The Genus Tospovirus: Emerging Bunyaviruses that Threaten Food Security.

The genus Tospovirus is unique within the family Bunyaviridae in that it is made up of viruses that infect plants. Initially documented over 100 years ago, tospoviruses have become increasingly important worldwide since the 1980s due to the spread of the important insect vector Frankliniella occidentalis and the discovery of new viruses. As a result, tospoviruses are now recognized globally as emerging agricultural diseases. Tospoviruses and their vectors, thrips species in the order Thysanoptera, represent a major problem for agricultural and ornamental crops that must be managed to avoid devastating losses. In recent years, the number of recognized species in the genus has increased rapidly, and our knowledge of the molecular interactions of tospoviruses with their host plants and vectors has expanded. In this review, we present an overview of the genus Tospovirus with particular emphasis on new understandings of the molecular plant-virus and vector-virus interactions as well as relationships among genus members.

Xylella fastidiosa Isolates from Both subsp. multiplex and fastidiosa Cause Disease on Southern Highbush Blueberry (Vaccinium sp.) Under Greenhouse Conditions.

Xylella fastidiosa is a xylem-limited gram-negative plant pathogen that affects numerous crop species, including grape, citrus, peach, pecan, and almond. Recently, X. fastidiosa has also been found to be the cause of bacterial leaf scorch on blueberry in the southeastern United States. Thus far, all X. fastidiosa isolates obtained from infected blueberry have been classified as X. fastidiosa subsp. multiplex; however, X. fastidiosa subsp. fastidiosa isolates are also present in the southeastern United States and commonly cause Pierce's disease of grapevines. In this study, seven southeastern U.S. isolates of X. fastidiosa, including three X. fastidiosa subsp. fastidiosa isolates from grape, one X. fastidiosa subsp. fastidiosa isolate from elderberry, and three X. fastidiosa subsp. multiplex isolates from blueberry, were used to infect the southern highbush blueberry 'Rebel'. Following inoculation, all isolates colonized blueberry, and isolates from both X. fastidiosa subsp. multiplex and X. fastidiosa subsp. fastidiosa caused symptoms, including characteristic stem yellowing and leaf scorch symptoms as well as dieback of the stem tips. Two X. fastidiosa subsp. multiplex isolates from blueberry caused more severe symptoms than the other isolates examined, and infection with these two isolates also had a significant impact on host mineral nutrient content in sap and leaves. These findings have potential implications for understanding X. fastidiosa host adaptation and expansion and the development of emerging diseases caused by this bacterium.

Ionome changes in Xylella fastidiosa-infected Nicotiana tabacum correlate with virulence and discriminate between subspecies of bacterial isolates.

Characterization of ionomes has been used to uncover the basis of nutrient utilization and environmental adaptation of plants. Here, ionomic profiles were used to understand the phenotypic response of a plant to infection by genetically diverse isolates of Xylella fastidiosa, a gram-negative, xylem-limited bacterial plant pathogen. In this study, X. fastidiosa isolates were used to infect a common model host (Nicotiana tabacum 'SR1'), and leaf and sap concentrations of eleven elements together with plant colonization and symptoms were assessed. Multivariate statistical analysis revealed that changes in the ionome were significantly correlated with symptom severity and bacterial populations in host petioles. Moreover, plant ionome modification by infection could be used to differentiate the X. fastidiosa subspecies with which the plant was infected. This report establishes host ionome modification as a phenotypic response to infection.

Genetic structure and molecular variability of Grapevine fanleaf virus populations.

