In situ hybridization and promoter analysis reveal that cotton leaf curl Multan betasatellite localizes in the phloem.

Begomoviruses (the family Geminiviridae) have either a monopartite or a bipartite (DNA A and DNA B) single-stranded DNA genome. DNA B contains the open reading frame for movement protein and enables some bipartite begomoviruses to invade non-phloem tissues, whereas monopartite begomoviruses are limited to the phloem. Betasatellite DNA replicates in association with some monopartite begomoviruses to produce severe symptoms and enhance replication of helper virus in some hosts. The cotton leaf curl Multan betasatellite (CLCuMuB) has been shown to substitute for the DNA B of a bipartite begomovirus by permitting systemic infection, but the tissue tropism and the role of betasatellites in releasing monopartite begomoviruses from the phloem to adjacent tissue has not been described. In this study, the tissue tropism of CLCuMuB and a monopartite helper virus, tomato leaf curl virus (ToLCV), has been investigated using in situ hybridization, promoter localization and analysis of transgenic 2ß plants, which contain a dimer of the CLCuMuB genome. In situ hybridization showed that CLCuMuB and ToLCV were localized into the vascular tissue of infected plants. In addition, CLCuMuB was detected only in the vascular tissue of 2ß transgenic plants after infection with ToLCV. ß-glucuronidase (GUS) expression under the ßC1 promoter was also limited to the phloem tissues. In conclusion, we showed for the first time that CLCuMuB localizes only in the phloem tissues and unlike the DNA B component, CLCuMuB is unable to release the monopartite helper virus out of phloem tissues.

Current status of viroid taxonomy.

Viroids are the smallest autonomous infectious nucleic acids known so far. With a small circular RNA genome of about 250-400 nt, which apparently does not code for any protein, viroids replicate and move systemically in host plants. Since the discovery of the first viroid almost forty-five years ago, many different viroids have been isolated, characterized and, frequently, identified as the causal agents of plant diseases. The first viroid classification scheme was proposed in the early 1990s and adopted by the International Committee on Taxonomy of Viruses (ICTV) a few years later. Here, the current viroid taxonomy scheme and the criteria for viroid species demarcation are discussed, highlighting the main taxonomic questions currently under consideration by the ICTV Viroid Study Group. The impact of correct taxonomic annotation of viroid sequence variants is also addressed, taking into consideration the increasing application of next-generation sequencing and bioinformatics for known and previously unrecognized viroids.

Nucleotide sequence diversity in Velvet tobacco mottle virus: a virus with a unique Australian pathosystem.

Velvet tobacco mottle virus (VTMoV) is a naturally occurring mirid-transmitted sobemovirus of native velvet tobacco (Nicotiana velutina) plants in the Australian arid zone. We have sequenced the coding region of a typical field isolate of VTMoV (isolate I-17-04, satellite-plus) and show that it differed by nine polymorphisms from the previously sequenced atypical 'satellite-minus' variant VTMoV-K1 (represented here as L-K1-04), while retaining the same genomic and amino acid sequence motifs. We also report that although L-K1-04 was confirmed to be free of detectable satellite RNA by gel electrophoretic assay, the satellite sequence was detected in it by RT-PCR assay. Nucleotide sequence variation among the RNA-dependent RNA polymerase open reading frames of 15 field and laboratory isolates identified four phylogenetic groups, but these did not show a pattern related to site or time of sampling. This result would be consistent with nucleotide sequence variants of VTMoV being dispersed widely by migrating adult mirid vectors.

Mutation rate in Velvet tobacco mottle virus varies between genomic region and virus variant but is not influenced by obligatory mirid transmission.

Genomic mutation in plant viruses of cultivated plants is known to be influenced by virus, host and vector, but the factors influencing mutation in viruses of native plants in natural ecosystems are rarely studied. We have tested the effect of mode of transmission on mutation in Velvet tobacco mottle virus (VTMoV), a mirid-vectored sobemovirus associated with Nicotiana velutina, an Australian native xerophyte growing in a region isolated from anthropogenic influences. Two variants of VTMoV (K1 and R17) were passaged monthly in the alternative experimental plant host, N. clevelandii, for 2 years, either by mechanical inoculation or by transmission with the mirid Cyrtopeltis nicotianae. Sequence variations were scored after 24 passages in regions of the genome containing the open reading frames (ORFs) for the P1 and coat protein (CP). The mean mutation rate was 6.83 × 10(-4) nt/site year, but a higher overall rate was observed for the K1 (satellite -) than the R17 (satellite +) variant. The P1 ORF showed a higher frequency of non-synonymous mutations than the CP. No clear association was found between either mutation site or mutation rate and the mode of transmission, indicating that obligatory mirid transmission had not exerted a specific bottle-neck effect on sequence variation during the experimental time frame. Failure to detect any sequence motifs linked to vector transmission suggests that a specific capsid-stylet interaction is not required for transmission by mirids.

