Transient expression of the coat protein of Apple chlorotic leaf spot virus inhibits the viral RNA accumulation in Nicotiana occidentalis

The coat protein of Apple chlorotic leaf spot virus (ACLSV-CP) plays a crucial role in infectivity and efficient viral RNA accumulation in host cells (J. Gen. Virol, 88, 2007). In this study, the effect of ACLSV-CP on viral RNA accumulation in Nicotiana occidentalis was investigated. The CP, CPm40 (an amino acid (aa) substitution of Ala to Ser at aa position 40), CPm75 (a substitution of Phe to Tyr at aa position 75), and CPm40m75 (two aa substitutions at positions 40 and 75) of ACLSV (P205) were transiently expressed in N. occidentalis leaves by agroinfiltration. Immunoblot analysis showed that CP and CPm40m75 accumulated in infiltrated tissues, in contrast to CPm40 and CPm75 which were not detected, suggesting that the stable accumulation of CP is important for effective viral RNA accumulation. However, co-agroinfiltration of an infectious ACLSV cDNA clone (pBICLSF) or pBICLSF-based CP mutants (pBICLCPm40, pBICLCPm75, and pBICLCPm40m75) with a vector expressing CP (pBE2113-CP) showed no viral genomic RNA accumulations were found in any leaves infiltrated with these constructs. The inhibition of ACLSV-RNA accumulation was found only in leaves co-expressed with CP protein, but not with a frame-shift mutant of CP, a movement protein (P50), and a frame-shift mutant of P50. Keywords: Apple chlorotic leaf spot virus, coat protein, protein stability, coat protein mediated resistance (CP-MR), agroinfiltratio

Remodeling of Airway Walls in Fatal Asthmatics Decreases Lymphatic Distribution; Beyond Thickening of Airway Smooth Muscle Layers

ABSTRACTBackgroundWe previously reported the phenotypic distribution patterns of airway smooth muscles in fatal asthmatics; Type I asthmatics with smooth muscle bundle thickening only in large airways and Type II in whole airways. We hypothesized that increased smooth muscle bundles in the airway walls would disrupt airway lymphatics to impair airway clearance in these fatal asthmatics.MethodsThe autopsy lungs of seven fatal asthmatics (three Type I, four Type II asthmatics) and five controls were examined by immunohistochemistry to reveal the lymphatics distributed in the airway walls. The total area of lymphatics around each cross-sectioned airway was measured and its airway radius was calculated using an image analyzer system. Finally, the distribution areas of lymphatics in the same level of airways of bronchial trees were compared among Type I, Type II asthmatics and controls.ResultsThe total area of airway lymphatics in each lung was found to be positively correlated with the airway radius (R). The distribution areas of lymphatics in larger airways (1.5 < R < 2.0 mm) of both types of asthmatics were significantly decreased than controls, and Type I asthmatics contained much less lymphatics than Type II asthmatics in these airways. The lymphatics around smaller airways (0.5 < R < 1.0 mm) were also reduced in both phenotypes of asthmatics without statistic difference between them. The airway lymphatics of these fatal asthmatics were observed to be interrupted by thickened muscle bundle layers, and by fibrotic tissues developed around these airways as well.ConclusionsThese results indicate that distribution of lymphatics were decreased in the airway walls of fatal asthmatics which contained muscle bundles and fibro-connective tissues both of which were augmented in these airway walls to disrupt lymphatics, impair airway clearance and accelerate mucosal edema which would cause refractory status of these patients

Effect of temperature on RNA silencing of a negative-stranded RNA plant virus: Citrus psorosis virus

Citrus psorosis virus (CPsV), genus Ophiovirus, causes a bark scaling disease of citrus. CPsV virions are kinked filaments with three negative-stranded RNA molecules (vRNA) and a 48 kDa coat protein. The effect of temperature on symptom expression, virus accumulation and RNA silencing was examined in sweet orange seedlings (Citrus sinensis) graft-inoculated with three different CPsV isolates and grown in a glasshouse at 26/18°C or 32/26°C (day/night). Most plants kept in the cooler glasshouse showed a shock reaction in the first flush with shoot necrosis, and then moderate to intense chlorotic flecking and spotting in young leaves, whereas plants incubated at 32/26°C did not exhibit shoot necrosis, and young leaf symptoms were milder. Virus titre estimated by ELISA and by northern and dot blot hybridization paralleled symptom intensity, with significantly higher virus accumulation in plants incubated at 26/18°C. The amount of CPsV-derived small RNAs (CPsV-sRNAs) slightly increased at 32/26°C, with the ratio of CPsV-sRNA/vRNA being higher at 32/26°C than at 26/18°C. These results suggest that (i) CPsV infection induces RNA silencing in citrus plants, (ii) symptom intensity is associated with virus accumulation, and (iii) temperature increase enhances the RNA silencing response of citrus plants and decreases virus accumulation.Facultad de Ciencias Exacta

