Production of polyclonal antibodies to a recombinant coat protein of potato virus Y.

The gene encoding the coat protein (CP) of a potato virus Y (PVY) was cloned into expression vector pMPM-A4Omega. PVY CP was expressed in Escherichia coli and the purified recombinant protein was used for raising rabbit polyclonal antibodies. The sera and antibodies were tested for the detection of PVY in the laboratory host Nicotiana tabacum cv. Petit Havana SR1 and in various cultivars of the natural host Solanum tuberosum by ELISA as well as by Western blots. The antibodies can be used for the detection of the whole strain spectrum of PVY by indirect plate trapped antigen ELISA and Western blot, but not by double antigen sandwich ELISA.

Nucleotide sequences of coat protein coding regions of six potato mop-top virus isolates.

Coat protein (CP) coding regions of six Potato mop-top virus (PMTV) isolates from the Czech Republic and Denmark (54-10, 54-11, 54-15, 54-19, Korneta and Pacov) were sequenced. Comparison of the obtained nucleotide sequences as well as alignment of the deduced amino acid sequences were performed. The obtained results showed that the isolates from different parts of Europe seem to have highly conserved coding regions which is unexpected for a viral RNA genome known for its high mutation rate. Thus considerable differences in virulence and significant variation in biological properties of these isolates should not be attributed to CP but to some other part of the genome.

The detection of recombinant, tuber necrosing isolates of Potato virus Y (PVY(NTN)) using a three-primer PCR based in the coat protein gene.

A simple and reliable procedure for reverse transcription-polymerase chain reaction (RT-PCR) detection and strain differentiation of Potato virus Y (PVY) was developed. Three primers were designed within the coat protein (CP) and nuclear inclusion protein b (NIb) region, exploiting a single base polymorphism identified as being present in all the recombinant PVY(NTN) isolates published. Samples infected with PVY produce a single band of 569 bp, while isolates belonging to PVY(NTN) strain give an additional band of 334 bp. The technique was tested on a collection of well-characterised isolates of PVY from a range of strains and was found to detect all of the isolates reported as belonging to the PVY(NTN) strain. All of the isolates detected possess a recombination event within the coat protein. Further sequence analysis revealed that all the recombinant PVY(NTN) isolates reported thus far would be detected using this assay, whilst isolates thought to be PVY(NTN) that do not possess the coat protein recombination event would not be detected.

Polyclonal antibodies to a recombinant coat protein of Potato virus A.

Specific mouse antibodies against a recombinant coat protein (CP) of Potato virus A (PVA) were produced. The PVA CP gene was cloned in an expression vector pMPM4omega. After expression in Escherichia coli the presence of the expressed CP was proved by Western blot analysis using polyclonal and monoclonal antibodies (MAbs). The expressed CP was purified by centrifugation in CsCl density gradient or on a sucrose cushion. The production of virus-like particles (VLPs) was proved by electron microscopy. The purified CP was used for preparation of a mouse antiserum which had a titer of 1:1024 in ELISA and reacted specifically in Western blot analysis and indirect plate-trapped antigen ELISA (PTA-ELISA).

Nucleotide sequences of 5'-terminal parts of coat protein genes of various isolates of NTN strain of potato virus Y.

Sequences of the first 300 nucleotides of coat protein (CP) genes of 7 isolates of NTN strain of potato virus Y (PVY, PVY(NTN)) were determined and compared with analogous published sequences of various isolates and strains of PVY. The sequence identity among the sequenced isolates ranged from 96 to 100%. The differences were found at different positions. The nucleotide sequence of this part of CP gene seems to be very conservative among the isolates tested that means that PVY(NTN) is the evolutionary youngest among all PVY strains.

Potato virus A detection by reverse transcription-polymerase chain reaction.

Simple and reliable procedure for sample preparation and reverse transcription-polymerase chain reaction (RT-PCR) detection of potato virus A (PVA) is described. PVA-specific primers used in the RT-PCR defined a target sequence of 321 bp and did not produce amplification product(s) with potato virus Y.

Monoclonal antibodies against potato virus A -- immunoblot analysis.

Monoclonal antibodies (MoAbs) against potato virus A (PVA) were examined in their reactivity with PVA and its denatured capsid protein (PVA-CP) bound to the nitrocellulose membrane. Five MoAbs reacted with native PVA, three of them also with PVA-CP. One MoAb gave no reaction in dot-blot test. In polyacrylamide electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) PVA-CP migrated as two major bands. In immunoblot analysis, two MoAbs reacted only with the slower band, one only with the faster one. We presume that those bands do not correspond to the intact CP but they do to truncated N- and C-terminal CP molecules, respectively, and that the corresponding epitopes reacting with MoAbs are localized near to both termini of CP molecules. After mild trypsinolysis of PVA particles no MoAb reacted with resulting "core" CP.