Complete nucleotide sequence of Velvet tobacco mottle virus isolate K1.

Velvet tobacco mottle virus (VTMoV) infects the native Australian plant Nicotiana velutina, which is endemic to central Australia. This virus is included in the genus Sobemovirus based on virion morphology and serological relationships. We report here the full genome sequence of VTMoV, attained using a genome-walking strategy with both degenerate and specific primers. This sequence confirms that VTMoV is a sobemovirus, with the same open reading frame (ORF) organisation as other described sobemoviruses. The VTMoV sequence is closest to those sobemoviruses isolated from monocotyledonous plants, although the narrow host range of VTMoV is limited to dicotyledonous plants.

Satellite DNA beta overrides the pathogenicity phenotype of the C4 gene of tomato leaf curl virus but does not compensate for loss of function of the coat protein and V2 genes.

We have investigated the ability of satellite DNA beta to complement mutations in the CP, V2 and C4 genes of the monopartite begomovirus, tomato leaf curl virus, which are potentially involved in movement. A mutation in the coat protein was not complemented by DNA beta. Mutations of the C4 and V2 genes attenuated and abolished symptoms, respectively. In the presence of the C4 mutant, but not the V2 mutant, DNA beta induced typical symptoms, confirming that the satellite encodes a dominant symptom determinant. In contrast to the C4 mutant, DNA beta did not enhance the viral DNA levels of the V2 mutant, suggesting that V2 is required for this phenomenon. The significance of these findings is discussed based on our present understanding of the functions of the viral genes and DNA beta.

Outcomes in 2748 patients referred to a colorectal two-week rule clinic.

OBJECTIVE: To assess the 3-year outcomes of a nurse-led, one-stop, 2-week rule (TWR) clinic for suspected colorectal cancer (CRC) in a large teaching hospital. METHOD: Data were collected prospectively from January 2002 to December 2004. In total, 2748 patients were seen over the 3-year period. The ratio of male:female subjects was 1190:1558 (43%:57%). Median age at presentation was 66 years (range 17-96). RESULTS: A total of 1363 (49.6%) nonconforming referrals were made; 1300 patients (47.3%) underwent flexible sigmoidoscopy during their initial assessment in clinic; 1439 patients (52.4%) underwent a barium enema during the course of their investigation; 2503 patients (91.1%) were seen within 14 working days. The median overall wait for the initial clinic appointment was 10 days. The annual number of patients seen was similar over the 3-year period. A total of 174 cancers (6.3%) were identified which accounted for 36.4% of all CRCs diagnosed during the study period. Nineteen cancers presented in the nonconforming group (1.6% of all non-conforming patients). Rectal tumours accounted for 59.8% (n = 104) of all cancers diagnosed while right-sided tumours accounted for only 10.9% (n = 19). Advanced tumours accounted for 73.0% (n = 127) of the total; 133 (76.4%) cancer patients underwent some form of surgical intervention. CONCLUSION: A specialist nurse-led, one-stop TWR clinic for suspected colorectal cancer is sustainable and can be run successfully with over 90% of referrals seen within the targeted time period. The proportion of non-conforming referrals was high and a large number of advanced and unstaged tumours was observed. Low numbers of proximal tumours were detected.

Discriminating Between Isolates of PSbMV Using Nucleotide Sequence Polymorphisms in the HC-Pro Coding Region.