Effect of temperature on RNA silencing of a negative-stranded RNA plant virus: Citrus psorosis virus

Citrus psorosis virus (CPsV), genus Ophiovirus, causes a bark scaling disease of citrus. CPsV virions are kinked filaments with three negative-stranded RNA molecules (vRNA) and a 48 kDa coat protein. The effect of temperature on symptom expression, virus accumulation and RNA silencing was examined in sweet orange seedlings (Citrus sinensis) graft-inoculated with three different CPsV isolates and grown in a glasshouse at 26/18°C or 32/26°C (day/night). Most plants kept in the cooler glasshouse showed a shock reaction in the first flush with shoot necrosis, and then moderate to intense chlorotic flecking and spotting in young leaves, whereas plants incubated at 32/26°C did not exhibit shoot necrosis, and young leaf symptoms were milder. Virus titre estimated by ELISA and by northern and dot blot hybridization paralleled symptom intensity, with significantly higher virus accumulation in plants incubated at 26/18°C. The amount of CPsV-derived small RNAs (CPsV-sRNAs) slightly increased at 32/26°C, with the ratio of CPsV-sRNA/vRNA being higher at 32/26°C than at 26/18°C. These results suggest that (i) CPsV infection induces RNA silencing in citrus plants, (ii) symptom intensity is associated with virus accumulation, and (iii) temperature increase enhances the RNA silencing response of citrus plants and decreases virus accumulation.Facultad de Ciencias Exacta

Individual Canine Airway Response Variability to a Deep Inspiration

In healthy individuals, a DI can reverse (bronchodilation) or prevent (bronchoprotection) induced airway constriction. For individuals with asthma or COPD, these effects may be attenuated or absent. Previous work showed that the size and duration of a DI affected the subsequent response of the airways. Also, increased airway tone lead to increased airway size variability. The present study examined how a DI affected the temporal variability in individual airway baseline size and after methacholine challenge in dogs using High-Resolution Computed Tomography. Dogs were anesthetized and ventilated, and on 4 separate days, HRCT scans were acquired before and after a DI at baseline and during a continuous intravenous infusion of methacholine (Mch) at 3 dose rates (17, 67, and 200 μg/min). The Coefficient of Variation was used as an index of temporal variability in airway size

Small airway hyperresponsiveness in COPD: relationship between structure and function in lung slices

The direct relationship between pulmonary structural changes and airway hyperresponsiveness (AHR) in chronic obstructive pulmonary disease (COPD) is unclear. We investigated AHR in relation to airway and parenchymal structural changes in a guinea pig model of COPD and in COPD patients. Precision-cut lung slices (PCLS) were prepared from guinea pigs challenged with lipopolysaccharide or saline two times weekly for 12 wk. Peripheral PCLS were obtained from patients with mild to moderate COPD and non-COPD controls. AHR to methacholine was measured in large and small airways using video-assisted microscopy. Airway smooth muscle mass and alveolar airspace size were determined in the same slices. A mathematical model was used to identify potential changes in biomechanical properties underlying AHR. In guinea pigs, lipopolysaccharide increased the sensitivity of large (>150 μm) airways toward methacholine by 4.4-fold and the maximal constriction of small airways

Myosin heavy chain and physiological adaptation of the rat diaphragm in elastase-induced emphysema

BACKGROUND: Several physiological adaptations occur in the respiratory muscles in rodent models of elastase-induced emphysema. Although the contractile properties of the diaphragm are altered in a way that suggests expression of slower isoforms of myosin heavy chain (MHC), it has been difficult to demonstrate a shift in MHCs in an animal model that corresponds to the shift toward slower MHCs seen in human emphysema. METHODS: We sought to identify MHC and corresponding physiological changes in the diaphragms of rats with elastase-induced emphysema. Nine rats with emphysema and 11 control rats were studied 10 months after instillation with elastase. MHC isoform composition was determined by both reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry by using specific probes able to identify all known adult isoforms. Physiological adaptation was studied on diaphragm strips stimulated in vitro. RESULTS: In addition to confirming that emphysematous diaphragm has a decreased fatigability, we identified a significantly longer time-to-peak-tension (63.9 ± 2.7 ms versus 53.9 ± 2.4 ms). At both the RNA (RT-PCR) and protein (immunocytochemistry) levels, we found a significant decrease in the fastest, MHC isoform (IIb) in emphysema. CONCLUSION: This is the first demonstration of MHC shifts and corresponding physiological changes in the diaphragm in an animal model of emphysema. It is established that rodent emphysema, like human emphysema, does result in a physiologically significant shift toward slower diaphragmatic MHC isoforms. In the rat, this occurs at the faster end of the MHC spectrum than in humans