To gain insights into the evolutionary mechanisms of Grapevine fanleaf virus (GFLV) from the genus Nepovirus, family Secoviridae, the sequences of the complete coding region of RNA2, including genes 2A(HP), 2B(MP) and 2C(CP), and partial sequence from the RNA1-encoded gene 1E(Pol) of 14 GFLV isolates from three naturally infected California vineyards were characterized. Phylogenetic analyses suggested two to three evolutionarily divergent lineages that did not reflect the vineyard origin of the isolates or an association with rootstock genotype or scion cultivar. Examination of the genetic variability of the California isolates alongside isolates worldwide, for which three RNA1 and 44 RNA2 coding sequences are available, revealed similar patterns of molecular evolution for the different regions within the GFLV genome but distinct selection constraints with the strongest pressure exerted on genes 2C(CP) and 2B(MP), an intermediate level of pressure exerted on gene 1E(Pol), and the weakest pressure exerted on gene 2A(HP). Some of the California isolates resulted from interspecies recombination events between GFLV and Arabis mosaic virus with crossover sites suspected in gene 1E(Pol) and identified in genes 2A(HP) and 2B(MP); and intraspecies recombination events inferred in the four target genes but most frequently observed within gene 2C(CP). This study suggested that purifying selection and recombination are important evolutionary mechanisms in the genetic diversification of GFLV.

Genetic Diversity of Prunus necrotic ringspot virus Isolates Within a Cherry Orchard in New York.

A survey for Prunus necrotic ringspot virus (PNRSV) in an orchard of Prunus cerasus cv. Montmorency and Prunus avium cv. Hedelfingen in New York by enzyme-linked immunosorbent assay indicated an eightfold higher infection rate in sour cherry (33%, 32 of 96) than in sweet cherry (4%, 6 of 136) trees. The presence of PNRSV was confirmed by reverse transcription-polymerase chain reaction and amplification of the coat protein (CP) gene in total RNA from infected leaf tissue. Latent infection was prevalent in the majority of trees infected (87%, 33 of 38), while a few of them exhibited shock symptoms or had severely reduced growth (13%, 5 of 38). Asymptomatic PNRSV-infected trees clustered in spatial proximity to symptomatic trees. Sequence analysis of the CP gene (675 bp) indicated a population structure consisting of one predominant molecular variant for 10 isolates and six minor molecular variants for seven isolates. A high sequence identity was found between the CP gene of PNRSV isolates from cherry trees and other isolates from diverse hosts and various geographic origins at the nucleotide and amino acid levels (88 to 100%). Phylogenetic analyses showed a clustering of PNRSV isolates from cherry trees in New York in the predominant group PV-96.

Risk factors for the development of rheumatoid arthritis.

There is increasing interest in attempting to understand what the risk factors are that lead to the development of rheumatoid arthritis (RA). Twin studies have proved a genetic role but also quantified the non-genetic risk. There is thus scope for identifying environmental predictors that might offer a strategy to prevent the disease. Changes in the female hormonal environment such as in pregnancy, breastfeeding and the use of the oral contraceptive (OC) pill appear to have a role. Of the traditional lifestyle exposures, cigarette smoking has been associated with a consistently increased risk that might also apply to the passive inhalation of smoke. Occupation probably has a minor influence, although exposure to silica dust is of aetiological importance. Recent studies have highlighted a role for diet, with suggestions that diets high in caffeine, low in antioxidants and high in red meat may contribute to an increased risk. The most plausible environmental exposure is infection and although several decades of study have produced few definitive candidate organisms, Epstein-Barr virus (EBV) remains an interesting target.

Characterization of a behaviorally active, gender-specific volatile compound from the male asparagus fly Plioreocepta poeciloptera.

Adult male asparagus flies exhibit typical calling behaviors (suggestive of pheromone production) during which they emit a single volatile compound that was identified as isopropyl (S)-5-hydroxyhexanoate. In laboratory bioassays, synthetic samples elicited an arrestant response in females, but did not appear to attract females. On the other hand, the synthetic material attracted conspecific males in olfactometer bioassays.

Application of Bruchin B to pea pods results in the up-regulation of CYP93C18, a putative isoflavone synthase gene, and an increase in the level of pisatin, an isoflavone phytoalexin.