The molecular diversity of 14 isolates of Pea seed-borne mosaic virus (PSbMV) from southern Australia, 13 previously described isolates from Pakistan, and a reference isolate from the United States have been studied to determine whether a relatively simple molecular diagnostic assay and classification scheme could be developed for this virus. The Australian isolates were placed into either pathotype P1 or pathotype P4 by bioassay on differential genotypes of Pisum sativum. The Pakistani isolates represented pathotypes P1, P4, U1, and U2, and an undetermined pathotype. The reference US isolate was pathotype P1. A reverse transcription-polymerase chain reaction (RT-PCR) assay based on an amplicon from the variable HC-Pro coding region of potyviruses was shown to distinguish PSbMV from seven other legume infecting potyviruses. Restriction fragment length polymorphisms (RFLPs) generated from the HC-Pro RT-PCR products of all 28 isolates using seven restriction endonucleases placed them into eight groups. A phylogenetic tree based on a Bray-Curtis similarity comparison placed the groups into three clusters. The groups and clusters had no clear association with either pathotype or geographic source. It is concluded that within the range of viruses and isolates tested, the RT-PCR-RFLP method will both specifically identify PSbMV and provide a simple, qualitative, and rapid means for placing PSbMV isolates into groups. Applications could include mapping and tracking isolates in space and time.

Variants of Coconut cadang-cadang viroid isolated from an African oil palm (Elaies guineensis Jacq.) in Malaysia.

Variants of Coconut cadang-cadang viroid have been identified in a plantation oil palm growing in Malaysia. Three size classes are described, comprising 297, 293, and 270 nt. Compared with the 296-nt form of coconut cadang-cadang viroid (CCCVd), all variants substituted C31 --> U in the pathogenicity domain and A175 --> C in the right-hand terminus. Other mutations and deletions accounted for the different sizes. These are the first sequences reported for variants of Coconut cadang-cadang viroid in a species other than coconut palm, and this is the first evidence that variants closely related to CCCVd occur outside the Philippines.

Importation of canid rabies in a horse relocated from Zimbabwe to South Africa.

In July 2003 a 2-year-old Thoroughbred colt was imported from Harare, Zimbabwe to the Ashburton Training Centre, Pietermaritzburg, South Africa. Five months after importation, the colt presented with clinical signs suggestive of rabies: it was uncoordinated, showed muscle tremors and was biting at itself. Brain tissue was submitted for analysis and the clinical diagnosis was confirmed by the fluorescent antibody test and reverse-transcription polymerase chain reaction (RT-PCR). Phylogenetic analysis of the nucleotide sequence of the cytoplasmic domain of the glycoprotein and the G-L intergenic region of the rabies virus confirmed it to be an infection with a canid rabies virus, originating from an area in Zimbabwe endemic for the domestic dog (Canis familiaris) and side-striped jackal (Canis adustus) rabies.

Gut-focused hypnotherapy normalizes disordered rectal sensitivity in patients with irritable bowel syndrome.

BACKGROUND: We have previously shown that hypnotherapy alters rectal sensitivity in some patients with irritable bowel syndrome. However, this previous study used incremental volume distension of a latex balloon, which might be susceptible to subject response bias and might compromise the assessment of compliance. In addition, the study group was symptomatically rather than physiologically defined. AIM: To assess the effect of hypnotherapy on rectal sensitivity in hypersensitive, hyposensitive and normally sensitive irritable bowel syndrome patients using a distension technique (barostat) that addresses these technical issues. METHODS: Twenty-three irritable bowel syndrome (Rome I) patients (aged 24-72 years) were assessed before and after 12 weeks of hypnotherapy in terms of rectal sensitivity, symptomatology, anxiety and depression. Normal values for sensitivity were established in 17 healthy volunteers (aged 20-55 years). RESULTS: Compared with controls, 10 patients were hypersensitive, seven hyposensitive and six normally sensitive before treatment. Following hypnotherapy, the mean pain sensory threshold increased in the hypersensitive group (P = 0.04) and decreased in the hyposensitive group, although the latter failed to reach statistical significance (P = 0.19). Normal sensory perception was unchanged. Sensory improvement in the hypersensitive patients tended to correlate with a reduction in abdominal pain (r = 0.714, P = 0.07). CONCLUSION: Hypnotherapy improves abnormal sensory perception in irritable bowel syndrome, leaving normal sensation unchanged.

First Report of Mundulla Yellows on Eucalyptus spp. Outside Australia.

