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.
- MeSH
- ELISA MeSH
- králíci MeSH
- monoklonální protilátky biosyntéza MeSH
- nemoci rostlin virologie MeSH
- Potyvirus imunologie MeSH
- protilátky virové biosyntéza MeSH
- rekombinantní proteiny imunologie MeSH
- Solanum tuberosum virologie MeSH
- tabák virologie MeSH
- virové plášťové proteiny imunologie MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- coat protein, Potato virus Y MeSH Prohlížeč
- monoklonální protilátky MeSH
- protilátky virové MeSH
- rekombinantní proteiny MeSH
- virové plášťové proteiny MeSH
Dahlia is an important ornamental crop in the Czech Republic where they have been grown for more than 150 years. New dahlia cultivars have been selected by Czech plant breeders. Virus diseases, including mosaic and stunt caused mostly by Dahlia mosaic virus, have been a problem. From 2003 to 2005, color breaking was observed in several dahlia cultivars of foreign and Czech origin. White stripes in blossoms were most frequently expressed in the second half of the flowering season. No symptoms are visible in flowers of white and yellow cultivars. It was difficult to characterize symptoms on leaves because most cultivars were infected simultaneously by Dahlia mosaic virus. Sap inoculations of Chenopodium quinoa produced local lesions after 5 to 7 days, followed by systemic chlorosis, necrosis of younger leaves, and death of the shoot apex, indicating possible Tobacco streak virus (TSV) infection (2). Spherical particles (25 to 30 nm) were observed in leaf-dip preparations of samples from experimentally infected C. quinoa plants and analyzed by using transmission electron microscopy. These particles became decorated when using immunoelectron microscopy with TSV IgG (Bioreba, Reinach, Switzerland and Neogen, Ayrshire, Scotland). Samples of 80 dahlia cultivars were tested for TSV infection by ELISA using commercially available kits (Bioreba and Neogen). Most of the samples were grown in a collection of dahlia cultivars of Czech and foreign origin and some were obtained from growers in the Czech Republic. Fifty six dahlia cultivars were shown to be TSV infected. ELISA also indicated a higher concentration of the virus in flowers. The identity of the virus isolated from symptomatic plants was confirmed by reverse transcription (RT)-PCR using total RNA extraction from symptomatic plants. RT-PCR (4), using a primer pair (1) derived from the coat protein gene sequence of TSV (3), was followed by electrophoresis on 1.0% agarose gels. Products of the predicted size (approximately 700 bp) were found in naturally infected dahlia plants (n = 10), systemically infected host plants C. quinoa (n = 10), and symptomatic Nicotina megalosiphon (n = 10) that scored as TSV positive by ELISA. No bands of this size were seen in negative controls. To our knowledge, this is the first detection of TSV in the Czech Republic. References: (1) A. I. Bhat et al. Arch. Virol. 147:651, 2002. (2) A. A. Brunt Plant Pathol. 17:119, 1968. (3) B. J. C. Cornelissen et al. Nucleic Acids Res.12:2427, 1984. (4) S. S. Pappu et al. J. Virol. Methods 4:9, 1993.
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- molekulární sekvence - údaje MeSH
- nemoci rostlin virologie MeSH
- RNA-viry klasifikace genetika izolace a purifikace MeSH
- rostlinné viry klasifikace genetika izolace a purifikace MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA * MeSH
- Solanum tuberosum virologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Complete genomes of three isolates of Potato virus S (PVS) were cloned and sequenced. The PVS ORF-1 was characterized for the first time. It encodes a putative replication protein (RPT) that shares the highest homology (about 52%) with that of Blueberry scorch virus (BlScV). ORF-1 motifs, characteristic for carlaviruses were found for methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRp). The complete sequence of PVS genome enabled to develop an immunocapture RT-PCR probing of the PVS genome. Using this system, the sequence variability of 11 genome zones was examined for 34 PVS isolates including 15 PVS-CS variants that caused a systemic infection in Chenopodium quinoa. A broad variability between PVS isolates and diverse sequence variants was found. cDNA fragments covering the coat protein (CP) leader and CP-coding region (approx. 420 bp) were pooled for PVS-O and Chenopodium-systemic PVS isolates (PVS-CS) and corresponding cDNA libraries were screened for sequence variants. Both cDNA pools differred mainly in the 5'-end of the CP gene. Methionine at the position 17 in combination with serine at the position 34 were frequently associated with the CS character of PVS. In general, hydrophobic and polar amino acids were characteristic for the positions 17 and 34, respectively in PVS-CS isolates. Genome probing and evolutionary distances suggested that the PVS-CS isolates analyzed were close to the ordinary European isolates of ordinary strain of PVS (PVS-O) but distant to the original Andean strain of PVS (PVS-A).
