PCR products of RNA of purified preparations.

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Fig.4.(A1-A4) Purified LMoV particles observed by transmission electron microscopy (TEM);(B1-B2)binding of rabbit anti-LMoV CP IgG on the virions of LMoV; (C1-C3) LMoV labeled with a rabbit anti-LMoV CP IgG and a 15nm goat anti-rabbit IgG colloidal gold conjugate. The classic black dots on the virions indicate that they are positively labeled with immuno gold.

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