Mundulla Yellows (MY) is a newly recognized lethal dieback of unknown etiology affecting Eucalyptus spp. in Australia (1). Progression of symptoms, unlike other disorders described for eucalypts, starts as foliar interveinal chlorosis on one branch followed by branch dieback advancing toward the main trunk. Epicormic shoots with symptomatic leaves typically develop proximal to the dying zone. Continuing dieback leads to tree death in 5 to 20 years. MY trees occur singly or in small foci and do not recover even if affected branches are removed. MY-like symptoms developed on seedlings grafted with bark patches from MY trees (1). There is no evidence for a fungal, phytoplasma, or other bacterial etiology. In a search for a putative virus-like agent we have identified an electrophoretic pattern (2) of several single-stranded RNAs with apparent size of 200 to 600 nt that is consistently associated with MY and absent from disease-free trees (unpublished). The RNAs are not yet characterized, but the pattern can be used as a preliminary confirmatory "fingerprint" for MY. We report here that MY-like symptoms occur in southern Spain and the Spanish island of Ibiza. RNA fingerprints closely resembling those of MY, were detected in symptomatic trees (4 of 4 of E. camaldulensis and 1 of 1 of E. globulus) from Valencia, Spain. MY therefore, appears not to be limited to Australia. References: (1) D. Hanold et al. Landscope 17(4):41, 2002. (2) J. W. Randles. Strategies for implicating virus-like pathogens as the cause of diseases of unknown etiology. Pages 315-332 in: Diagnosis of Plant Virus Diseases, CRC Press, Boca Raton, FL, 1993.

Genomic heterogeneity in Pea seed-borne mosaic virus isolates from Pakistan, the centre of diversity of the host species, Pisum sativum.

A range of isolates of Pea seed-borne mosaic virus (PSbMV) was compared in the segments of the genome representing the partial NIb/CP/UTR and the partial P1-Pro/HC-Pro coding regions. Nucleotide and amino acid sequences, and a phylogenetic analysis of the CP region, divided isolates with available sequence information into two groups, one representing pathotype 4, the other pathotype 1. The pathotype 1 group showed greater diversity than the pathotype 4 group. A comparison of 14 isolates, S6 (a pathotype 4 isolate), US (a pathotype 1 isolate) and 12 isolates from Pakistan, by ribonuclease protection assay (RPA) using cRNA transcripts of the cloned partial NIb/CP/UTR regions of the S6, US and Pakistani isolate PK9 placed them into three distinct phylogenetic groups. RPA with a partial P1-Pro/HC-Pro cRNA probe identified a greater level of variation which was too high to be used for generating an overall phylogeny. Thus, RPA identified greater molecular diversity in PSbMV than described hitherto. We conclude that, in addition to the pathotypes 1 and 4 typified by US and S6 respectively, isolates of PSbMV from Pakistan include previously unrecognised molecular variants, and this accords with our previous recognition of new pathotypes from Pakistan.

The genome organisation and taxonomy of Sugarcane striate mosaic associated virus.

Sugarcane striate mosaic associated virus (SCSMaV) has slightly flexuous 950 nm x 15 nm filamentous particles and is associated with sugarcane striate mosaic disease in central Queensland, Australia. We report the full sequence of its RNA genome, which comprises 5 open reading frames representing the polymerase, movement function proteins encoded in a triple gene block and coat protein. Phylogenetic analyses based on either the full nucleotide sequence, the polymerase protein, or the coat protein all placed SCSMaV in an intermediate position between the genera Foveavirus and Carlavirus, but outside both genera. In addition, the absence of a sixth open reading frame excludes it from the genus Carlavirus, and the coat protein is approximately half the size of the type member for the genus Foveavirus. Although SCSMaV was most closely allied to Cherry green ring mottle virus by genome analysis, the two viruses are morphologically and biologically dissimilar. SCSMaV may therefore represent a new plant virus taxon.

Characterization of Na+-coupled glutamate/aspartate transport by a rat brain astrocyte line expressing GLAST and EAAC1.

D-aspartate (D-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. A-asp influx is Na+-dependent with Km = 5 microm and Vmax = 0.7 nmoles x min(-1) x mg protein-1. Influx is sigmoidal as f[Na+] with Na+Km approximately 12 microm and Hill coefficient of 1.9. The cells establish steady-state D-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase in Vmax, with no change in Km. At initial [D-Asp] = 10 microm, RBA take up more than 90% of total D-Asp, and extracellular levels are reduced to levels below 1 microm. Ionophores that dissipate the Delta(mu)Na+ inhibit gradient formation. Genistein (GEN, 100 microm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4alpha-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na+-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), L-Asp, and L-Glu, but not by D-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells.

