Plum Pox Virus Isolates Research Articles

Identification of Plum pox virus Determinants Implicated in Specific Interactions with Different Prunus spp.

ABSTRACT The characterization of pathogenic properties of two infectious clones of Plum pox virus (PPV) isolates, pGPPV (D group) and pGPPVPS (M group), was investigated in their woody hosts (seedlings of Prunus spp.). The two clones differed in their ability to infect plum and peach cultivars, from no infection to local and systemic infection. The phenotype determinants were located with a set of chimeric viruses from the two clones. In plum, determinants of systemic infection were located in a genomic fragment encoding the P3 and 6K1 proteins, which might influence genome amplification or virus movement. The capacity of pGPPVPS to induce stable local and systemic infections in peach was not located accurately and might be influenced by multiple determinants carried by different regions of the genome, excluding those encoding the protein 1, the majority of helper component, nuclear inclusions a and b, and coat protein. We conclude that PPV infections of plum and peach are governed by different determinants.

Existence of two serological subclusters of Plum pox virus, strain M

A large-scale serological characterisation of Plum pox virus (PPV) isolates was carried out with 19 monoclonal antibodies (MAbs), including the universal MAb5B and the following strain-specific MAbs: AL (specific to PPV-M), 4DG5 (specific to PPV-D), TUV and AC (specific to PPV-C), and EA24 (specific to PPV-EA). The study involved 108 PPV isolates of different geographical origin (Albania, Bulgaria, Cyprus, Czech Republic, Egypt, France, Germany, Greece, Italy, Hungary, Moldova, Romania, Slovakia, Spain, Turkey and Yugoslavia) and hosts (almond, apricot, peach, plum and cherry). The inter- and intra-strain serological relationships of PPV isolates were evaluated by DASI-ELISA. High serological variability was detected, not only between strains, but also among isolates of the same strain. Computer-assisted analysis of serological data support the hypothesis of the existence of two distinct subclusters, denoted PPV-M1 and PPV-M2, which seem to prevail in Mediterranean and Eastern–Central European countries, respectively.

PROPERTIES OF TWO MONOCLONAL ANTIBODIES SPECIFIC TO THE CHERRY STRAIN OF PLUM POX VIRUS

Two monoclonal antibodies (MAbAC and MAb- TUV) raised against the sweet cherry isolate of Plum pox virus (PPV-SwC) were tested against homologous and heterologous PPV strains from different geographical origins. Both MAbs proved to be PPV-C (Cherry) strain-specific. They reacted in DASI-ELISA with PPVSwC and the sour cherry isolate of PPV (PPV-SoC) from Nicotiana benthamiana, but not with any of additional 44 PPV isolates tested belonging to the Dideron, Marcus and El Amar strains. The strain specificity of both MAbs was also assessed by Western blot and immuno electron microscopy. As indicated by competitive ELISA, the two MAbs are elicited by different epitopes.

Experimental evaluation of apricot genotypes for resistance to Plum pox virus

The reaction of 19 Slovak apricot cultivars and hybrids (breeding program of Research Breeding Station at Vesele) to infection by M isolates of plum pox virus (PPV-M) was evaluated. The genotypes were inoculated by grafting to naturally infected plum trees in the field and by chip-budding in the glasshouse. Monitoring of PPV infection was done over a 3 year period by visual inspection and DAS-ELISA. In the third year of evaluation the RT-PCR assay was also applied. The tested apricot genotypes differed in their reaction to PPV infection. Most of them developed mild or severe symptoms on leaves in the first year and/or next two consecutive years after artificial inoculation. The four Slovak apricot genotypes Veharda, Vemina, VS 15811 and VS 157/8 were resistant to PPV-M infection.

The Differential Reactivity of Two Polyclonal Antisera to Plum Pox Virus Isolates Collected at Different Stages After Immunization

ABSTRACTTwo polyclonal antisera to plum pox virus isolates, PPV-C and PPV-A, raised after a single immunization were collected at different stages after injection and serologically studied using electro-blot-immunoassay and indirect ELISA. Antisera, collected one week after the single injection, reacted strongly with the respective homologous capsid protein, but weakly or not at all with the core capsid protein. Batches of both antisera obtained 3, 4, 5, 6 and 7 weeks after injection reacted as well as with the core capsid proteins, indicating a continuously increase in the proportion of antibodies to the core capsid proteins. Comparing the antisera collected 1 week after immunization and N-termini antibodies to PPV-C and PPV-A, respectively, demonstrated that both types of antisera possess strain specificity and confirmed our previously findings on the serological relationships between the PPV isolates studied.

