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.

Sequence analysis of the Czech potato mop-top virus (PMTV) isolate Korneta-Nemilkov.

The nucleotide sequence was determined for Czech potato mop-top virus (PMTV) isolate Korneta-Nemilkov, found in the potato field situated in South Bohemia. The nucleotide and amino acid sequences were compared with other PMTV isolates available in databases. The sequence identity was always >99% when Czech isolate RNA 2 and RNA 3 sequences were compared with each of the 3 Danish isolates and with Sw isolate, and slightly lower when compared to Scottish isolates. Similarity of deduced proteins was 100% for 5 out of 6 proteins used in comparison of Czech isolate with Danish isolate 54-15. The only difference between 2 isolates was found in coat protein (CP) gene. Interestingly, the CP of the Czech isolate seems to be 100% identical to the one of Sw, while many changes were found in the region encoding TGBp2, TGBp3 and cysteine-rich protein (CRP) for these 2 isolates. The lowest similarity scores were found when comparing the Czech isolate CRP with CRP of Scottish isolates.

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.

Partial antigenic characterization of potato virus S (Andean strain) by monoclonal antibodies.

Four mouse monoclonal antibodies (MoAbs)) against potato virus S Andean strain (PVSA) were tested. While MoAbs 2 and 3 reacted only with complete virions and were apparently specific for epitopes dependent on quaternary structure, MoAbs 1 and 4 appared to be conformation independent and reacted with exposed regions on native virions as well as on the surface of dissociated coat protein subunits. This seems to be an evidence of metatope existence. The results of competitive binding tests together with reaction patterns of individual MoAbs suggest that the used MoAbs reacted with at least two different epitopes on PVSA particles or polypeptide subunits. Immunoblot analysis of proteolytically cleaved PVSA capsid protein (CP) confirmed close proximity of epitopes recognized by MoAbs 1 and 4. Anti-PVS polyclonal antibody recognized both intact CP and its natural or artificial digest, while the MoAbs bound to intact CP only. These results indicate that the surface virus-specific epitopes are located near the terminus of CP molecule as it is characteristic for potyviruses.

Optimum conditions for the storage of potato virus S.

The effect of storage conditions on the serological activity of two strains of potato virus S (PVS), Andean and the ordinary, was studied by ELISA. Virus purificates, infected leaves and their homogenates, stored in lyophilized, frozen and dissolved form at various temperatures were tested. Virus purificates were most stable in lyophilized form, their activity decreased after 9 months only by 20-30%. Also non-purified virus was most stable as a lyophilized leaf homogenate, its activity decreased after 12 months by 30%. When lyophilized leaves were stored, the virus activity dropped after 12 months by 45%. Both the Andean and the ordinary strain of PVS behaved similarly during storage under the conditions tested.

A comparative study of the effect of triazine herbicides on alcohol dehydrogenases isolated from various sources.

The studied herbicides (terbutylazine, simazine) inhibit the activity of plant, animal, and yeast alcohol dehydrogenases. The inhibition constant Ki for alcohol dehydrogenase (ADH) isolated from peas and bakers' yeast equals approximately 10(-4) M, and that for ADH isolated from horse liver is of the order of 10(-5) M. The character of inhibition for all the herbicides studied for the reaction catalyzed by pea, liver, and yeast ADH is always noncompetitive toward ethanol and competitive with respect to NAD. The inhibition constants for the enzyme isolated from peas are pH independent. The interaction constants found for terbutylazine and simazine and for o-phenanthroline, nicotinamide, and ATP indicate that the herbicides are bonded through the metal component of the enzyme, similar to the nicotinamide part of NAD. The interaction constant less than unity found for the herbicide-ATP system indicates that the bonding site in the active center of the enzyme is different for the herbicides and the adenine part of NAD.