The cylindrical inclusion gene of Turnip mosaic virus encodes a pathogenic determinant to the Brassica resistance gene TuRB01.

The viral component of Turnip mosaic virus (TuMV) determining virulence to the Brassica napus TuRB01 dominant resistance allele has been identified. Sequence comparisons of an infectious cDNA clone of the UK 1 isolate of TuMV (avirulent on TuRB01) and a spontaneous mutant capable of infecting plants possessing TuRB01 suggested that a single nucleotide change in the cylindrical inclusion (CI) protein coding region (gene) of the virus was responsible for the altered phenotype. A second spontaneous mutation involved a different change in the CI gene. The construction of chimeric genomes and subsequent inoculations to plant lines segregating for TuRB01 confirmed the involvement of the CI gene in this interaction. Site-directed mutagenesis of the viral coat protein (CP) gene at the ninth nucleotide was carried out to investigate its interaction with TuRB01. The identity of this nucleotide in the CP gene did not affect the outcome of the viral infection. Both mutations identified in the CI gene caused amino acid changes in the C terminal third of the protein, outside any of the conserved sequences reported to be associated with helicase or cell-to-cell transport activities. This is the first example of a potyvirus CI gene acting as a determinant for a genotype-specific resistance interaction.

Resistances to turnip mosaic potyvirus in Arabidopsis thaliana.

The responses of a collection of Arabidopsis thaliana ecotypes to mechanical inoculation with turnip mosaic potyvirus were assessed. The virus induced characteristic severe symptoms of infection in systemically infected plants. Resistance was found in four ecotypes: Bay-0, Di-0, Er-0, and Or-0. Enzyme-linked immunosorbent assay results of the resistant ecotypes suggested that ecotypes Di-0, Er-0, and Or-0 actually consist of mixed genotypes with resistances acting at different levels in the virus life cycle. Another form of resistance was found in ecotype Bay-0, for which several lines of evidence indicated an interference with viral cell-to-cell movement in the inoculated leaves.

Infectivity of turnip mosaic potyvirus cDNA clones and transcripts on the systemic host Arabidopsis thaliana and local lesion hosts.

Full-length cDNA clones of turnip mosaic virus were assembled under the control of T7 or 35S promoter. The 35S or nopaline synthase terminator signals were introduced downstream the full length cDNA controlled by 35S promoter. Both the capped in vitro transcripts from T7 controlled template, and the cDNAs from 35S controlled plasmids were infectious on Arabidopis thaliana plants according to systemically induced symptoms and to ELISA assays. The cDNAs from 35S controlled plasmids induced local lesions in Chenopodium amaranticolor and Chenopodium quinoa plants. A spontaneous silent C/T transition, giving rise to an additional SpeI restriction site, not present in the original viral RNA template, was used as a marker of the origin of infection.

Characterization of typical pepper-isolates of PVY reveals multiple pathotypes within a single genetic strain.

Potato virus Y (PVY) isolates originally coming from infected pepper plants, were biologically and genetically characterized, especially in comparison with PVY potato-isolates. Pepper PVY isolates could be differentiated from potato isolates in their host range, aphid transmission efficiencies, Mab serology, and genetic status. The genetic distances estimated for PVY pepper-isolates, based on their restrictotypes with five restriction enzymes and on their coat protein gene sequences, indicated that they form a single genetic strain with different pathotypic properties. This situation is essentially different to that of PVY potato-isolates.

Strains of Turnip mosaic potyvirus as defined by the molecular analysis of the coat protein gene of the virus.

Turnip mosaic virus (TuMV) is a member of the potyvirus genus with a wide host range and highly variable in its biological characteristics. Analysis of the CP gene sequences from databases, combined with the experimental analysis of the CP gene of further isolates, using data derived from sequence or restriction analysis, has allowed the genetic classification of 60 TuMV isolates or sequences. Two main genetic clusters MB (mostly Brassica isolates) and MR (mostly Radish isolates) were found, together with several apparently independent lineages. Isolates in the latter could be grouped as Intermediate between Brassica and Radish clusters (IBR) or outside Brassica and Radish clusters (OBR), according to their genetic distance to the main clusters. The genetic diversity of TuMV isolates deposited in the databases was increased with the sequences of the CP gene of seven selected isolates, mainly belonging to IBR or OBR groups. There was a correlation between the MR genetic cluster and JPN 1 serotype.

