Inouye, Narinobu

Maeda, Takanori

Okayama University Digital Information Repository

DIFFERENTIATION OF TWO SEROTYPES OF CUCUMBER MOSAIC VIRUS IN JAPAN BY F(ab')2 ELISA WITH CROSS-ABSORBED ANTIBODIES*

Okayama University Scientific Achievement Repository

Ber. Ohara Inst. landw. Biol.， Okayama Univ. 19: 149-157 (1987) DIFFERENTIATION OF TWO SEROTYPES OF . CUCUMBER MOSAIC VIRUS IN JAP AN BY F (ab') 2 ELISA WITH CROSS-ABSORBED ANTIBODIES申Takanori MAEDA and Narinobu INOUYE INTRODUCTION Cucumber mosaic virus (CMV) has a very wide host range including both herbaceous and woody plants and causes economically important diseases of many crops throughout the world. In Japan， CMV isone of the most common viruses occurring on vegetable and ornamental crops. CMV isolates found in Japan are serologically classified into two distinct groups， designated Y and P serotypes 4，11). The determination of CMV serotypes has been done by a gel double-diffusion test. In this test， purified or partially purified virus preparations are usually used as antigens because this test needs relatively higher concentrations of antigens to develop reactions. Recently， the various forms of enzyme-linked immunosorbent assay (ELISA) have been applied to detect many plant viruses as very sensitive procedures. The application of ELISA for identifying virus serotypes previously has been repoi:ted for some plant viruses 2， 7，13，17) including CMV10， 12， 14). However， differentiation of CMV serotypes by ELISA seems to be unreliable when crude sap from infected plants or virus samples of unknown concentration are used， as ELISA values for each strain may vary depending on antibody specificities and antigen concentra-tions used in the tests. This paper describes the differentiation of two J apanese serotypes of CMV by F( ab' h ELISA with cross-absorbed antibodies with heterolo-gous strains and its application to serotyping of CMV isolates obtained from Japan. MA TERIALS AND METHODS 1. Viruses The yellow strain of CMV (CMV-y)16) and the strain from Zinnia elegans (CMV-Z)ll) were used as the standard strains for Y and P Institute for Agricultural and Biological Sciences， Okayama University， Kurashiki， 710 Japan Receved July 3， 1987. * Supported in part by Grant-in-Aid for Developmental Scientific Research (No. 61860∞5) from the Ministry of Education， Science and Culture of Japan. 150 T. Maeda and N. Inouye serotypes 4)， respectively. Viruses were propagated in Nicotiana tabacum cv. White Burley and were purified by the method previously describedll). CMV isolates obtained from various plants in Japan were maintained in N. tabacum cv. White Burley or in N. clevelandii after serial local lesion transfers on Chenopodium quinoa. 2. Production 0/ Antisera Antisera to CMV -Y and CMV -Z were prepared in rabbits. Each animal received four intramuscular injections of purified virus (tota110 mg) emulsified with an equal volume of Freund' s complete adjuvant at 2-week intervals and was bled at 120 days after the initial injection. Both antisera to CMV -Y and CMV -Z had homologous and heterologous titers of 1 : 256 and 1 : 128， respectively， in double-diffusion tests. Spurs were formed between the precipitin lines of two serotypes in reciprocal combinations of antisera and antigens. The antisera were absorbed with insolubilized healthy tobacco proteins before use3). 3. Absoゆtion0/ Antiseraωth Heterologous Strains and Preparation 0/ lmmunoglobulin The antiserum to each serotype was diluted ten fold with phos-phate-buffered saline (PBS) and purified heterologous virus was added to the antiserum (5mg of virus/ 1m 1 of undil uted antiserum). After incubation for lhr at 37"(;， the reaction mixtures were centrifuged at 65， 000 xg for 1. 