Bruchins, mono and bis (3-hydroxypropanoate) esters of long chain alpha,omega-diols, are a recently discovered class of insect elicitors that stimulate cell division and neoplasm formation when applied to pods of peas and certain other legumes. Differential display analysis resulted in the identification of an mRNA whose level was increased by the application of Bruchin B to pea pods. The corresponding amplification product was cloned and sequenced and a full length cDNA sequence was obtained. This cDNA and the gene from which it was derived were assigned the name CYP93C18 based upon sequence similarities to the cytochrome P450 mono-oxygenase CYP93C subfamily, which contains isoflavone synthase genes from legumes. RNA gel blots and quantitative RT-PCR demonstrated that expression of CYP93C18 increased within 8 h of bruchin treatment to a maximum of 100-200-fold of the level in untreated pods, and then declined. The up-regulation of CYP93C18 was followed by an increase in the level of the isoflavone phytoalexin, pisatin. Pisatin was detectable in the bruchin-treated pods after 16 h and reached a maximum between 32 h and 64 h. This, the first report of induction of phytoalexin biosynthesis by an insect elicitor, suggests that Bruchin B not only stimulates neoplasm formation, but also activates other plant defence responses.

Cloned cattle derived from a novel zona-free embryo reconstruction system.

As the demand for cloned embryos and offspring increases, the need arises for the development of nuclear transfer procedures that are improved in both efficiency and ease of operation. Here, we describe a novel zona-free cloning method that doubles the throughput in cloned bovine embryo production over current procedures and generates viable offspring with the same efficiency. Elements of the procedure include zona-free enucleation without a holding pipette, automated fusion of 5-10 oocyte-donor cell pairs and microdrop in vitro culture. Using this system, zona-free embryos were reconstructed from five independent primary cell lines and cultured either singularly (single-IVC) or as aggregates of three (triple-IVC). Blastocysts of transferable quality were obtained at similar rates from zona-free single-IVC, triple-IVC, and control zona-intact embryos (33%, 25%, and 29%, respectively). In a direct comparison, there was no significant difference in development to live calves at term between single-IVC, triple-IVC, and zona-intact embryos derived from the same adult fibroblast line (10%, 13%, and 15%, respectively). This zona-free cloning method could be straightforward for users of conventional cloning procedures to adopt and may prove a simple, fast, and efficient alternative for nuclear cloning of other species as well.

Coordination between donor cell type and cell cycle stage improves nuclear cloning efficiency in cattle.

Several studies have shown that both quiescent and proliferating somatic donor cells can be fully reprogrammed after nuclear transfer (NT) and result in viable offspring. So far, however, no comparative study has conclusively demonstrated the relative importance of donor cell cycle stage on nuclear cloning efficiency. Here, we compare two different types of bovine fetal fibroblasts (BFFs) that were synchronized in G(0), G(1), and different phases within G(1). We show that for non-transgenic (non-TG) fibroblasts, serum starvation into G(0) results in a significantly higher percentage of viable calves at term than synchronization in early G(1) or late G(1). For transgenic fibroblasts, however, cells selected in G(1) show significantly higher development to calves at term and higher post-natal survival to weaning than cells in G(0). This suggests that it may be necessary to coordinate donor cell type and cell cycle stage to maximize overall cloning efficiency.

Effects of follicular size of cytoplast donor on the efficiency of cloning in cattle.

In cattle, oocytes obtained from follicles smaller than 3 mm in diameter can undergo maturation in vitro, progressing to MII and undergoing fertilization, but are developmentally incompetent. Cytoplasts were prepared from in vitro matured oocytes aspirated from small (1-3 mm) or large (6-12 mm) follicles and fused to serum starved mural granulosa cells. Following activation, reconstructed embryos were cultured for 7 days and classified G1 to G4, before being processed for nuclei counting or transferred to synchronized recipients. Oocytes from small follicles had lower rates of polar body extrusion (59.6 vs. 69%; 731/1230 vs. 608/857) and fusion (71.4 vs. 78.8%; 360/497 vs. 364/465; P < 0.06). There were no differences in total rate of blastocysts development (60 vs. 59.8%; small vs. large), or any grade classification. A significant interaction was detected between follicle size and embryo grade with G3 embryos from small follicles having a greater cell number. Developmental competence of G1 and G2 embryos did not differ at day 27 (48 vs. 46%; 16/33 vs. 17/37; small vs. large). Although there were no differences in fetal size between the two groups, differences in allantois length (53 vs. 86 mm; small vs. large; P < 0.002) and allantois width (9.5 vs. 13 mm; small vs. large; P < 0.06) were seen. No differences in survival to term (2/13 in each group) were observed. These results indicate that cytoplasts from follicles of 1-3 and 6-12 mm in diameter are equally developmentally competent when used in a nuclear transfer procedure.