Validation of the VITEK2 and the Advance Expert System with a collection of Enterobacteriaceae harboring extended spectrum or inhibitor resistant beta-lactamases.

The susceptibility testing accuracy of the VITEK2 system and the ability of the Advance Expert System (AES) to provide interpretive readings were evaluated against 86 extended spectrum (ESBL) and 6 inhibitor-resistant-TEM (IRT) beta-lactamases producing Enterobacteriaceae clinical isolates. VITEK2 MICs of 12 beta-lactams were compared with those obtained by the standard NCCLS microdilution technique. The overall essential agreement ( +/- 1 log dilution) was 87.8%. Discrepancies were mainly observed with cefepime (30.3% of total number of discrepancies), ceftazidime (21.2%), and cefotaxime (15.1%). MIC discrepancies were slightly higher in CTX-M- (14.4%) than in TEM- (12.5%) or SHV- (11.9%) type ESBL producers and were rare in IRT producers (1.4%). Overall interpretive agreement was 92.5% and minor, major, and very major errors were 5.4%, 1.7%, and 2.1%, respectively. The AES was able to identify an ESBL phenotype in 85 out of 86 isolates (98.8%) and an IRT phenotype in all 6 isolates harboring these enzymes, thus reducing very major errors to 0.9%. The VITEK2 system, in conjunction with the AES software, is a reliable tool for detection of ESBL or IRT producing Enterobacteriaceae isolates.

An appraisal of different methods for the detection of the walnut strain of cherry leafroll virus.

Three methods were evaluated for the detection of cherry leafroll virus: ELISA, dot-blot and reverse transcriptional polymerase chain reaction (RT-PCR). Dot-blot and RT-PCR were carried out in crude plant extracts without any further RNA purification. Dot-blot hybridization using a 32P-labelled DNA probe was as sensitive as previously reported ELISA results for cherry leafroll virus detection. The most sensitive method was RT-PCR, which amplified a specific fragment of 448 bp from the 3' untranslated region of both viral genomic RNAs. RT-PCR was used to detect cherry leafroll virus in infected walnut buds and twigs.

Amplification and cloning of a long RNA virus genome using immunocapture-long RT-PCR.

A rapid and easy method was developed in order to amplify long fragments of the genome of potato virus Y (PVY), a virus possessing a 10-kb genomic RNA. The method of immunocapture-RT-PCR was adapted, by using thermostable DNA polymerases with proofreading activity and the proper buffers and cycles, to amplify almost the whole genome of PVY in two fragments (5.6 and 4.3 kb) without purifying virions nor viral RNA. Both fragments were cloned subsequently and their ends sequenced. The method is applicable to the rapid cloning and molecular characterization of the genomes of many other RNA viruses.

A method combining immunocapture and PCR amplification in a microtiter plate for the detection of plant viruses and subviral pathogens.

A method for the detection of RNA viral and subviral plant pathogens was developed that combines pathogen partial purification by solid-phase adsorbed antibodies, reverse transcriptional-polymerase chain reaction (RT-PCR) and quantitation of the amplified products by fluorescence. The reverse transcription of the RNA is performed directly on the retained material without any previous thermal or chemical disruption of the virus particles. The whole procedure can be carried out in a microtiter plate. Its validity has been successfully confirmed for the detection of bean yellow mosaic virus, cherry leafroll virus, cucumber mosaic virus, citrus tristeza virus, grapevine fanleaf virus, potato leafroll virus, pepper mild mottle virus, and tomato spotted wilt virus, as well as the satellite RNA of cucumber mosaic virus and potato spindle tuber viroid. In this procedure virus-specific antibodies can be replaced by monoclonal antibodies against double-stranded RNA, thus offering the possibility of detection when no specific virus antibodies are available, or immunological methods are difficult to use (i.e., subviral pathogens like satellite-RNAs or viroids). The method described has the typical sensitivity of assays based on the polymerase chain reaction, it is not more laborious than ELISA, and an equivalent degree of automation is possible.

