Amino acid substitutions within the Cys-rich domain of the tobacco etch potyvirus HC-Pro result in loss of transmissibility by aphids.

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.

Helper component mutations in nonconserved residues associated with aphid transmission efficiency of a pepper isolate of potato virus y.

ABSTRACT The aphid transmission properties of a pepper isolate of potato virus Y belonging to the pathotype 1-2 (PVY 1-2) have been characterized. PVY 1-2 was not transmitted in plant-to-plant experiments, although purified virus particles were efficiently transmitted when supplemented with heterologous helper component (HC) of the transmissible isolate PVY 0 AT through membrane acquisition assays, indicating that its coat protein was functional in transmission. Additionally, virions of PVY 1-2 were able to bind to different HCs in in vitro binding assays. Analysis of the sequence of the PVY 1-2 HC gene and comparison with that of PVY 0 AT revealed 19 nucleotide differences, but only 2 resulted in amino acid changes, one of which induced a change of charge. Neither of these two amino acid changes occurred within the cysteine-rich domain, nor did they coincide with conserved motifs of the HC protein known to be involved in aphid transmission and which are present in all known potyvi-ruses. However, both changes are located in positions highly conserved among PVY strains. The possible role of both mutations on the activity of the PVY 1-2 HC in aphid transmission is discussed.

Serological characterization of Hungarian plum pox virus isolates.

Three Hungarian (no. 2, 4 and 9), and a Moldavian (K) plum pox virus isolates were compared with a characterized Spanish isolate (5.15) by RT-PCR, ELISA, dot-blot and Western blot analysis. Monoclonal antibodies prepared against the external, intermediate and internal sequences of the coat protein of the Spanish isolate were able to differentiate the four isolates. Hungarian isolate No. 2 proved to be serologically identical to the Spanish isolate, while No. 4 showed appreciable differences and No. 9 could be recognized only by the monoclonal antibodies representing the intermedial and internal parts of the coat protein. K isolate showed a more distant relationship to other isolates. Our experiment provided the first demonstration of the presence of D type isolates in Hungary.

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.

Production and characterization of monoclonal antibodies to plum pox virus and their use in differentiation of Mediterranean isolates.

Monoclonal antibodies (MAbs) specific to plum pox virus (PPV) were prepared by fusing myeloma cell lines to spleen cells of mice immunized with purified virus, including virus prepared with protease inhibitors to preserve the integrity of the coat protein (CP). The characterized MAbs could be used in ELISA to differentiate several Mediterranean PPV isolates differing in their geographical origin and CP size. At least seven antigenic sites could be established based on the recognition pattern and competition binding analysis, and the epitopes could be classified in three groups by Western blot analysis of intact and trypsin digested virus particles. By means of electron microscopy the epitopes could be seen to be located on the surface of the virus particles.

Detection of cauliflower mosaic virus (CaMV) in single aphids by the polymerase chain reaction (PCR).

The detection for the first time of a plant virus in a single aphid by the high sensitivity polymerase chain reaction (PCR) technology is reported. The application of PCR for the detection of viruses in their vectors will aid the understanding of the complex virus-vector relationship and therefore allow the development of new approaches for control of spread of plant virus diseases.