5hr to remove virus-antibody complexes. This absorption was repeated four times. IgG was isolated from the native and the cross-absorbed antisera by ammonium sulfate precipitation followed by DEAE-cellulose column chromatography. F(ab')2 fragments of IgG were prepared by digesting IgG with pepsin (1 : 60，000， Sigma) 1 ).4. F(ab')2 ELISA F(ab'h ELISA was performed essentially as described by Barbara and Clark 1 ). Wells of polystyrene microtiter plates (Nunc-Immuno plate U 1， Inter Med) were incubated with F(ab')2 (2μg/ml，180μl/well) in 0.05M sodium carbonate buffer， pH 9.6， for 3hr at 30"(;. Plant extracts were prepared by homogenizing tissues with PBS containing O. 02 % sodium azide， O.05% Tween-20， 2% polyvinylpyrrolidone (mol wt 40， 000) and 0.2% bovine serum albumin (PBS/Tween/PVP/BSA) . The homogenate was filtered through a filter paper (Whatman No. 2) . Purified virus or plant extract at l00-fold dilution in PBS/Tween/ PVP/BSA was added to the F(ab')2 coated wells and was then incubated for 16hr at 4 t. After washing the wells， IgG from native or cross-absorbed antiserum was added as detecting antibodies and incubated for 3 hr at 30"(;. Differentiation of Serotypes of Cucumber Mosaic Virus by ELISA 151 Protein A (Nakarai Chem. Co.， Kyoto) was conjugated simultaneously with horseradish peroxidase (Type トC，264u/mg， RZ : 3.29， Toyobo Co.， Osaka) and BSA at a ratio of 0.5 : 1 : 0.75 (w/w) by the periodate oxidation procedurel). Following the addition of protein A conjugate diluted 1 : 4，000， the plates were incubated for 3hr at 30t; and then the enzyme substrate (0. 5g/ l o-phenylenediamine in O. 025M sodium acetate buffer， pH 5.5， containing 0.06% hydrogen peroxide)was added. The reaction was stopped by the addition of 50μlof 3M sulphuric acid to each well after 30min at 30t; and absorbance of reacted mixture was measured at 490nm. RESUL TS AND DISCUSSION 1. Reactions 01 Two Serotypes against Homologous and Heterologous Antibodies in F(ali)2 ELISA Two serotype of CMV were compared in F(ab')2 ELISA against homologous and heterologous antibodies. Purified CMV -Y and CMV -Z at concentrations of ranging from 0.01 to 10μg/ml were added to the wells ???-e・g 0.5 m .0 2 .0 〈1.0 ~ A B 。10 0.1 0.01 Virus concentration (μgJm2 ) 10 0.1 0.01 FIG. 1. Comparison of F(ab'h ELISA values for two serotypes of CMV in homologous and heterologous systems. Purified CMV-Y(serotype Y) and CMV-Z (serotype P) were added to the wells of microplates precoated with F(ab'lz (2μg/ml) prepared from antiserum to CMV-Y (A) orCMV-Z (8) andthenhomologouslgG (2flg/ml) tocoatedantibodywas reacted. The antibodies bound on trapped viruses were detected by protein A -peroxidase conjugate at a dilution of 1 : 8αm 152 T. Maeda and N. Inouye precoated with F( ab') 2 fragments of respective antibodies. After incuba-tion， homologous IgG (2μg/ml) to coated F(ab'h was added to react with the trapped viruses. The bound antibodies on trapped viruses were detected with protein A -peroxidase conjugate. ELISA values for each serotype were higher in homologous than those in heterologous combina-tions of antibody and virus within the range of virus concentrations tested (Fig. 1). In this system， the antibodies to CMV-Y and CMV-Z could differentiate homologous from heterologous serotype. However， the difference in ELISA values between the two serotypes was less compared with those of direct DAS ELISA 10). Indirect ELISA has been observed to exhibit lower specificity in differentiating among serologically related viruses or strains of virus than direct ELISA 8.18). This may be attributed to the differences in specificities of detecting antibodies used in the two forms of ELISA. In direct ELISA， antibody reactivity may be reduced after enzyme conjugtion and this may reduce the extent of cross-reactions in heterologous systems as suggested by Koenig 7 ). On the other hand， indirect ELISA has a wider specificity than direct ELISA because of using native antibodies 8 • 18). 2. Reaction 01 Two Seroty.