Direct PCR detection of foot-and-mouth disease virus.

A PCR assay for the detection and characterization of foot-and-mouth disease virus was developed. The procedure allows RT-PCR amplification following direct adsorption of viral suspensions to microtiter plates, avoiding previous steps of phenol-extraction or heating. Using this procedure, FMDV-specific (based on 3D gene sequences), as well as serotype-specific (based on VP1 gene sequences) amplification were achieved for viral samples of serotypes A, O and C, either from cell culture supernatants or from lesions of infected animals. The assay allowed detection of around 15 PFU, being 500-fold more sensitive than a conventional indirect ELISA. This new method constitutes a simple, rapid and efficient alternative for the diagnosis and characterization of FMDV by PCR.

First Report of Grapevine Virus A in Spain.

Grapevine trichovirus A (GVA), a flexuous, filamentous, phloem-limited virus with an approximately 7.3-kbp RNA genome, is widespread in grapevines showing symptoms of leafroll and/or rugose wood. The virus can be mechanically inoculated to Nicotiana benthamiana and N. clevelandii. A field survey of diseased Vitis vinifera white and red cultivars was carried out in Pontevedra (northwest Spain) during the autumn of 1993. We detected the presence of GVA in vines showing leafroll symptoms by an immunocapture-reverse transcription-polymerase chain reaction (PCR) method (2) with GVA-specific primers (1). Bands of the expected size of 430 bp were obtained with extracts from petioles and stem bark as reaction substrates. To verify these results, Northern (RNA) blots with double-stranded (ds) RNAs isolated from grapevines were prepared. Hybridization was positive in two out of 10 samples analyzed. The probe was a 32P-labeled 430-bp PCR product amplified from extracts of N. benthamiana plants infected with GVA strain Is151 (gift of A. Mina-fra). The specificity of this probe was confirmed in dot blot hybridization, as a positive signal was obtained with extracts from GVA-inoculated N. benthamiana, but not with extracts of phosphate buffer-inoculated N. benthamiana, turnip mosaic potyvirus-inoculated Arabidopsis thaliana, or potato potyvirus Y-inoculated N. xanthi plants. The probe did not hybridize to dsRNAs extracted from enzyme-linked immunosorbent assay-positive GLRaV-III-infected grapevines. GVA has been identified in other Mediterranean countries, but to our knowledge this is the first report of the detection of GVA in Spain. References: (1) A. Minafra and A. Hadidi. J. Virol. Methods 47:175, 1994. (2) G. Nolasco et al. J. Virol. Methods 45:201, 1993.

Identification of new isolates of Turnip mosaic virus that cluster with less common viral strains.

Turnip mosaic virus (TuMV) was found infecting cultivated brassicas and wild and cultivated ornamental Brassicaceae plants in different regions of Spain. Five new TuMV isolates, originating from different host plant species (Brassica cretica, Brassica juncea, Brassica napus, Eruca vesicaria subsp. sativa and Sisymbrium orientale), have been identified. The nucleotide sequences of the coat protein (CP) genes of the five isolates were determined. Phylogenetic analysis of the CP sequences showed that the five isolates grouped into two different clusters. The three isolates from the central region of Spain clustered with a previously reported Pisum sativum isolate from southeastern Spain, whereas the other two isolates from the eastern region clustered with two Italian and two Greek isolates. Both clusters were genetically distinct and belonged to the multi-lineage group OBR. The OBR group contains mainly TuMV isolates from hosts other than Brassica spp. and Raphanus sativus and mostly originating from Mediterranean countries. These new sequences provide further phylogenetic resolution of the OBR group. Although new TuMV isolates have been found in Spain, they were not associated with any serious disease outbreaks.

Kinetics of the mediated transport and the passive net flux of phenylalanine across the rat small intestine in vivo.