The efficacy of ergometry determined heart rates for flatwater kayak training.

The aim of this study was to investigate the use of incremental ergometry determined heart rate training intensities for the control of kayak ergometer and open water kayak training. Eight well-trained male kayakers completed a maximal incremental exercise test on an air-braked kayak ergometer for the determination of LT(1) (the power output at which blood lactate concentration increased by > or = 1 mmol x L(-1)), the associated heart rate (HR-LT(1)), VO(2)peak, maximal heart rate and maximal aerobic power. Subjects then performed 20 min trials of kayak ergometry (E), open water kayaking in a single kayak (K1) and open water kayaking in a four-seat kayak (K4) at HR-LT(1). During the three trials, heart rate was continuously measured, and blood lactate concentration, rating of perceived exertion (RPE) and stroke rate were determined every 5 min. In all trials, exercise at HR-LT(1) resulted in stable blood lactate concentrations and a stable RPE. Comparison of the three trials demonstrated that the only difference was for RPE, which was lower in (K4) than in (E), (p < 0.05). The results demonstrate that the prescription of HR-LT(1) elicits similar blood lactate concentrations during kayak ergometer and open water kayak training in both single and team boats.

The long-term outcome of glaucoma filtration surgery.

PURPOSE: To determine the long-term outcome of glaucoma filtration surgery in preserving vision. Visual loss from progressive glaucomatous damage and from complications of surgery, both short and long term, were included. METHODS: A retrospective, community-based, longitudinal study of residents of Olmsted County, Minnesota, who were newly diagnosed with open-angle glaucoma between 1965 and 1980 and underwent filtration surgery in these or subsequent years through 1998. Intraocular pressure (IOP), visual acuity, visual fields, and progression to legal blindness were monitored. Kaplan-Meier analysis was used to determine the cumulative probabilities of changes in these parameters. RESULTS: 73 eyes of 49 patients underwent conventional filtration surgery. Analysis of the first eye having surgery revealed a mean preoperative IOP of 27.6 +/- 8.5 mm dropping to 16.7 +/- 5.6 mm at year one, and remaining in this range throughout follow-up (14.7 +/- 3.0 mm at 10 years; with or without use of medications). The probability of progression to blindness was 46% at 10 years after surgery, as calculated by Kaplan-Meier analysis. Eyes going blind had a postoperative IOP equal to or lower than those not becoming blind (14.0 +/- 4.4 vs. 15.4 +/- 3.0 at postoperative year 10). Eyes going blind had more advanced field loss at the time of surgery, with scotomas above and below the horizontal axis, than eyes not going blind, which had scotomas in only one hemifield. Three patients developed late bleb leaks; two patients developed endophthalmitis. The probability of undergoing cataract surgery was 37% by 10 years postoperatively, which did not differ significantly from the cohort of patients not undergoing surgery at a comparable time point. CONCLUSIONS: Filtration surgery was associated with a 54% probability of preservation of vision from progression to legal blindness at 10 years after surgery. Patients becoming blind had more advanced field loss at the time of surgery; IOP was similar between those going blind and those retaining vision.

Results of cystometry and urethral pressure profilometry in dogs sedated with medetomidine or xylazine.

