Detection of a New and Unusual Isolate of <i>Plum pox virus</i> in Plum (<i>Prunus domestica</i>)

Abstract

Plum pox virus (PPV) isolate 3174-01 was detected by triple-antibody sandwich enzyme-linked immunosorbent assay using the universal PPV monoclonal antibody (MAb) 5B as the secondary antibody, and by reverse-transcription polymerase chain reaction (RT-PCR) using primers that amplify a 243-bp fragment in the C-terminus of the coat protein coding region. The restriction sites RsaI and AluI were absent from this fragment, which is a feature unique to PPV-C isolates. The restriction sites in 3174-01 were replaced by GTAA/GTGA and GGCA, respectively. There was 95 to 99, 94, 91, and 92 to 94% identity of the 243-bp fragment of 3174-01 with the corresponding region of the strains C, D, EA, and M, respectively. Attempts to detect the virus by RT-PCR using strain C-specific primers in three different approaches were unsuccessful. All molecular techniques assessed in attempting to strain type isolate 3174-01 gave negative results, or results inconsistent for D or M in the case of P3-6K₁ restriction fragment length polymorphism analysis. Isolate 3174-01 reacted in Western blot assay with MAb 5B, with an estimated molecular mass of 32 kDa. No reaction was observed with D-, M-, EA-, or C-specific monoclonal antibodies in Western blot or enzyme-linked immunosorbent assay. The molecular and serological data seem to indicate that PPV isolate 3174-01 does not belong to any of the recognized strains of PPV.