The kinetics of L-phenylalanine absorption by rat jejunum, in vivo, has been studied with luminal perfusion (0.68 ml/min) during successive periods at different substrate concentrations. The non-saturable passive component, measured by inhibiting the active transport with 60 mM methionine, was a linear function of the substrate concentration with an apparent mass-transfer coefficient of 1.42 nmoles/cm/min/mmoles/l. The transport component, estimated from the difference between total absorption and the passive component, displays saturation kinetics with an apparent transport constant (Km) of 7.5 mM and maximal transport rate (Vmax) of 107 nmoles/cm/min. Active transport seems to be the main component in absorbing phenylalanine proceeding from the digestion of food proteins.

Absorption kinetics for leucine, cycloleucine and alpha-aminoisobutyric acid by rat small intestine in vivo.

The intestinal absorption kinetics of three neutral amino acids, leucine, cycloleucine and alpha-aminoisobutyric acid, has been studied in rat jejunum in vivo, with luminal perfusion during successive periods, by measuring the passive component and the active transport. The mass-transfer coefficients of the passive process, are similar for the three amino acids and increase with the perfusion rate. The transport component, obtained from the difference between total absorption and passive diffusion, shows saturation kinetics and also increases with the perfusion rate. The apparent Michaelis constants, Km, and the maximal transport rates for the three amino acids have been determined. The Km values are greater than those reported for in vitro studies, a result imputable to greater thickness of the unstirred layers in vivo and to the unequal signification of the constant in both conditions. Passive flux has proved to be an important component for in vivo absorption, even at low substrate concentrations (1-5 mM), so that its evaluation cannot be neglected for the calculation ot the kinetic constants of the mediated transport.

Inhibition of sugar active transport across rat intestine in vivo by cadmium.

Galactose absorption by rat jejunum perfused in vivo is inhibited by 0.5 mM Cd2+. This effect is explained by impairment of the phlorizin-sensitive sugar transport system, as Cd does not modify the absorption of L-sorbose or that of galactose in the presence of 0.5 mM phlorizin. Cd inhibition is observed as early as in the 1st minute, does not increase by previous exposure of the mucosa to the metal and does not decrease after washing with saline solution, but it is entirely reversed by EDTA or dithioerythritol. Results agree with a Cd2+ binding to HS- groups of membrane proteins at the brush border, appertaining or functionally related to the phlorizin-sensitive and Na+ dependent transport system for sugars.

Role of -SH groups in rat sugar intestinal transport in vivo.

The effect of p-chloromercuribenzoic acid (pCMB), either alone or in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), on the 1 mM galactose absorption by in vivo perfused rat intestine has been studied. At 0.25 mM concentration, pCMB inhibits galactose absorption in about 32% but it does not modify the absorption of this sugar when the transport is blocked by 0.5 mM phlorizin, or that of the non-transportable monosaccharide derivative 2-deoxy-D-glucose. This shows that only the active transport component of galactose absorption is inhibited. A 2 min preexposure period is required for the inhibition to appear. The inhibition was not reversed by washing with saline solution even when it contained 0.5 mM dithioerythritol, 10 mM cysteine or 5 and 10 mM EDTA. The simultaneous exposure to 0.25 pCMB and 0.25 mM DTNB inhibits the total galactose entry in about 50%, an effect higher than the one exerted by each reagent separately and close to the one obtained with 0.5 mM phlorizin. Our results, in vivo, confirm the importance of the thiol groups in the cotransport of Na+ and sugar. As DTNB is an SH-reagent of lesser liposolubility than pCMB, the existence of two populations of sulfhydryl groups related to sugar transport which differ in their location within the brushborder membrane and in accessibility from the intestinal lumen, is suggested.

Active transport of sugars in tortoise intestine.

The tortoise intestine capability for active transport of sugars has been studied in vitro at 30 degrees C, using labelled sugars. A release of glucose from the glycogen stores of the intestinal wall to the medium took place throughout the incubation period of the sacs. An active transport of 14C-D-glucose against a concentration gradient from the mucosal to the serosal compartment was evident, whereas no such activity could be detected for 14C-D-galactose. The tissue oxygen uptake was 36% higher with glucose than with galactose in the medium.