Identification and Differentiation ofPrunusVirus Isolates that Cross-React with Plum Pox Virus and Apple Stem Pitting Virus Antisera

Abstract

Prunus virus isolates (PVIs) were found that cross-reacted with plum pox virus (PPV) polyclonal antisera in immunosorbent electron microscopy, immunogold labeling, and Western blot analysis. The positive reactions in Western blot analysis were not affected when bovine serum albumin, fetal calf serum, glycerol, D-glucose, or skimmed (defatted) milk were used as blocking agents. Monoclonal antibodies to PPV reacted with the PVIs in Western blot analysis. The PVIs and some PPV isolates also cross-reacted in immunosorbent electron microscopy and Western blot analysis with apple stem pitting virus polyclonal antisera. The RNAs associated with the PVIs were analyzed to evaluate their homology with PPV RNA. The double-stranded RNA (dsRNA) profile of the PVIs showed that there were 3 to 5 dsRNA species ranging in size from 2.7 to 7.4 x 10 6 Da. Oligonucleotide sequences corresponding to the 3' noncoding region, the 5' noncoding region, and the 3' terminus of the coat protein coding sequence of a non-aphid-transmissible strain of PPV (PPV-NAT) were used as probes in hybridization studies and as primers in reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The PVIs did not react. RT-PCR analysis using PPV-specific primers from the conserved 5' terminus of the coat protein coding sequence of PPV gave negative results with all PVIs. These analyses of the viral genome indicate that the PVIs are not isolates of PPV. Further studies to determine if the PVIs were members of the potyvirus group were negative. No evidence of associated cytoplasmic cylindrical inclusions or reaction with a potyvirus-specific monoclonal antibody was observed. The PVI particles ranged in size from 740 to 791 nm in length, and 10.4 to 14.4 nm in width. The coat protein subunit molecular mass ranged from 48 to 56 kDa, and can be distinguished from PPV by Western blot analysis. The implications of the serological cross-reactions are discussed.