OBJECTIVE: To compare effects of medetomidine and xylazine hydrochloride on results of cystometry and micturition reflexes in healthy dogs and results of urethral pressure profilometry (UPP) in sedated and conscious dogs. ANIMALS: 20 dogs. PROCEDURES: Urodynamic testing was performed 6 times in each dog (3 times after administration of xylazine [1 mg/kg of body weight, IV] and 3 times after administration of medetomidine (30 microg/kg, IM). Before each episode of sedation, UPP was performed. Heart and respiratory rates and indirect blood pressures were recorded prior to and 5, 10, 20, and 30 minutes after injection of sedative. Cystometry measurements included threshold volume, threshold pressure, and tonus limb. The UPP measurements included maximal urethral closure pressure (MUCP), functional profile length, and, in male dogs, plateau pressure. RESULTS: Mean MUCP was decreased markedly in xylazine- and medetomidine-sedated dogs. Xylazine and medetomidine also decreased plateau pressure in male dogs. The MUCP measurements were consistent among days for conscious and xylazine-sedated dogs but were inconsistent for medetomidine-sedated female dogs. The proportion of valid cystometry measurements was greater for xylazine (39 of 60) than for medetomidine (27 of 60). Cystometry was considered invalid when bladder pressure reached 30 cm H2O without initiation of a micturition reflex. CONCLUSIONS AND CLINICAL RELEVANCE: Medetomidine and xylazine have similar effects on measurement of UPP and cystometry. Medetomidine was less consistent among days for UPP in female dogs and produced fewer valid cystometry tests, compared with xylazine. For urodynamic evaluations, medetomidine administered IM cannot be substituted for xylazine administered IV.

Bruchins: insect-derived plant regulators that stimulate neoplasm formation.

Pea weevil (Bruchus pisorum L.) oviposition on pods of specific genetic lines of pea (Pisum sativum L.) stimulates cell division at the sites of egg attachment. As a result, tumor-like growths of undifferentiated cells (neoplasms) develop beneath the egg. These neoplasms impede larval entry into the pod. This unique form of induced resistance is conditioned by the Np allele and mediated by a recently discovered class of natural products that we have identified from both cowpea weevil (Callosobruchus maculatus F.) and pea weevil. These compounds, which we refer to as "bruchins," are long-chain alpha,omega-diols, esterified at one or both oxygens with 3-hydroxypropanoic acid. Bruchins are potent plant regulators, with application of as little as 1 fmol (0.5 pg) causing neoplastic growth on pods of all of the pea lines tested. The bruchins are, to our knowledge, the first natural products discovered with the ability to induce neoplasm formation when applied to intact plants.

Lipid composition of the extracellular matrix of Botrytis cinerea germlings.

Six simple lipid classes (mono-, di- and tri-acylglycerols, free fatty acids, free fatty alcohols and wax esters) were identified by TLC in the extracellular matrix of Botrytis cinerea germlings and the molecular components of each class were characterized using GC-MS. The relative amounts of fatty acids and fatty alcohols within each lipid class were determined by GC-FID. Over all the lipid classes, the most abundant saturated fatty acids were palmitic (ca. 30%) and stearic acid (ca. 22%). Palmitoleic and oleic acids made up ca. 21% and 24% (respectively) of the free fatty acids, while erucic (ca. 4.1%) and linoleic (ca. 3.6%) acids were the most abundant unsaturated fatty acids in the acylglycerides. The acylglycerides also contained almost 35% long chain fatty acids (C20:0 to C28:0). Six fatty acids were identified which had odd-numbered carbon chain lengths (C15:0, C17:0, C19:0, C21:0, C23:0 and C25:0). Of these, pentacosanoic acid made up almost 14% of the fatty acids in the acylglycerides. Three methyl-branched chain fatty acids, namely isopalmitic, isoheptadecanoic and anteisopalmitic, were identified in the ECM, all in small amounts. Of the fatty alcohols identified, only palmityl and stearyl alcohols were found in the free form (ca. 57% and 43%, respectively) but arachidyl alcohol (ca. 47%) and 1-octacosanol (ca. 30%) were the most abundant fatty alcohols found in the wax ester fraction.

Studies on the measurement of phospholipase A2 (PLA2) and PLA2 inhibitor activities in ram semen.