ρ白 withCross-absorbed Antibodies in F(al/)2 ELISA The use of ELISA for differentiating plant viral serotypes has been reported 2， 7，13，14，17，18). However， differentiation of very closely related serotypes such as strains of CMV by ELISA seems to be unreliable when unknown concentrations of virus preparations are used as antigens because ELISA values may vary depending on virus concentrations， antibody specificities and reaction conditions used. To overcome this， cross-absorbed antibodies with heterologous strains were examined to detect selectively two serotypes. Purified viruses were reacted in F(ab')2 coated wells and then homologous cross-absorbed antibodies (IgG fraction) to coated F(ab')2 were added to the wells. To determine the optimum concentration of cross-absorbed antibodies， various concentrations of antibodies ranging from 2μg to 20μg/mlwere tested against homologous serotypes. Relatively high ELISA values were obtained at higher concen-trations of IgG up to 10μg/ml However， only weak color developments were observed at lower concentrations of IgG. Low activities of the cross-absorbed IgG apparently due to elimination of common antibodies to both serotypes after cross-absorption. As 10μg/mlof IgG concentration seemed to be reasonable for enough color development， this concentration of IgG was adopted for al further experiments. The ability of this system for selective detection of two serotypes of CMV was examined using 153 purified viruses. As shown in Fig. 2， positive reactions were observed with homologous combinations and gave only weak reactions with heterologous systems. To determine the suitability of this procedure for differentiation of two serotypes using crude sap， preparations from the tobacco plants infected with several strains belong to Y or P type were tested at a sap dilution of 1 : 100. The homologous ELISA values for al the strains tested were higher than those of heterologous reactions (Table 1) and we obtained similar results to those from the experiments employing purified preparation of CMV. This method has some advantages over gel diffusion test. This may be useful for rapidly differentiation of CMV serotypes using crude sap from field-collected samples. Differentiation of Serotypes of Cucumber Mosaic Virus by ELISA B A Z Y 0.5 ????? ?????』〈0.01 0.1 0.1 0.1 10 Vil'l5∞ncentration (μ81m2 ) 10 。FIG. 2. Selective detection of two serotypes of CMV in F(ab'h ELISA with cross-absorbed antibody. Purified CMV-Y (serotype Y) and CMV-Z (serotype P) were added to the wells of microplates precoated with F(ab')2 prepared from antiserum to CMV-Y (A) orCMV-Z (B) and then homologous IgG (10μg/ml) absorbed with heterologous serotype was reacted. Other details as in Fig. 1. 154 T. Maeda and N. Inouye TABLE 1. Reaction of sorne CMV isolates in F(ab')z ELISA with cross -absorbed antibodies. Serotype Isolate Cross -absol悦 dantibodies to Y type P type CMV -Y 0.87* * 0.09 O. 76 0.12 Y 2 0.98 O. 18 3 O. 73 0.11 CMV -Z 0.12 1. 02 4 0.09 0.89 P 5 0.11 O. 79 6 0.12 0.69 * Crude sap frorn infected tobacco plants was used as antigens at a dilution of 1 : 100. Other details as in Fig. 2. -・ Absorbancevalues at 490nrn for undiluted reacted substrate. Each value is the average for two separate experirnents. 3. Serotyping 01 CMV Isolates Found in Ja_μn by F( ali) 2 ELISA with Cross-absorbed Antibodies The above experiments demonstrated that two serotypes of CMV could be differentiated from each other by F(ab')2 ELISA with cross-absorbed antibodies. One hundred and twenty-four CMV isolates obtained from 38 plant species in 20 families collected in various districts in Japan were serotyped by this method. The serotypes of some isolates have already been determined by a gel double-diffusion test previously 5. 6. 9 • 11). As listed in Ta ble 2 and 3， 115 isola tes belong to Y and 9 to P serotypes. It was very interesting that al the 3 isolates obtained from Petasites japonicuswere P type. Tochihara and Tamura 15) also reported an isolate of P type from this species. This fact may strongly suggest that the P serotype of CMV has an affinity to P. japonicus. 155 Differentiation of Serotypes of Cucumber Mosaic Virus by ELISA T ABLE 2. Detection of Y serotypes of CMV from various plants in J apan Number of isolates Host plant L田 alityAmaryllidaceae Mie， Ooita Okayama Okayama Tokyo Fukuoka， M阻Mie， Miyazaki， Okayama Fukuoka， Gunma， bhikawa， Kagawa， M問(5 ).