- MeSH
- Carlavirus chemie genetika MeSH
- genom virový * MeSH
- molekulární sekvence - údaje MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- sekvenční analýza DNA MeSH
- sekvenční seřazení MeSH
- Solanum tuberosum virologie MeSH
- virové proteiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- virové proteiny MeSH
DNA microarray assay has become a useful tool for gene expression studies. Less frequent is its application to detection of viruses or diagnostics of virus diseases. Here we show design of a microscope slide-based microarray assay for simultaneous identification of several potato viruses. Different primer pairs were designed or adopted to obtain specific amplicons from six potato viruses: Potato virus A (PVA), Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY), Potato mop-top virus (PMTV) and Potato leaf-roll virus (PLRV). Purified viral DNA probes were spotted on a microscope slide coated with poly-L-lysine. The same primers were used for preparation of fluorochrome-labeled targets. The latter were denatured and hybridized on the microarray slide (chip). An example of simultaneous assay of two pathogens is given and possibilities of practical application of this type of assay are discussed.
- MeSH
- DNA primery genetika MeSH
- DNA virů analýza izolace a purifikace MeSH
- nemoci rostlin virologie MeSH
- Potexvirus genetika izolace a purifikace MeSH
- Potyvirus genetika izolace a purifikace MeSH
- rostlinné viry genetika izolace a purifikace MeSH
- sekvenční analýza DNA MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody statistika a číselné údaje MeSH
- sekvenční homologie MeSH
- senzitivita a specificita MeSH
- Solanum tuberosum virologie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA primery MeSH
- DNA virů MeSH
Twenty potato virus Y (PVY) isolates were characterized. They represented two strains only, PVY(O) (three isolates) and PVY(N) (17 isolates). However, application of serological and molecular genetic methods led to a more complicated characterization. For example, five isolates induced necrotic symptoms on tobacco plants typical of PVY(N), despite reacting as PVY(O) serologically. Moreover, the PVY isolates were not identical according to molecular genetic properties. Typical PVY(NTN) PCR products were observed for 14 isolates, but five of them (Hr 220-5, Hr 387-7, Nord 242, Syn1Scot, and 41-97) did not produce potato tuber necrotic symptoms in infected cultivars. An immunocapture reverse transcription-polymerase chain reaction (RT-PCR) probing was developed using a set of 24 primer pairs derived from eight regions of the PVY genome. Using this method, five out of seven PVY(NTN) isolates including the Czech standard PVY(NTN) from the potato cv. Nicola were found to be identical. However, two PVY(NTN) isolates and all the other probed PVY samples showed unique patterns, suggesting specific differences at the nucleotide level. This method enabled specific identification of individual isolates variability even within different PVY strains.
- MeSH
- DNA primery genetika MeSH
- listy rostlin virologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí metody MeSH
- Potyvirus klasifikace genetika izolace a purifikace MeSH
- senzitivita a specificita MeSH
- sérotypizace MeSH
- Solanum tuberosum virologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA primery MeSH
Reaction conditions specific for reverse taranscription-polymerase chain reaction (RT-PCR) of potato virus Y strain NTN (PVYNTN) were used to amplify a 394 bp fragment of the P1 gene from selected PVY isolates with the aim to study the PVY variability within this genomic region. The P1 gene fragment from the Nicola isolate (Nicola P1/1 clone) was sequenced and characterized by temperature-gradient gel electrophoresis (TGGE). The Nicola P1/1 clone differed from that from the Hungarian isolate by double point mutation resulting in two changes at the deduced amino acid level. The clone showed simple transition from double-stranded to single-stranded form with two characteristic melting end points of about 41 degrees C and 48 degrees C. A more complicated TGGE pattern was, however, found for the whole P1 cDNA library of the Nicola isolate, suggesting accumulation of some minor sequence variants of PVY in this isolate. Based on the TGGE pattern, 46 degrees C was selected as the standard temperature for electrophoretic analysis of heteroduplex DNAs formed with the Nicola P1/1 DNA as reference. More than 40 other PVY isolates from PVYN group were analysed using this method. In most cases only minor fractions of electrophoretically distinguishable DNA heteroduplexes were found, however, in most isolates of PVYN-Wilga type, mixtures of the major sequence variants were observed. Two of these variants from the hybrid 220-5 (Czech Republic) were sequenced. Both of them differed from the Nicola P1/1 clone by 6 point mutations, which led to several changes at the amino acid level.
