Characterization and Serological Detection of Four Closterovirus‐like Particles Associated with Leafroll Disease on Grapevine

Abstract

Four serologically unrelated closterovirus-like particles (GLRV-1, GLRV-2, GLRV-3 and GLRV-4) were isolated in our laboratory from leafroll diseased grapevines. Polyclonal antibodies raised against these particles were useful for their characterization and their detection m infected plants.The coat proteins of these four serotypes were characterized by a SDS-PAGE after denaturation, followed by a transfer on nitrocellulose sheet and itnmunoprinting using the specific polyclonal antibodies. The capsid of GLRV-1, GLRV-2, GLRV-3 and GLRV-4 contains a single protein species with molecular weight of about 39 Kd, 26 Kd, 43 Kd and 36 Kd respectively. No serological relation was found between tbese four filamentous particles either by ELISA, immuno electron microscopy or immunoblotting experiments.Serological analysis of many grapevines originating from tbe Middle East and Europe sbowed a ver)' close association between the presence of GLRV-1, GLRV-2, GLRV-3 and GLRV-4 antigens, and leafroll symptoms on Vitis vinifera Pinot Noir. This association was confirmed by testing sytnptomless and diseased grapevines collected in the field, and by serological analysis of heat treated plants originally infected by GLRV-1 and GLRV-3, which are the most widespread antigens detected in leafroll infected grapevines. ZusammenfassungCharakterisierung und serologischer Nachweis von vier mit der Blattrollkrankheit der Rebe assoziierten Closteroviren Vier serologisch nicht verwandte Glosteroviren (GLRV-1, GLRV-2, GLRV-3 und GLRV-4) wurden aus blattrollkranken Reben isoliert. Polyklonale Antikorper, die gegen diese Partikel gewonnen wurden, konnten mit Erfolg zu deren Charakterisierung und Nacbweis in infizierten Pflanzen eingesetzt werden. U.S. Copyrigh, Clearance Center Code Su.e^eni: 093 1 -1 785/90/3003-0205S02.50/0 206 ZiMMERMANN, BASS, LEGIN and WALTER

Expression of Grapevine leafroll-associated virus 3 coat protein gene in Escherichia coli and production of polyclonal antibodies

“…GLRaV-3, the type-member of the genus Ampelovirus (Closteroviridae), is the best characterized among the viruses of the grapevine leafroll virus complex. The coat protein (CP), encoded by ORF 6 (Ling et al, 1997;Martelli et al, 2002), has a predicted molecular mass of approximately 35 kDa (Ling et al, 1997), estimated at 41.6 kDa (Rigotti et al, 2006) and 43 kDa (Zimmermann et al, 1990) by SDS-PAGE and Western blot. According to Ling et al (1997), the difference between the predicted molecular mass on the basis of the deduced amino acid sequence and estimated values from SDS-PAGE lies within acceptable range.…”

Establishment of a relationship between grapevine leafroll closteroviruses 1 and 3 by use of monoclonal antibodies

“…The procedure was essentially the same as that described by Zimmermann et al (1990a). IgGs were purified from the polyclonal antiserum by the rivanol precipitation method described by Hardie & Van Regenmortel (1977).…”

“…The procedure used was essentially the same as that described by Zimmermann et al (1990a). After sucrose gradient centrifugation, the virus fractions recorded at 254 nm were collected with an ISCO-640 fractionator and dialysed overnight at 4ЊC in 0·1 M Tris-HCl pH 8·2, containing 0·01 M MgCl 2 .…”

“…These have been named grapevine leafroll-associated viruses 1-7 (Gugerli et al, 1984;Hu et al, 1990a;Zimmermann et al, 1990a;Boscia et al, 1995;Choueiri et al, 1996). Among these, GLRaV-1 and -3 are the most frequently detected in leafroll-infected grapevines worldwide (Walter & Martelli, 1997).…”

Serological Methods to Confirm Expression of Coat Protein Gene From an Iranian Isolate of Cucumber Mosaic Virus in Escherichia coli

“…Detection and prevention are the critical steps in the control of plant viruses. Detection in a large number of samples is still done by serological methods due to their robustness and perhaps low cost (8,9). After the advent of recombinant DNA techniques, expression of viral genes in E. coli has been an important strategy for obtaining largescale recombinant proteins that can be used for production of virus-specific antibodies (10-13).…”