Effect of temperature on plum pox virus infection.

One of the key factors of progress of an epidemic is the duration of virus availability for a vector in plants, which could be influenced by temperature. Using five epidemiologically different isolates of Plum pox virus (PPV) we studied the effect of temperature on the virus infectivity, intensity of disease symptoms and virus accumulation in Nicotiana benthamiana plants as determined by a double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA). No differences in infectivity and intensity of disease symptoms between the five isolates were observed at 17 degrees C. However, they differed in their capacity to infect and multiply in the plant at higher temperatures. The temperature of 32 degrees C was inhibitory to the multiplication of all the five PPV isolates studied. Fewer plants were infected and a significantly decreased amount of virus antigen was detected at 30 degrees C. The natural PPV recombinant BOR-3 isolate showed a greater temperature tolerance compared to other PPV isolates tested. We conclude that adaptation to higher temperatures may favour the epidemiological impact of PPV.

Molecular variability of the P3-6K1 genomic region among geographically and biologically distinct isolates of Plum pox virus.

To evaluate molecular variability in the 5' region of the Plum pox virus (PPV) genome, a fragment spanning the C-terminal part of P3, the complete 6K1 and start of the CI gene in 12 Slovak and French PPV isolates was amplified in IC-RT-PCR and sequenced. Computer analysis of obtained and previously published sequences showed rather substantial differences among isolates of PPV-M and D subgroups (percentage of nucleotide identity ranged from 85.65 to 87.68%). There was a relatively low percentage of heterogeneity among isolates within the same subgroup. Amino acid multiple sequence alignment showed a total of 14 conserved subgroup-specific amino acid positions among PPV-M and D isolates in the examined region. The geographical origin of the isolates was not correlated with their phylogenetic clustering. No reasonable correlation could be established between amino acid substitutions and host species and/or biological properties of isolates. The present IC-RT-PCR detection method targeting a 5' region of the PPV genome, followed by simple RFLP subgroup determination, could enhance conventional CP-based techniques, especially with peculiar isolates. Using this approach, the Slovak isolate BOR-3 was found to be a natural recombinant between a D and an M PPV-type.

Pathogenicity determinants in the complex virus population of a Plum pox virus isolate.

Several subisolates were separated from a single Plum pox virus (PPV) isolate, PPV-PS. In spite of an extremely high sequence conservation (more than 99.9% similarity), different subisolates differed largely in pathogenicity in herbaceous hosts and infectivity in woody plants. The severity of symptomatology did not seem to correlate with virus accumulation. Sequence analysis and site-directed mutagenesis demonstrated that single amino acid changes in the helper component (HC) protein caused a drastic effect on virus symptoms in herbaceous hosts and notably modified virus infectivity in peach seedlings. These results indicate that HC variation might play an important role in virulence evolution of natural plant virus infections. Moreover, the analysis of Potato virus X (PVX)-HC chimeras showed that the identified HC amino acid changes had parallel effects on the severity of symptoms caused by PPV and on HC-induced enhancement of PVX pathogenicity, indicating that HC functions in potyvirus symptomatology and in synergism with other viruses have overlapping determinants.

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.

Evidence of a Naturally Occurring Recombinant Isolate of Plum pox virus from Slovakia.

