Identification and characterization of a new member of the genus Luteovirus from cherry.

The complete genomic sequence of a new virus from cherry trees was determined. Its genome is 5857 nt long and resembles that of members of the genus Luteovirus in its genomic organization and nucleotide sequence. Based on the species demarcation criteria for luteoviruses, the virus represents a new luteovirus species. Furthermore, a 47-nt-long inverted repeat was found at the 3' end of its genome. The virus has been provisionally named cherry-associated luteovirus (ChALV) and is the fourth member of the family Luteoviridae reported to naturally infect woody plants.

Molecular analysis of gooseberry vein banding associated virus.

UNLABELLED: The intraspecies variability of gooseberry vein banding associated virus (GVBaV) was analyzed by using 5 complete and 9 partial sequences and compared with other badnavirus species. GVBaV was recognized to be a monophyletic and very homogeneous species with up to 94% identities in distinct proteins. Analysis of non-synonymous and synonymous substitution ratios (dNS/dS) revealed higher values for ORF4 in comparison with other genes. This could reflect different evolutionary pressure upon this ORF. A highly variable region with possible diagnostic value has been localized in the intergenic region of the virus. KEYWORDS: badnavirus; complete genome; phylogeny.

New in vitro method for evaluating antiviral activity of acyclic nucleoside phosphonates against plant viruses.

A new method was developed for testing antiviral compounds against plant viruses based on rapidly growing brassicas in vitro on liquid medium. This method enables exchange of media containing tested chemicals in various concentrations and simultaneous evaluation of their phytotoxicity and antiviral activity. While using ribavirin as a standard for comparison, phytotoxicity and ability of the acyclic nucleotide analogues (R)-PMPA, PMEA, PMEDAP, and (S)-HPMPC to eliminate ssRNA Turnip yellow mosaic virus (TYMV) were evaluated by this method. Double antibody sandwich ELISA and real-time PCR were used for relative quantification of viral protein and nucleic acid in plants. Ribavirin had the most powerful antiviral effect against TYMV. On the other hand, (R)-PMPA and PMEA had no antiviral effect and almost no phytotoxicity compared to the control. (S)-HPMPC and PMEDAP showed moderate antiviral effect, accompanied by higher phytotoxicity. The tested compounds can be screened within 6-9 weeks in contrast to the 6 months for traditionally used explants on solid medium. The method enables large-scale screening of potential antivirals for in vitro elimination of viruses from vegetatively propagated crops and ornamentals.

First Report of Blueberry red ringspot virus in Highbush Blueberry in the Czech Republic.

A collection of highbush blueberry (Vaccinium corymbosum L.) cultivars planted in the field for propagation in South Bohemia was surveyed in May and July of 2009 for the occurrence of detrimental viruses. A total of 67 plants of 10 cultivars (Berkeley, Burlington, Blue Crop, Bluetta, Darrow, Duke, Gila, Jersey, Late Blue, and Northland), were observed for typical Blueberry red ringspot virus (BRRV) symptoms that appear as reddish ring spots and blotches on stems and fruits, exclusively on the upper surface of the older leaves but not the underside. Samples of leaves were collected and maintained at -20°C until used for DNA extraction, then assayed for BRRV infection using PCR. Controls originated from the same blueberry cultivars in vitro. DNA was extracted from leaf tissue with a NucleoSpin Plant II kit for isolating genomic DNA according to the manufacturer's instructions (Macherey-Nagel, Düren, Germany). Primer pair BRRV15/16, which amplified fragments of the reverse transcriptase gene (1), was used in PCR for BRRV detection. The program used for PCR amplification was 94°C for 2 min, followed by 35 cycles at 94°C for 30 s, 49°C for 30 s, and 70°C for 45 s, followed by a final extension at 70°C for 5 min. The total PCR volume of 25 µl contained 20 ng of DNA, 200 µmol liter-1 dNTPs, 0.5 µl of each primer BRRV15 and BRRV16 (20 pmol µl-1), 75 mM Tris-HCl pH 8.8, 20 mM (NH4)2SO4, 0.01% Tween 20, 2.5 mM MgCl2, 2.5 U of Taq Purple DNA polymerase, and stabilizers (Top-Bio Ltd., Prague, Czech Republic). Amplifications were conducted in an MJ Research (Waltham, MA) thermocycler. Aliquots (4 µl) of each PCR product were analyzed by electrophoresis in tris-acetate-EDTA buffer. No BRRV symptoms were observed on the plants in early spring, yet BRRV was detected in one symptom-free bush of cv. Darrow by PCR. In July, typical symptoms developed on that and another cv. Darrow bush that was also positive by PCR. DNA fragments of the expected sizes were amplified from total nucleic acid samples of both infected blueberry bushes using primers BRRV15/16, while no amplification products were detected in plants without symptoms. The amplicons obtained with primers BRRV15/BRRV16 were sequenced and revealed 97.5%-nt identity to the BRRV putative reverse transcriptase gene (GenBank Accession No. AF404509). The 845 nt of the amplicon has been deposited at GenBank under Accession No. HM107773. The disease was likely introduced in infected planting material, since no highbush blueberry plantations exist in the vicinity and V. corymbosum is not native to the Czech Republic. In conclusion, to our knowledge, this is the first report of Blueberry red ringspot virus (genus Soymovirus, family Caulimoviridae) in V. corymbosum L. in the Czech Republic. Symptom observation and PCR testing for BRRV should therefore, be incorporated into the certification scheme for highbush blueberry in the Czech Republic. Reference: (1) J. J. Polashock et al. Plant Dis. 93:727, 2009.

