MOLECULAR TECHNIQUES FOR PLUM POX POTYVIRUS (PPV) DETECTION AND CLONING OF THE GENOMIC RNA FROM AN ATYPICAL STRAIN: PPV EL AMAR

A dot-blot molecular hybridization assay using ³²P-labelled cRNA probes has been developped for PPV detection. Probes corresponding to PPV D non-structural protein genes detected both serotypes PPV D and PPV M with an identical sensitivity (5 pg of purified virus per assay). These probes detected with maximal sensitivity all the isolates maintained in our laboratory, except for an Egyptian isolate, PPV El Amar, which was only poorly detected. A cDNA bank to the genomic RNA of PPV El Amar has been prepared, and the 3' half of the genome has been sequenced. A divergency level of 20% has been observed between the partial nucleotide sequence of PPV El Amar and the corresponding sequences of the three other strains of PPV already sequenced. A single tube PCR test has been developped for PPV detection, with a sensitivity of 10 fg of purified viral RNA, this test proved to be more sensitive than both molecular hybridization and ELISA assays. The test proved also to be polyvalent, an amplified fragment being obtained from all the PPV isolates tested, including PPV El Amar. Presence or absence of an Rsal restriction site in the amplified fragment allows to distinguish two groups of PPV isolates.