Eleven monoclonal antibodies specific to plum pox potyvirus (PPV) coat protein were obtained by hybridoma technology from Spanish PPV isolates. In addition, two monoclonal antibodies specific for PPV cylindrical inclusions (CIP non‐structural proteins) were obtained. The monoclonal antibodies specific for PPV coat protein were assayed by DASI ELISA against 81 PPV isolates. At least nine different epitopes were found and 21 distinct serological patterns of reaction (serogroups) were established using nine selected monoclonal antibodies against the collection of PPV isolates, indicating the high variability of coat protein among PPV isolates. Changes in epitope composition were observed after aphid and mechanical transmission, indicating the occurrence of mixtures of isolates in field trees. Monoclonal antibody 5B reacted with all PPV isolates assayed, with very high affinity, using DASI ELISA. This method was compared with immunocapture‐PCR on field samples in spring, and showed very good coincidence of results. The efficiency of PPV detection can be slightly increased using monoclonal antibodies specific to cylindrical inclusions mixed with monoclonal antibodies against structural proteins, and using mixtures of monoclonal antibodies against different epitopes of coat protein. ELISA‐I and immunoprinting‐ELISA were able to detect CIP and PPV in extracts and tissue section, respectively, of woody plants. Two monoclonal antibodies offer the possibility of distinguishing between Marcus and Dideron PPV types (M or D). These D‐specific monoclonal antibodies can be used in routine tests with high affinity.

Oligomeric forms of the HC-Pro protein of the tobacco etch potyvirus (TEV) have been analyzed by analytical ultracentrifugation and single-particle electron microscopy combined with three-dimensional (3D) reconstruction. Highly purified HC-Pro protein was obtained from plants infected with TEV by using a modified version of the virus that incorporates a histidine tag at the HC-Pro N terminus (hisHC-Pro). The purified protein retained a high biological activity in solution when tested for aphid transmission. Sedimentation equilibrium showed that the hisHC-Pro preparations were heterogenous in size. Sedimentation velocity confirmed the previous observation and revealed that the active protein solution contained several sedimenting species compatible with dimers, tetramers, hexamers, and octamers of the protein. Electron microscopy fields of purified protein showed particles of different sizes and shapes. The reconstructed 3D structures suggested that the observed particles could correspond to dimeric, tetrameric, and hexameric forms of the protein. A model of the interactions required for oligomerization of the HC-Pro of potyviruses is proposed.

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.

Potyviruses are non-persistently transmitted by aphid vectors with the assistance of a viral accessory factor known as helper component (HC-Pro), a multifunctional protein that is also involved in many other essential processes during the virus infection cycle. A transient Agrobacterium-mediated expression system was used to produce Plum pox virus (PPV) HC-Pro in Nicotiana benthamiana leaves from constructs that incorporated the 59 region of the genome, yielding high levels of HC-Pro in agroinfiltrated leaves. The expressed PPV HC-Pro was able to assist aphid transmission of purified virus particles in a sequential feeding assay, and to complement transmission-defective variants of the virus. Also, HC-Pro of a second potyvirus, Tobacco etch virus (TEV), was expressed and found to be functional for aphid transmission. These results show that this transient system can be useful for production of functionally active HC-Pro in potyviruses, and the possible uses of this approach to study the mechanism of transmission are discussed.

We examined the role of several amino acid residues located at the N-terminus of the tobacco etch potyvirus (TEV) helper component-proteinase (HC-Pro) in virus transmissibility by aphids. Site-directed mutagenesis resulted in changes affecting amino acids that appear highly conserved among a number of potyviruses. The TEV HC-Pro amino acid residues Gly343, Val345, Ala346, Ile348, Pro355, Lys358, and Ile359 were arranged within a Cys-rich domain in a region dispensable for TEV infectivity. Two HC-Pro mutants (TEV-P355R and -K358N) exhibited a drastically reduced rate of aphid transmission whereas other mutants (TEV-G343D, -V345E, -A346H, -I348D, and -P355L) were completely unable to be aphid transmitted. In contrast, the I359M mutation had no effect on aphid transmissibility of TEV. This lack of transmissibility did not appear to be due to large differences in the amounts of both coat protein (CP) and HC-Pro in infected tobacco plants. Our results indicated that these amino acid residues likely play a highly conserved role in aphid transmission among potyviruses.