The approved screening protocol for the field is the PPV Enzyme-Linked ImmunoSorbent Assay (ELISA). The work instruction is available upon request. The work instruction describes detection of PPV using the ELISA kit from Agdia Inc., which detects six known PPQ strains/subgroups: PPV-C, PPV-D, PPV-EA, PPV-M, PPV-Rec, and PPV-W in leaves, fruit, and flowers.
In Progress / Literature-based Diagnostics:
Indicator Hosts: Prunus persica cv. GF305, P. tomentosa, Chenopodium foetidum, Pisum sativum, and others can be inoculated and monitored for diagnostic symptom development.
Antibodies: Monoclonal antibody 5B-IVIA (Cambra et al., 1994) allows for the universal detection of PPV. Strain-specific monoclonal antibodies have also been developed for both the D and M strains/serotypes (Cambra et al., 1994; Boscia et al., 1997). These enzymes, however, are not suited for differentiating other serotypes (e.g., El-Amar and Cherry) (Candresse et al., 1998). Polyclonal and monoclonal antibodies have also been produced for the PPV-C and PPV-EA strain (Boscia et al., 1997; Crescenzi et al., 1997; Myrta et al., 1998; Myrta et al., 2000).
ELISA: Clark and Adams (1977) developed the first ELISA test and included Plum pox virus as an application of this technology to plant viruses. Strain-specific antibodies are available (see section above).
Lateral Flow Device: Mumford et. al. (2010) describe a lateral flow device for on site detection of PPV. The on site kit, which contains a one-step lateral flow device and a simple, bottle extraction system, can give a result in three minutes.
Immunochromatographic Assay: Byzova et al. (2010) raised two monoclonal antibodies that recognized strains PPV-D, M, and C. The authors developed a 10-minute immunochromatographic assay for PPV with a detection limit of 3 ng/ml. The assay demonstrated good compatibility with the data obtained via ELISA.
Molecular: Sequence analysis of PCR fragments corresponding to the C-terminal part of the PPV coat protein gene has allowed identification of a molecular polymorphism correlated to serotype of the PPV isolates (Candresse et al., 1994; Candresse et al., 1995). Initial results have indicated that an RsaIrestriction fragment length polymorphism (RFLP) located in this region could be used for PCR amplification, to discriminate between D and M serotypes of PPV (Bousalem et al., 1994; Wetzel et al., 1991). More recently, a cluster of non-coding, third-base mutations on five consecutive codons located around the RsaI RFLP site was found to show excellent correlation with the viral serotype (Candresse et al., 1995). This observation was used as a basis for direct PCR typing of isolates belonging to the D and M serotypes of PPV (Candresse et al., 1994).
Levy and Hadidi (1994) utilized a simple and rapid procedure for processing PPV infected plant tissue (Gene Releaser) for use with a specific 3' non-coding region RT-PCR assay. The 3' non-coding region was used, because Asian Prunus latent potyvirus, a newly identified latent potyvirus in Prunus spp., may react positively with PPV-coat protein primers, in Southern blot hybridization with a PPV coat protein clone, and in ELISA with PPV polycolonal antiserum (Hadidi and Levy, 1994).
Immunocapture PCR (IC-PCR), reverse transcriptase PCR (RT-PCR), RT-PCR with RFLP, PCR-ELISA, print-capture PCR, and integrated RT-PCR/nested PCR have been used to detect PPV and to type strains/serotypes of PPV (Wetzel et al., 1991; Wetzel et al., 1992; Olmos et al., 1996; Olmos et al., 1997; Olmos et al. 1999; Poggi Pollini et al., 1997; Hammond et al., 1998; Nemchinov et al., 1998; Staniulis et al., 1998; Szemes et al., 2001; Glasa et al., 2005; Papayiannis et al., 2007; Olmos et al., 2008). Szemes et al. (2001) developed a RT-PCR/nested PCR technique for the simultaneous detection of PPV-D, M, EA, and C.
Faggioli et al. (1998) compared three different techniques to prepare PPV viral RNA for RT-PCR: 1) an immunocapture technique using a specific antiserum, 2) a silica-capture method using a non-specific matrix, and a simple and rapid RNA extraction. All three techniques allowed for the successful amplification and detection of PPV, but the silica capture method was less effective.
Candresse et al. (1998) compared an indirect double antibody sandwich ELISA using monoclonal antibodies for PPV-D and PPV-M with specific PCR assays or RFLP analysis of PCR fragments. Overall, the authors found an excellent correlation between the results of the ELISA and PCR assays for PPV-D and PPV-M. IC-PCR has been shown to be about a thousand times more sensitive than ELISA (Candresse et al., 1995).
Sanchez-Navarro et al. (2005) developed a multiplex RT-PCR for the detection of eight stone fruit viruses, including PPV. Jarosova and Kundu (2010) used a single-tube multiplex RT-PCR to detect PPV, Prune dwarf virus, and Prunus necrotic ringspot virus. Both methods included an internal control.
Real-Time PCR: Schneider et al. (2004) developed a real-time, fluorescent, RT-PCR reaction assay for the detection of PPV in the Smart Cycler (Cepheid). Varga and James (2005) developed real-time multiplex assay utilizing SYBR Green technology to detect and differentiate PPV-D and PPV-M types in woody and herbaceous plants. Olmos et al. (2005) used Taqman technology in real-time assay for the universal detection and quantification of PPV in plant material and aphid vectors. The sensitivity of the real-time RT-PCR assay was 100 times higher than nested RT-PCR and 1000 times higher than ELISA and conventional RT-PCR.
In the United States, the disease was recorded in Pennsylvania in 1999 followed by New York and Michigan in 2006. The disease has since been eradicated in Michigan and Pennsylvania. Eradication efforts are continuing in New York (Levy et al., 2000).
Seven strains of PPV (D, M, El-Amar, C, W, T, and Rec) have been identified worldwide based on their biological, serological and molecular properties to date. PPV-M and PPV-D are the most widespread. All occurrences in the United States have been identified as strain D (PPV-D).