- MeSH
- elektroforéza v polyakrylamidovém gelu metody MeSH
- genetická variace MeSH
- genom virový * MeSH
- heteroduplexní analýza MeSH
- komplementární DNA analýza MeSH
- molekulární sekvence - údaje MeSH
- Potyvirus genetika MeSH
- RNA virová analýza genetika MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- Solanum tuberosum virologie MeSH
- vysoká teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- komplementární DNA MeSH
- RNA virová MeSH
Complex formation between different antisense RNAs directed against either plus-strand or minus-strand sequences of the potato spindle tuber viroid (PSTVd) was studied using temperature-gradient gel electrophoresis and immunochemical detection with an antibody specific for double-stranded RNA. Short minus-strand sequences were directed against the upper central conserved region (UCCR) of plus-strand viroid replication intermediates, a plus-strand corresponding to the left half of the rod-like secondary structure (VL+) against minus-strand replication intermediates. It was shown that antisense RNA forms complexes with the corresponding target RNA only with low yield during incubation at low (physiological) temperatures but with high yield during in vitro transcription of the target RNA when the antisense RNA is already present in the solution. The antisense RNA sequences were integrated into Solanum tuberosum L. by Agrobacterium tumefaciens transformation. Antisense RNA expression in vivo was analyzed by Northern analysis. Infection tests were performed using the transgenic potato lines in order to evaluate their degree of resistance against PSTVd infection. Although some lines showed a significant inhibition of viroid accumulation, a high variability of viroid infection in different transgenic potato lines was obtained. Since strongly infected plants were observed in all transgenic lines 6 to 8 weeks post inoculation, a threshold concentration of viroid, overcoming the antisense effect has to be assumed. When the rate of viroid accumulation was tested using agroinfection assays on leaf discs, a stronger antisense effect could be achieved.
- MeSH
- Agrobacterium tumefaciens genetika metabolismus MeSH
- antisense RNA biosyntéza genetika metabolismus MeSH
- dvouvláknová RNA analýza chemie metabolismus MeSH
- genetická transkripce MeSH
- geneticky modifikované rostliny genetika MeSH
- molekulární sekvence - údaje MeSH
- monoklonální protilátky imunologie MeSH
- RNA virová antagonisté a inhibitory metabolismus MeSH
- sekvence nukleotidů MeSH
- Solanum tuberosum genetika virologie MeSH
- transformace genetická MeSH
- viroidy genetika fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antisense RNA MeSH
- dvouvláknová RNA MeSH
- monoklonální protilátky MeSH
- RNA virová MeSH
The effects of DNA oligonucleotides complementary to potato spindle tuber viroid (PSTVd) on viroid infection were investigated. The oligonucleotides were used to form hybrids with PSTVd in the infection mixture. A 75% reduction of viroid infection was found when an oligonucleotide complementary to nucleotides 79-110 of PSTVd was hybridized with PSTVd at a molar ratio of approximately 5000:1, respectively. A total inhibition of PSTVd infection was observed using an oligonucleotide complementary to nucleotides 42-78 at the same molar excess of DNA over PSTVd, although a 200-fold molar excess was found to be sufficient for the complete blocking of infection by PSTVd. This oligonucleotide caused a significant reduction (about 83%) of viroid infection even if the hybridization was done at a low (30 degrees C) temperature. Shorter oligonucleotides containing 22 and 15 bases corresponding to position 42-62 and 63-78, respectively, exhibited a significant effect only at a high (80 degrees C) initial temperature of molecular hybridization. Heteroduplexes formed between PSTVd RNA and antisense DNA were found to be less stable in a crude nuclease extract from tomato leaves as compared with PSTVd RNA alone. RNase H was demonstrated to cleave the molecular hybrids in vitro.
- MeSH
- antisense oligonukleotidy terapeutické užití MeSH
- deoxyribonukleasy MeSH
- DNA * MeSH
- molekulární sekvence - údaje MeSH
- nemoci rostlin mikrobiologie MeSH
- sekvence nukleotidů MeSH
- Solanum tuberosum MeSH
- viroidy účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antisense oligonukleotidy MeSH
- deoxyribonukleasy MeSH
- DNA * MeSH
Potato virus A (PVA) was purified from mechanically infected plants Nicotiana tabacum cv Samsun by high speed centrifugation and subsequent isopycnic density gradient centrifugation in CsCl gradient. From different procedures tested 0.05 mol/l phosphate buffer (PBS) pH 8 seemed optimal for virus purification and 0.1 mol/l borate buffer pH 8.0 for virus storage; other pH of PBS, Tris and Hepes buffers were less suitable. Additives preventing virus aggregation were beneficial. The average yield ranged about 40 mg virus protein per kg starting material. The purified virus has remained serologically active after 30 days storage.
- MeSH
- jedovaté rostliny MeSH
- rostlinné viry izolace a purifikace MeSH
- tabák mikrobiologie MeSH
- virologie metody MeSH
- Publikační typ
- časopisecké články MeSH