Specific detection of D- and M-isolates of plum pox virus by immunoenzymatic determination of PCR products

Molecular techniques based on the polymerase chain reaction (PCR) can provide rapid and sensitive diagnosis of plum pox virus (PPV), the causal agent of the devastating `sharka' disease of stone fruit trees. The present study compared routine polymerase chain reaction (PCR) procedures against a new system, PCR-ELISA (Boehringer Mannheim), which enables immunoenzymatic detection of PCR products. The results show that this hybridisation system ensures fast and more sensitive detection of PPV associated with stone fruit trees and herbaceous hosts. Strain-specific capture probes were also designed to identify the two major PPV isolates, D and M, without subsequent restriction fragment length polymorphism analysis of the PCR products. Optimisation of all parameters involved in the PCR-ELISA procedure are discussed and its advantages reported.

Prunus tomentosa as a Diagnostic Host for Detection of Plum Pox Virus and Other Prunus Viruses.

The efficacy of seedlings of Prunus persica cv. GF 305, P. persica cv. Siberian C, and P. tomentosa (Nanking Cherry) as diagnostic indicators of plum pox infection, and of P. tomentosa for other Prunus viruses was evaluated by graft-inoculation with eight different strains or isolates of plum pox virus (PPV) representative of the Marcus (M) and Dideron (D) serogroups; and one isolate each of Prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), and sour cherry green ring mottle virus (GRMV). The initial PPV symptoms that developed in P. tomentosa within 30 days after inoculation were chlorotic banding along the midrib spreading to lateral veins from the leaf base upward, giving the appearance of a chlorotic oak-leaf pattern. Symptoms caused by PPV-M could be distinguished from those caused by PPV-D. Virus titers in infected P. tomentosa and GF 305 were higher than those in Siberian C when measured by triple antibody sandwich enzyme-linked immunosorbent assay. Infections by PNRSV, PDV, and GRMV were evident with the first flush of vegetative growth.

Characterization of the Sour Cherry Strain of Plum Pox Virus

Properties of the sour cherry isolate of plum pox virus (PPV) were investigated by reverse transcription-polymerase chain reaction (RT-PCR), restriction fragment length polymorphism (RFLP), molecular hybridization, nucleotide sequencing, ultrathin sectioning of infected tissue, and graft transmission to different cherry rootstocks. Analysis of RT-PCR-amplified cDNA product from infected tissue with primers for the 3' noncoding region (3'-NCR) of the PPV genome and molecular hybridization of the amplified product with a labeled PPV cRNA probe verified that the potyvirus infecting sour cherry trees (Prunus cerasus) in Moldova is an isolate of PPV. RFLP analysis of RT-PCR products from infected tissue with specific primers for the 3'-terminal region of the PPV coat protein (CP) gene revealed that the sour cherry isolate of PPV is a unique strain of PPV and a prototype of a new group that contains neither the RsaI nor the AluI restriction site. These results were confirmed by nucleotide sequencing analysis. Nucleotide sequencing of the 3'-NCR and the region coding for the 3'-terminal fragment of the PPV CP gene showed about 93% identity to that of other PPV isolates. RT-PCR assays of tissue extracts from three sour cherry cultivars demonstrated that sour cherry PPV was distributed systemically in sour cherry trees and infected leaf, bark, root, flower, fruit, and seed tissues. The virus was successfully transmitted by chip bud grafting to rootstocks of P. avium (sweet cherry) and P. mahaleb.

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

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.

Transmission by aphids of a naturally non-transmissible plum pox virus isolate with the aid of potato virus Y helper component.

