thermodynamic methods. In the present work we sequenced the entire PVS genome including the whole replicase gene and developed a system of molecular probing of PVS, where the whole PVS genome was divided into eleven zones, each probed with three primer pairs derived from ordinary PVS sequence using immunocapture RT PCR. 23 PVS isolates were probed including 14 so-called Andean variants of PVS, showing systemic infection on Chenopodium quinoa. In addition to these isolates 16 isolates originating from German potato cultivars included in ecological agriculture systems were analyzed. Among them 4 isolates (Yuguima, Peruanita, Clivia and Blaue mandel) were found to show systemic infection in the indicator. Our analyses revealed a broad variability of PVS throughout the whole PVS genome with characteristic probing cDNA fragment patterns for individual isolates. All European isolates did not show positive reactions with primers derived from Andean PVS in many positions, suggesting principal divergence of Central European isolates from published Andean variants. For detailed analyses we selected a zone covering the 7K-CP leader-coat protein (CP) region within a fragment of approximately 420 bp. TGGE analysis showed complex transition patterns for Andean and ordinary PVS populations with melting points in the range from 42 to 50oC. Deviating sequence variants were selected from each group from cDNA libraries using heteroduplex methods and sequenced to determine possible conserved sites having either specificity for Andean or ordinary characters of PVS. It was found that both cDNA pools differ in distinct nucleotide positions especially within the 5' end of the coat protein gene, suggesting population shifts and divergence of these two groups of sequences. For instance the G or C nucleotide at position 232 in PVS-O group is replaced by the A-nucleotide in all Chenopodium systemic (CS) PVS isolates (PVS-CS). Distinct mutations also accumulated to high frequencies in other positions and in total 23 such positions were revealed. Some specific amino acid combinations were revealed at the amino acid level in these two groups. Especially methionine at position 17 in combination with serine at position 34 are frequently associated with the CS character of PVS. As shown in the consensus sequence, hydrophobic and polar amino acids are characteristic for positions 17 and 34 respectively in CS variants. Evolutionary distances show that CS-variants evolved most probably from ordinary European PVS and are unrelated to original Andean PVS. The project was supported by the grant NAZV QF 3109 and by GA AS CR project IBS5051014.
Molecular characterization of two ampeloviruses in the Czech Republic
Petr Komínek, Marcela Bryxiová
Research Institute of Crop Production, Drnovská 507, Prague-Ruzyně, 16106, Czech Republic
A prebasic propagation material of grapevine, held in the Research Station of Viticulture, Karlštejn, Central Bohemia, Czech Republic, was screened for the presence of viruses. Large numbers of vines were found to display symptoms of leafroll disease - the downrolling of leaf blades and the premature autumn discoloration of leaves. Dormant canes were taken from tested grapevines during winter, and phloem scrapings were used as tested tissue. DAS-ELISA and RT-PCR methods were used to identify the causal agent of the disease. Grapevines were tested by DAS-ELISA for the presence of Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2, Grapevine leafroll-associated virus 3 (GLRaV-3), Grapevine leafroll-associated virus 5, Grapevine leafroll-associated virus 6, Grapevine leafroll-associated virus 7, and also for the presence of other viruses which can cause diseases of grapevine wood, Grapevine virus A (GVA) and Grapevine virus B. Two ampeloviruses, GLRaV-1 and GLRaV-3 were found to be widely spread in tested grapevines. Both viruses were detected by DAS-ELISA. Several primer pairs for amplification of important parts of the genome and the detection of those viruses were designed and utilized in RT-PCR. PCR fragments corresponding to the Heat shock protein 70 gene of those two viruses were cloned and sequenced. Obtained sequences are now available in GenBank under accession numbers AY644650 (GLRaV-1) and AY424408 (GLRaV-3). A phylogenetic tree was constructed and identity of our virus isolates and their classification to the genus Ampelovirus was confirmed. In addition, GVA was found to be present in a prebasic propagation material of grapevines, but occurred mostly in grapevines without symptoms of leafroll disease. Other viruses were not found in a prebasic propagation material of grapevine. The work was supported by grant number 522/01/D131 of Czech Science Foundation.
Sequential variability among different isolates of potato mop-top virus (PMTV)
T. PEČENKOVÁ1, T. MORAVEC1, M. FILIGAROVÁ1, P. DĚDIČ2 and N. ČEŘOVSKÁ1
1Department of Virology, Institute of Experimental Botany, Czech Academy of Sciences, Na Karlovce 1a, Prague 6, 160 00, Czech Republic, 2Institute for Potato Research, Havlíčkův Brod, Czech Republic
We determined the entire nucleotide sequence of coding regions of a Danish PMTV isolate 54-15 and compared it to other known and sequenced isolates of PMTV. Many nucleotide and amino acid changes were found in parts of the RNA coding for triple gene block (TGB) proteins and part of the RNA coding for the read-through region of the coat protein (CP). These regions from two other isolates, the weak one 54-10 and the strong one 54-19, were sequenced. Only two amino acid changes were found that correlated with the subdivision of isolates according to symptom development into weak and strong subgroups. Although the sequence comparisons indicate a high genetic stability of PMTV populations, the remarkable change was found in the newly sequenced isolates - the replacement of the AUG start codon of the fourth gene of the TGB encoding RNA, coding for a cystein-rich protein, by the less efficient GUG start codon. Phylogeny analysis confirmed that the three Danish isolates are closely related to each other. This research was supported by the grant No.522/04/1329 of the Grant Agency of Czech Republic.