Extraction with Tris-citrate or Tris-NaCl-EGTA improved the yield of phospholipase A2 (PLA2) from ram semen by 40-50 fold over the previously recommended method of extraction by dilute (0.18 N) sulphuric acid. The enzyme activity in the citrate extract deteriorated more rapidly than in Tris-NaCl-EGTA. The semen PLA2 activity was optimum at pH 8.0, heat sensitive at 70 degrees C for 30 min, activated by Ca2+ (although approximately 60% activity was also found in the absence of calcium) and did not exist as a pro-enzyme. The semen PLA2 activity was equally distributed among the sperm and seminal plasma (SP) components of ram semen. However, the low levels of PLA2 activity in the SP of vasectomised rams tend to suggest that PLA2 in the SP fraction may have originated from testicular or epididymal secretions or leakage, from sperm. PLA, in sperm exists as a large molecular weight aggregate, whereas in SP it is present as a smaller aggregate. In addition to PLA2, semen also contained PLA2 inhibitor activities. Inhibition was observed against PLA2s from bee venom, pig pancreas and oviductal extracts. The inhibitory activity is presumed to be due to a large molecular weight protein as the inhibitor activity was not extracted in a chloroform:methanol (2:1; v/v) mixture, it was non-dialysable, precipitated by 10% trichloroacetic acid and destroyed by proteases. The inhibitor activity was distributed in various molecular weight fractions of sperm, SP and SP from vasectomised rams.

Stress leak point pressures and urethral pressure profile tests in clinically normal female dogs.

OBJECTIVE: To develop a stress leak point pressure (LPP) test for dogs, determine LPP for continent female dogs, and determine urethral pressure profile (UPP) values for nonanesthetized, continent female dogs. ANIMALS: 22 continent female dogs weighing from 21 to 29 kg. PROCEDURE: A standard UPP test and a modification of the LPP test used in women were performed on all dogs. On 3 occasions, dogs underwent UPP testing while awake. They then were anesthetized with propofol, and LPP was measured at bladder volumes of 75, 100, and 150 ml. For LPP tests, abdominal pressure was applied by inflating a human blood pressure cuff placed around the dog's abdomen. LPP were recorded through a urethral catheter (bladder LPP) and a rectal balloon catheter (abdominal LPP). RESULTS: Mean +/- SD and median maximal urethral closure pressure was 110.1+/-20.2 and 109.0 cm water, respectively. Mean bladder LPP for the 75, 100, and 150 ml bladder volumes was 172.4 cm water. Significant differences among LPP for the 3 bladder volumes were not detected. CONCLUSIONS: Stress LPP can be recorded in female dogs.

Vision-specific function and quality of life after cataract extraction in south India.

PURPOSE: To assess visual and overall patient function after intracapsular (ICCE) and extracapsular (ECCE) cataract extraction in rural South India. SETTING: Aravind Eye Hospital, Tirunelveli, Tamil Nadu, South India, and the Dana Center for Preventive Ophthalmology, Wilmer Eye Institute, Baltimore, Maryland, USA. METHODS: This study evaluated preoperative visual acuity and demographic information and postoperative visual acuity and functional status measures in 71 patients having ECCE with posterior chamber intraocular lens (IOL) placement and 73 patients having ICCE with aphakic spectacle correction at Aravind-Tirunelveli Eye Hospital, Tamil Nadu, India. The principal outcomes assessed were visual acuity; quality-of-life score (possible range 0 to 100%); visual function measurement (possible range 0 to 100%). RESULTS: Patients in the ECCE group scored 10.17 (P = .0001) points higher than those in the ICCE group on the visual function scale after adjustment for differences in age, sex, level of education, marital status, residence, and type of employment. The ECCE group scored 7.69 points higher on visual function when adjusting for the differences in best corrected visual acuity, which was also better in the ECCE group. In the quality-of-life assessment, 77.1% in the ECCE group and 46.6% in the ICCE group scored 90% or better (OR 3.85; P = .006). CONCLUSIONS: Patients in rural south India having ECCE with posterior chamber IOL implantation obtained better postoperative visual function, quality of life, and visual acuity than those receiving ICCE with aphakic spectacle correction. These differences, which were not significantly affected by adjustment for age, sex, education, marital status, type of residence, and occupation, indicate that ECCE is clearly superior to ICCE.