Two Spanish plum pox virus (PPV) isolates, 5.15 and 3.3, were used in transmission experiments involving the aphid vector Myzus persicae, with woody and herbaceous host plants. These isolates differ in the size of their coat protein (CP) and sequence analysis revealed that isolate 3.3 has a 15 amino acid deletion near the N terminus of the CP, affecting the same positions as in a previously reported non-aphid-transmissible PPV isolate from Germany. Aphid transmission experiments showed that isolate 5.15 was transmitted from infected plants whereas isolate 3.3 was not. In contrast, both isolates were readily aphid-transmitted when acquired through artificial membranes from purified virus preparations supplemented with purified helper component (HC) obtained from potato virus Y-infected plants. This indicates that non-transmissibility of isolate 3.3 may be due to a defect in the HC rather than in the CP.

Comparison of Three Methods for Assessing Plum Pox Virus Variability: Further Evidence for the Existence of Two Major Groups of Isolates

AbstractTwenty‐eight plum pox virus isolates from several European and Mediterranean countries were compared by electrophoretic mobility of their coat protein subunit measured by Electroblot Immuno‐Assay (EBIA), antigenic properties of the N‐ and C‐terminal parts of the coat protein and restriction fragment length polymorphism (RFLP) and analysis of polymerase chain reaction (PCR) amplification of the C‐terminal part of the coat protein gene. Similar results were obtained by each of the three methods. These confirm the existence of the two major subgroups of PPV, and which we now propose to designate PPV‐D and PPV‐M, respectively.

Detection of Double‐Stranded RNA (DSRNA) in Crude Extracts of Virus‐Infected Plants by Indirect ELISA

AbstractAn antiserum against polyinosinic‐polycytidylic acid (In‐Cn) was used to detect double‐stranded RNA (dsRNA) by indirect ELISA (ELISA‐I). DsRNA from cucumber mosaic virus (CMV) and plum pox virus (PPV)‐infected plants was detected using different types of extracts. The pH of the extraction buffer was very important in dsRNA detection, the highest optical density values being obtained at pH 6 or in aqueous extracts. Extracts heated at 80°C for 2 min showed increased optical density values compared with unheated extracts. DsRNA from Nicotiana benthamiana plants infected with each of six PPV isolates was readily detected by ELISA‐I 50 days after inoculation. ELISA values then obtained with the In‐Cn antiserum were generally higher than those obtained by double antibody sandwich ELISA using an antiserum to virus coat protein. Purified dsRNA from the same infected plants showed no visible band, but it produced a fluorescent background when analysed by polyacrylamide gel electrophoresis.

Nucleotide sequence of the coat protein gene of the Skierniewice isolate of plum pox virus (PPV).

The coat protein (CP) gene of the Skierniewice isolate of plum pox virus (PPV-S) has been amplified using the reverse transcription--polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide sequence of the gene and the deduced amino-acid sequence of PPV-S CP were compared with those of other PPV strains. The nucleotide sequence showed very high homology to most of the published sequences. The motif: Asp-Ala-Gly (DAG), important for the aphid transmissibility, was present in the amino-acid sequence. Our isolate did not react in ELISA with monoclonal antibodies MAb06 supposed to be specific for PPV-D.

Differentiation of Mediterranean plum pox virus isolates by coat protein analysis

Six isolates of plum pox potyvirus from different Mediterranean countries were compared by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE), peptide mapping and Western blotting after improved purification of virions using a protease inhibitor cocktail that reduced coat protein degradation. One isolate (Spanish isolate 3.3 from plum) differed from the others in possessing a smaller coat protein (approximately 34 instead 36 kDa) with a possible deletion in the surface‐exposed amino‐terminal region. Infectivity of the viruses after proteolysis, assessed using a local lesion host, was significantly reduced. Protease digestion conditions were established to generate a 28 kDa resistant core of the viral coat protein. Such conditions (longer incubation times or an increase in the enzyme concentration) differed from the milder ones reported for other potyviruses. Implications of the results in relation to the production and screening of virus‐specific monoclonal antibodies are discussed.

Detection of plum pox virus in leaves and aphids by SIBA and DAS-ELISA assays

The slot-immunobinding assay (SIBA) was adapted for detection of plum pox virus (PPV) and compared with DAS-ELISA. SIBA was easy to perform and as sensitive as DAS-ELISA in detection of various PPV isolates in herbaceous and woody plants, but not in aphids (Myzus persicae).