Identification of sugarcane striate mosaic-associated virus by partial characterization of its double-stranded RNA.

ABSTRACT Sugarcane striate mosaic (ScSM)-affected sugarcane leaves contain a disease-associated 9-kilobase (kb) double-stranded RNA (dsRNA), usually together with 6- and 2.6-kb dsRNAs. The purified 9-kb dsRNA was amplified by the randomly primed polymerase chain reaction (PCR) and cloned. The nucleotide sequences of three separate regions, representing about 2.55 kb (28%) of the dsRNA sequence, were found to have significant similarities to viruses in the genera Capillo-, Carla-, Fovea-, Potex-, Poty-, Tricho-, and Tymovirus. Greatest overall similarity was found to apple stem pitting virus, with less similarity to blueberry scorch virus and potato virus M. A standard virus purification procedure was used to identify slightly flexuous filamentous particles that copurified with the disease-associated RNA. Particle modal lengths were approximately 950 and 1,900 nm with a diameter of 15 nm. Preparations contained a 51-kDa putative capsid protein and a 9-kb single-stranded RNA with a probable 3' polyadenylate tract. These ScSM-associated virus particles differ physically from viruses in existing genera because of their relative rigidity, length, and putative coat protein size. Reverse-transcription PCR with a primer pair designed from the sequenced segments amplified a 820-base pair fragment from ScSM-affected but not healthy sugarcane plants.

Na+-coupled alanine transport in LLC-PK1 cells: the relationship between the Km for Na+ at low [Alanine] and potential dependence for the system.

Analysis of the mechanistic basis by which sodium-coupled transport systems respond to changes in membrane potential is inherently complex. Algebraic expressions for the primary kinetic parameters (Km and Vmax) consist of multiple terms that encompass most rate constants in the transport cycle. Even for a relatively simple cotransport system such as the Na+/alanine cotransporter in LLC-PK1 cells (1:1 Na+ to substrate coupling, and an ordered binding sequence), the algebraic expressions for Km for either substrate includes ten of the twelve rate constants necessary for modeling the full transport cycle. We show here that the expression of Km of the first-bound substrate (Na+) simplifies markedly if the second-bound substrate (alanine) is held at a low concentration so that its' binding becomes the rate limiting step. Under these conditions, the expression for the KNam includes rate constants for only two steps in the full cycle: (i) binding/dissociation of Na+, and (ii) conformational 'translocation' of the substrate-free protein. The influence of imposed changes in membrane potential on the apparent KNam for the LLC-PK1 alanine cotransporter at low alanine thus provides insight to potential dependence at these sites. The data show no potential dependence for KNam at 5 micron alanine, despite marked potential dependence at 2 mm alanine when the full algebraic expression applies. The results suggest that neither translocation of the substrate-free form of the transporter nor binding/dissociation of extracellular sodium are potential dependent events for this transport system.

Specific identification of coconut tinangaja viroid for differential field diagnosis of viroids in coconut palm.

ABSTRACT Tinangaja is a widespread lethal disease of putative viroid etiology affecting coconut palm on the island of Guam. Determination of its distribution and mode of spread requires a simple and reliable diagnostic procedure that is specific for the associated coconut tinangaja viroid (CTiVd). A method of extracting tissue followed by analytical agarose gel electrophoresis for CTiVd detection has been developed and used to identify the viroid in leaf samples of suspect symptomatic palms growing in the field. Two-dimensional polyacrylamide gel electrophoresis showed that the viroid band contained circular molecules that are typical for viroids. Confirmation of the identity of CTiVd and detection of low levels of viroid below the threshold of detection by agarose gel electrophoresis was achieved either by diagnostic oligonucleotide-probe (DOP) hybridization assay or by reverse-transcription polymerase chain reaction (RT-PCR) with the oligonucleotide probe as one of the two PCR primers. RT-PCR was not substantially more sensitive than DOP-hybridization assay. This procedure also was applicable to coconut cadang-cadang viroid (CCCVd), and oligonucleotide probes designed to be specific for either CTiVd or CCCVd distinguished between these two viroids in coconut leaf extracts. This strategy provides a rapid and specific indexing procedure for the two characterized viroids of coconut palm and will be applicable to further studies on the viroid-like sequences previously reported in tropical monocotyledons.