Sharka, caused by the Plum pox virus (PPV), is the most detrimental viral disease of stone fruit trees worldwide. Two main groups of PPV isolates have been identified, PPV-M and PPV-D (1). Natural variability of Slovak PPV isolates in Prunus hosts has been recently evaluated (3). The PPV isolate BOR-3 was isolated in the summer of 1996 from a 5-year-old apricot tree, cv. VS 123/9, in an orchard in western Slovakia. The host apricot tree was propagated from seed; hence infection via aphid transmission from the immediate surroundings is highly probable. In order to retain its original properties, the isolate was transmitted by chip budding from a diseased tree to seedlings of Prunus persica, cv. GF 305. In contrast to other PPV isolates collected from the same location, infection of GF 305 with BOR-3 was either symptomless or produced only weak symptoms. On the other hand, sap-infected young seedlings of P. insititia × P. domestica St. Julien No. 2 presented leaf chlorotic spots and rings similar to those produced by other tested Slovak PPV isolates. Serological typing with group-specific MAbs (1) and restriction fragment length polymorphism of RT-PCR products amplified from the C terminus of the capsid protein (CP) gene demonstrated that BOR-3 is a member of PPV-M group (3). However, nucleotide (nt) sequence analysis of the P3-6K1 genomic region (GenBank accession AF357541) has shown a strong similarity with PPV-D group isolates (reaching 97%). To obtain more information, a 1846-nt long 3 genomic region encompassing the C-terminus of NIb, CP and 3 noncoding region (NCR) was subsequently sequenced (GenBank accession AY028309). The sequence comparison revealed that the BOR-3 isolate was the most identical to the PPV isolate ð6 (GenBank accession S57404) isolated more than 10 years ago in former Yugoslavia, which is about 550 km from the Slovak focus. The ð6 isolate is known to be the only PPV RNA recombinant at present (2). Nucleotide sequence identity in 3'NIb+CP+NCR region between BOR-3 and ð6 isolates reached >98%, contrary to 96% identity with PPV-M isolates (PS, SK68) and 88% identity with PPV-D isolates (Dideron, Rank). Further, the potential recombination site in the BOR-3 genome has been located in the C-terminus of NIb gene near nt position 8450 (numbered according to PPV-PS isolate, AJ243957). This site corresponds to the location of the recombination crossover identified in the PPV-ð6 isolate (2). Despite geographical and temporal distinctiveness of the two PPV isolates, an identical origin of ð6 and BOR-3 cannot be excluded. The hypothesis that these PPV isolates are the product of two independent recombination events at a recombination hot spot in the PPV genome situated near the C terminus of NIb gene also should be considered. Based on these data, the PPV BOR-3 isolate is a product of a natural homologous RNA recombination event between an M and D isolate. Evidence of homologous RNA recombination in this isolate significantly enriches current knowledge on the occurrence of recombination among potyviruses and emphasizes the role of RNA recombination in viral evolution and adaptation. This report indicates that occurrence of recombinants within PPV isolates infecting perennial crops might not be as rare as previously suggested (4). References: (1) T. Candresse et al. Phytopathology 88:198, 1998. (2) M. T. Cervera et al. J. Gen. Virol. 74:329, 1993. (3) M. Glasa et al. Acta Virol. 42:226, 1998. (4) F. Revers et al. J. Gen. Virol. 77:1953, 1996.

Identification of a pathogenicity determinant of Plum pox virus in the sequence encoding the C-terminal region of protein P3+6K(1).

A full-length genomic cDNA clone of a plum pox potyvirus (PPV) isolate belonging to the M strain (PPV-PS) has been cloned downstream from a bacteriophage T7 polymerase promoter and sequenced. Transcripts from the resulting plasmid, pGPPVPS, were infectious and, in herbaceous hosts, produced symptoms that differed from those of virus progeny of pGPPV, a full-length genomic cDNA clone of the D strain PPV-R. Viable PPV-R/-PS chimeric viruses were constructed by recombination of the cDNA clones in vitro. Analysis of plants infected with the different chimeras indicated that sequences encoding the most variable regions of the potyvirus genome, the P1 and capsid protein coding sequences, were not responsible for symptom differences between the two PPV isolates in herbaceous hosts. On the contrary, complex symptomatology determinants seem to be located in the central region of the PPV genome. The results indicate that a genomic fragment that encodes 173 aa from the C-terminal part of the P3+6K(1) coding region is enough to confer, on a PPV-R background, a PS phenotype in Nicotiana clevelandii. This pathogenicity determinant also participates in symptom induction in Pisum sativum, although the region defining the PS phenotype in this host is probably restricted to 74 aa.

Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the d and m serotypes of plum pox potyvirus.

ABSTRACT Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D- and PPV-M-specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M-specific monoclonal antibody were found to belong to the M serotype using the PCR-based assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific monoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes recognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates.

[Differential thermal behavior of various strains of cucumber mosaic virus. Hypothesis of a pleiotropic mechanism connecting various properties]. / Comportement thermique différentiel de certaines souches du virus de la mosaïque du concombre. Hypothèse d'un mécanisme pléiotropique reliant plusieurs propriétés

Pseudorecombinat viruses are constructed by mixing RNA coming from two CMV strains differentiated by their thermosensitivity. The pseudorecombinant shows thermal properties analogous to the parental strain diving RNA number 3. In order to explain the numerous properties coded by RNA 3 a pleiotropic effect is considered.