Investigating the sensitivity of a fluorescence-based microarray for the detection of fruit-tree viruses.

An oligonucleotide microarray for the detection of some fruit-tree viruses was designed and its theoretical detection limit was assessed using Cy3-labelled oligonucleotides. The real sensitivity of the microarray was compared for different kinds of fluorescently labelled targets: (a) cDNA and PCR amplified targets, (b) PCR amplified targets labelled using three different labelling methods. In the first case (a), the number of viral cDNA molecules was below the assessed detection limit of the microarray and only PCR amplified targets were detected. A second comparison (b), done on 3 selected viruses, included indirect labelling, the direct incorporation of labelled-dUTPs, and the use of Cy3-labelled primer. The targets labelled most intensively were produced by the Cy3-primer labelling (2 of 3 viruses) or by the indirect labelling method (1 of 3 viruses), the weakest signal showed targets labelled directly (all 3 viruses). The use of Cy3-primer labelling involved the simplest preparation and the lowest cost, however occasional weak cross-hybridization appeared. The indirect labelling method was of the highest specificity. The probes hybridizing near the 3-end of the targets showed the lowest intensities of fluorescent signal.

Complete nucleotide sequence of Radish mosaic virus RNA polymerase gene and phylogenetic relationships in the genus Comovirus.

The 3'-terminal part of RNA1 genome segment of Radish mosaic virus (RaMV) including complete RNA polymerase gene was sequenced. The 207 amino acids long polymerase is matured from a polyprotein precursor by cleavage at putative Q/H site by viral protease. The alignment of available amino acid sequences of RNA polymerase genes of comoviruses revealed a closest (55%) identity of RaMV to Red clover mottle virus (RCMV).

Multiplex RT-PCR detection of four aphid-borne strawberry viruses in Fragaria spp. in combination with a plant mRNA specific internal control.

The principal aphid-borne viruses infecting Strawberry (Fragaria spp.) Strawberry crinkle virus (SCV), Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV) can cause serious crop losses. In this paper, a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) method is described for the simultaneous detection of all four viruses in combination with a plant mRNA specific internal control which can be used as an indicator of the effectiveness of the extraction and RT-PCR. In total, 18 strawberry isolates infected naturally were analysed by this method. Every combination of RNA virus was able to be detected and a full complement of all four viruses were found together in three isolates, all taken from wild strawberry (Fragaria chiloensis (L.) Duch.) in Chile. The upper detection limit for the four viruses was at an extract dilution of 1/200. The broad applicability of the RNA specific internal control primers-which produced a PCR fragment of the expected size in 25 of 27 plant species tested-combined with improvements, made in extraction methods described provides potentially a standard method for comparable RT-PCR analyses in a wide variety of plant species.

Mixed infection of black currant (Ribes nigrum L.) plants with Blackcurrant reversion associated virus and rhabdovirus-like particles with symptoms of black currant reversion disease.

Black currant plants of cvs. Black Smith and Karlstejnský dlouhohrozen showing symptoms of severe Russian (R) form of black currant reversion disease (BCRD) were found in 1999-2000 in the Czech Republic. Five selected plants of both cultivars originating from two distant loci were tested by polymerase chain reaction (PCR) for presence of the Blackcurrant reversion associated virus (BRAV), the causal agent of BCRD. In all plants, virus-specific 215 nt cDNA fragments proving the presence of BRAV were obtained. Moreover, in two of those five black currant plants, rhabdovirus-like particles were found in ultrathin sections by electron microscopical examinations. The particles measured 200-347 nm by 64-90 nm. They occurred mostly within nuclei of parenchyma cells of vascular bundles as single particles, rafts of particles, but also in aggregates. They were found also in the perinuclear space and occasionally directly in the cytoplasm. Clusters of particles either within the nucleus or in the perinuclear space were membrane-bound. We bring evidence on the occurrence of the severe (R) form of BCRD and the first evidence of BRAV in the Czech Republic.

The vitrification of in vitro fertilized cow blastocysts by the open pulled straw method.