Monoclonal Antibodies Suitable for Plum Pox Virus Determination

Monoclonal antibodies (MAbs) to plum pox virus (PPV) were obtained after immunization of BALB/c mice with purified PPV-W isolate. Spleen cells from a mouse showing a high serum titer were used for fusion with Sp2/0-Ag14 myeloma cells. Culture supernatants were screened for specific antibody production against PPV-W isolate using indirect ELISA. A total of six stable hybridoma lines producing MAbs of IgG class were obtained. All four PPV isolates tested (W, A, D and M) can be distinguished by these MAbs. Two highly efficient antibodies were chosen for practical purposes, and their applicability in PPV diagnostics has been studied since 1989 in parallel with polyclonal antibodies and commercial kits (Boehringer, Bioreba). The experiments have proven comparable sensitivity with a detection limit ranging between 10 and 50 ng of virus per ml sample. In routine diagnostics of plums, peaches and apricots our MAbs ranked at least as high as the commercial kits.

3'-Terminal sequence of the plum pox virus PS and o6 isolates: evidence for RNA recombination within the potyvirus group

The sequence of the 3'-terminal 1768 nucleotides of the PS and ŏ6 isolates of plum pox virus (PPV) has been determined and compared with that of the equivalent regions of other PPV isolates sequenced previously. The sequenced region is part of the PPV open reading frame encoding the last 186 amino acids of the NIb protein and the coat protein (CP, 330 amino acids), followed by a non-coding region of 220 nucleotides and a poly(A) tail. PPV-PS and PPV(-)ŏ6, just like PPV-El Amar, show rather high levels of nucleotide diversity in the sequence encoding the C-terminal region of the NIb protein (19.4 to 31%) and the N terminus of CP (22.8 to 41.1%) when compared with PPV-Rankovic, PPV-D and PPV-NAT, whereas the level of diversity in the rest of the CP sequence and the 3' non-coding region is low (8 to 10.8% and 5.5 to 7.7%, respectively). However, the first 429 sequenced nucleotides of PPV(-)ŏ6 are very similar to those of the PPV-Rankovic, PPV-D and PPV-NAT isolates, whereas the rest of the sequence clearly resembles PPV-PS. Thus, PPV(-)ŏ6 seems to be the result of a natural recombination event between two wild strains of PPV. To our knowledge this is the first evidence of homologous RNA recombination (a process which could play an important role in the evolution of RNA viruses) within the potyvirus group.

The complete nucleotide sequence of plum pox virus RNA.

The complete nucleotide sequence of the RNA of an aphid non-transmissible plum pox virus (PPV-NAT) isolate has been determined from five overlapping cDNA clones. cDNA prepared by primer extension was used to determine the 5' terminus. The assembled RNA is 9741 nucleotides in length, excluding a 3' terminal poly(A) sequence. One large open reading frame starts at nucleotide positions 36 to 38 and is terminated with an UAG codon at positions 9522 to 9524. The putative start codon is located at positions 147 to 149. The encoded polyprotein has a predicted Mr of 353.8K. Comparison of cistrons from tobacco vein mottling virus and tobacco etch virus with those predicted for PPV-NAT indicated a similar genome organization. A highly conserved sequence of 12 nucleotides was found in the 5' non-coding region of these three potyviruses. The potential polyadenylation signal from yeast (UAUGU) was found in the 3' non-coding region of PPV-NAT and several other members of the potyvirus group.

Experimental Transmission of Plum Pox Virus (PPV) to Prunus Mahaleb and Prunus avium

AbstractSharka caused by plum pox virus (PPV) is a disease spread in France since 1970, and causing severe damages essentially on apricot but also on plums and peach.Cherry is generally considered as not infected by PPV. Experimental transmissions by chip budding or aphids allowed to show that 3 isolates of PPV can multiply inside three cherry rootstocks (P. Mahaleb cv.‘SL 64′, P. avium cv.‘F 12‐1′, and P. avium*P. pseudocerasus cv. ‘Colt') (Tables 1 and 2).But generally, the virus remained localized to the infection site and disappeared quickly (Table 3). Typical symptoms of chlorotic ringspot or vein clearing are also limited to the leaves probed by the aphids.The fact that no translocation was detected is discussed.