EFFICIENT CLONING OF CDNA FROM GRAPEVINE LEAFROLL-ASSOCIATED VIRUS-4 AND DEMONSTRATION OF PROBE SPECIFICITY BY THE VIRAL ANTIBODY

Using a random-PCR method, a cDNA clone (LR4) was constructed from the replicative form dsRNA of grapevine leafroll-associated virus 4 (GLRa V-4). Northern blot analysis showed hybridization of LR4 to dsRNA in a n extract of a Thompson Seedless grapevine clone from which GLRaV-4 wa s isolated originally by Hu et al. (1990). The cDNA clone was sequence d and shown to be specific to GLRaV-4 by reverse-transcription-PCR usi ng GLRaV-4 particles enriched by the virus antibody coupled to magneti c beads. Reverse-transcniption-PCR was used successfully to screen dif ferent varieties of grapevines for the virus. Western blot analysis of GLRaV-4 extracts from different varieties of infected grapevines reve aled two distinct species of capsid protein with estimated Mr of eithe r 35 500 or 38 000 depending on the variety used. Both proteins reacte d with polyclonal as well as monoclonal antibodies. (C) 1998 Elsevier Science B.V. All rights reserved.