In 6 replicates, a total of 450 immature oocytes recovered from 144 slaughterhouse-derived bovine ovaries were matured and fertilized in vitro, then cultured for 7 to 9 d on a granulosa cell monolayer in tissue culture medium 199 (TCM-199) supplemented with fetal calf serum. Of 126 blastocysts (28% of oocytes cultured), 117 (26% of oocytes cultured) were vitrified in Hepes/bicarbonate-buffered TCM-199 medium and 20% fetal calf serum, with ethylene glycol and dimethylsulfoxid as the cryoprotectants. After thawing in 1.2 mL holding medium with 0.25-M sucrose and after 1 min in holding medium with 0.15-M sucrose, blastocysts were cultivated in vitro for 24 h. The re-expansion rate of blastocysts was 69.2% (81 blastocysts), and 39.5% (32 blastocysts) were hatched. Re-expansion and hatching rates differed between the blastocysts vitrified on 7 and 8+9 days (74.6% and 46% vs. 62% and 29%, respectively). After transfer to recipient cows, 3 out of 6 were diagnosed by ultrasonography as pregnant. Three calves were born from 18 transferred embryos (16.7%). The open pulled straw (OPS) method seems to be a convenient, simple and effective method for cryopreservation of 7 to 9 d bovine embryos.

Septic arthritis and osteomyelitis of the wrist: reconstruction with a vascularized fibular graft.

A case of spontaneous staphylococcus arthritis of the wrist with associated carpal and distal radius osteomyelitis is reported. Following sequential debridements and a 6-week course of parenteral antibiotics, an extensive defect was bridged with a vascularized fibular autograft to achieve a successful fusion. There was no donor site morbidity or recurrent infection. Follow-up radiographs 41 months later demonstrated complete incorporation and hypertrophy of the graft.

Strawberry vein banding virus--definitive member of the genus Caulimovirus.

The complete DNA sequence (7876 nucleotides) of strawberry vein banding virus (SVBV) has been determined. Seven open reading frames are detected that potentially code for proteins of calculated weight 37.8; 18.3; 16.6; 56.0; 81.1; 59.0 and 12.6 kDa, respectively. Their position on the viral genome is the same as that of the corresponding proteins on the cauliflower mosaic virus (CaMV) genome. Phylogenetic analysis based on the amino acid sequence of this protein shows a closer relationship of SVBV with CaMV, figwort mosaic virus and carnation etched ring virus than with other caulimoviruses.

Occurrence of nepoviruses in Rubus species in the Czech Republic.

The occurrence of arabis mosaic virus (AMV), raspberry ringspot virus (RRV), tomato black ring virus (TBRV), strawberry latent ringspot virus (SLRV) and cherry leaf roll virus (CLRV) in cultivated and wild plants of raspberry and blackberry has been studied in the Czech Republic in 1993-1996. Five hundred and seventy samples were collected at 51 localities and assayed by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The results represent the first evidence on the occurrence of AMV, RRV, TBRV and SLRV in cultivated Rubus species in the Czech Republic. Isolates AMV M20 and TBRV ML15 which were successfully transmitted by mechanical inoculation and characterized by reactions of differential host plants and by electron microscopy are the first isolates from Rubus from this territory. CLRV was not detected in either cultivated or wild Rubus species.

Detection and isolation of nepoviruses on strawberry in the Czech Republic.

Arabis mosaic, strawberry latent ringspot, tomato black ring and raspberry ringspot nepoviruses were monitored using double sandwich enzyme-linked immunosorbent assay (DAS-ELISA) in 18 cultivars of strawberry Fragaria x ananassa Duch. in the Czech Republic. Arabis mosaic and strawberry latent ringspot viruses were detected, isolated and characterized on differential host plants and by electron microscopy. Both viruses were purified and antisera to them were prepared.

Comparison of the Czech Scrophularia isolate with the Italian Anagyris strain of scrophularia mottle virus.

Scrophularia mottle virus (ScMV) was newly found in the Czech Republic in Scrophularia nodosa L. plants. The Czech isolate (ScMV-C) was serologically identical and similar in symptoms and host range to the Italian Anagyris strain (ScMV-I). Nicotiana tabacum L. cv. Samsun, Nicotiana glutinosa L., Nicotiana tabacum L. cv. White Burley, Physalis floridana Rybd. and Cucumis sativus L. are described as new host plants of ScMV. Double-stranded RNA patterns and the isoelectric point of this virus are characterized.

The occurrence of mixed infections of turnip mosaic, cauliflower mosaic and cucumber mosaic viruses in winter oilseed rape from the territory of Uzbekhistan.

Turnip mosaic, cauliflower mosaic and cucumber mosaic viruses (TuMV, CaMV and CMV) were isolated and identified from winter oilseed rape plants from Uzbekhistan. This is the first record on the occurrence of CaMV and of mixed infection of rape with the three aforementioned viruses from the territory of the former Soviet Union.

Characterization, purification and serology of the Czechoslovak isolate of radish mosaic virus.

The radish mosaic virus 1 (RMV 1) isolate was characterized according to the reaction of differential test plants and then purified by the chloroform-butanol method. UV-analysis of sucrose density gradients at 254 nm revealed three components. The values of A260/280 ratio were 1.42 for the top, 1.49 for the middle and 1.67 for the bottom component, respectively. Electron microscopy of the purified virus revealed isometric particles about 30 nm in diameter. Two antisera were prepared with the titre 1:1024 and 1:2048, respectively. The RMV 1 antigen formed a distinct precipitation line with the antiserum against the California neo-type strain of RMV.