， Miyagi ( 4 ).Miyazaki. Nagano. Nara. Okayama (4 ) M問 (2) Species Na町iS5凶 spp.(narcissus) 2 HitJ町出trumsp. (amaryllis) 1 A戸)Cynaceae Nerium indicum( seet -scented oleander) 1 Balsaminaceae lmpatiens bal岨mina(garden balsam) 1 Caryophyllaceae Stellaria media (chickweed) 2 Chenopodiaceae Stinacia oleracea (spinach) 3 Commelinaceae 白.mmelinacommunis ( dayf10wer ) 20 Family Fukuoka Mie. Nara. Okayama Okayama Mie Okayama Fukuoka ( 2 ). Kumamoto (4 ). M陀 (3). Okayama ( 2 ).S副tama.Shimane ( 2 ) Okayama Mie ( 2 ).Miyagi， Okayama. Shimane. Yamaguchi Fukuoka Niigata (2) Okayama Nagano ( 2 ) Mie. Miyagi. Okayama (2) Fukuoka. Gunma Mie ( 2 ). Miyagi， Okayama Mie， Okayama. Tokushima Fukuoka ( 5 ).Kagoshima ( 4 ).Okayama ( 2 ).Saga ( 3 ) Mie. Okayama (2) Okayama Miyazaki. Okayama. Tokyo Okayama Fukuoka. Mie ( 2 ).Okayama Fukuoka. Miyagi， Okayama Fukuoka. Okayama Fukuoka. Saga Fukuoka Fukuoka. M阻2 ?????????ーChry皿 nthemummorifolium ( chrysanthemum ) Dahlia sp. (dahlia) Zinnia elegan$ ( zinnia ) BrassIca叫 mμ'st巾 (chinensisgroup) B oleracea (cabbage) Catsella bursa -μs如巾Rath仰凶 sativw(Japanese radish) Compositae Cruciferae ?????。，???????，????????ーCucurbita maIima (pumpkin) CucumiJ却 t.v凶 (cucumber)Trichosanthes cucumer別 d白Gentiana scabra var.仇'ergenZea mays ( COrn ) Glycine ma.x ( soybean ) Ph前回lusangularis (adzuki bean) R 四 Igaris( snap bean ) 的'gnasinensis ( cowpea ) Li/ium X elegans ( Thunberg liIy ) L langiflorum (Easter lily ) Cucurbitaceae Gentianaceae Graminineae Leguminosae Liliaceae ????????????????T円。tisspp. (hairly tod liIy ) Plumbaginaceae Limonium sin凶 tum(statice) Scrophulariaceae Antirrhinum majus (9na凶ragon)Paulownia tomento.岨(paulownia ) Catsi，佃 mannuum (sweet pepper) Lycopenicon escuw山 m (tomato) Petun凶 Xhybrida (petunia ) Sola制 m melongena (eggplant) 品川neodora (winter daphne ) Zingiber mioga Solanaceae Thymelaeaceae Zingiberaceae Figures in parentheses indicate number of isolates . • 156 T. Maeda and N. Inouye TABLE 3. Detection of P serotyses of CMV from various p1ants in Japan F .凶IyHost plant 勾則"Number of 泊d嶋田 Loeality CotnposItae Petasites ja.如nicus(1apanese butterbur) 3 Gunma， Okayama， Saitama 1 Nagano Cucurbitaceae Leguminosae Orchidaceae Solanaceae Violaceae Zinnia elegans (zinnia) Cucumis .sativus (cucumber) Phaseolus anguUlris (adzuki bean) Calanthe discolor Lycopersicon e.即 lentum(tomato) Viola X wittrockiana (garden pansy) SUMMARY Mie Miyagi 1 Okayama 1 Miyagi 1 Okayama Two serotypes of cucumber mosaic virus (CMV) designated Y and P types in J apan were tested against homologous and heterologous anti-bodies in F(ab')2 ELISA. The ELISA values of homologous reactions were higher than those in heterologous combinations. The use of cross-absorbed antibodies with heterologous serotypes as detecting anti-bodies was demonstrated to be valuable in differentiating two serotyes of CMV in F( ab')2 ELISA using crude sap from infected plants. One hundred and twenty-four isolates of CMV obtained from 38 plant species in 20 families collected in Japan were serotyped by this method. One hundred and fifteen isolates belonged to Y and 9 to P serotypes. A1l3 strains isolated from Peωsites japoniωs were P type strains. REFERENCES 1. Barbara，D. 1.and Clark， M. F. 1982. A simple indirect ELISA using F(ab'lz fragments of immunoglobulin. J. gen. Virol. 58 : 315-322. 2. Barbara，D. 1.， Clark M. F.， Thresh，J. M. and Casper， R.1978. Rapid detection and serotyping of prunus necrotic ringspot virus in perennial crops by enzyme-linked immunosorbent assay. Ann. Appl. Biol. 90 : 395-399. 3. Clark， M， F， and Bar-Joseph， M. 1984. Enzyme immunosorbent assay in plant virology. In Methods in Virology. 7 : 51-85. 4. Hanada， K. and Tochihara， H. 1980. Genetic analysis of cucumber mosaic， peanut stunt and chrysanthemum mild mottle viruses. Ann. Phytopath. Soc. Japan. 46 : 159-168. 5. lino， N.， Inouye， N. and Maeda， T. 1987. 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DIFFERENTIATION OF TWO SEROTYPES OF CUCUMBER MOSAIC VIRUS IN JAPAN BY F(ab')2 ELISA WITH CROSS-ABSORBED ANTIBODIES*

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