In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells.

Orf, which is caused by orf virus (ORFV), is distributed worldwide and is endemic in most sheep- and/or goat-raising countries. RNA interference (RNAi) pathways have emerged as important regulators of virus-host cell interactions. In this study, the specific effect of RNAi on the replication of ORFV was explored. The application of RNA interference (RNAi) inhibited the replication of ORFV in cell culture by targeting the ORF025 gene of ORFV, which encodes the viral polymerase. Three small interfering RNA (siRNA) (named siRNA704, siRNA1017 and siRNA1388) were prepared by in vitro transcription. The siRNAs were evaluated for antiviral activity against the ORFV Jilin isolate by the observation of cytopathic effects (CPE), virus titration, and real-time PCR. After 48 h of infection, siRNA704, siRNA1017 and siRNA1388 reduced virus titers by 59- to 199-fold and reduced the level of viral replication by 73-89 %. These results suggest that these three siRNAs can efficiently inhibit ORFV genome replication and infectious virus production. RNAi targeting of the DNA polymerase gene is therefore potentially useful for studying the replication of ORFV and may have potential therapeutic applications.

Delayed Bitterness of Citrus Wine Is Removed Through the Selection of Fining Agents and Fining Optimization.

"Delayed bitterness" (DB) in citrus wine is caused by limonoids and determines the acceptability to consumers. In this study, a variety of fining agents, specifically gelatin, agar, chitosan, bentonite, the crosslinking agent polyvinylpyrrolidone (PVPP), diatomite, and casein, were evaluated for their ability to lower DB in citrus wine. Factorial experiments with three factors at four levels (L 3 4 ) and with two factors at three levels (L 2 3 ) were used to determine the optimal effect. We found that a mixture of agar (125 mg/L) and gelatin (30 mg/L) not only decreased the limonoid concentration and clarified the liquor, but also increased the precipitation content, retention rate of ascorbic acid, and antioxidant capacity. After treatment, the quality of the citrus wine was improved, and a few volatile chemical compounds were lost. We determined that agar and gelatin were the best fining agents for reducing DB in citrus wine.

Role of autophagy in cellular response to infection with Orf virus Jilin isolate.

Autophagy is a conserved catabolic process of the cell, which has been described to be involved in the development of various viral diseases. However, the role of autophagy in Orf virus (ORFV) replication remains unknown. In this study, we provide the first evidence that ORFV infection triggered autophagy in primary ovine fetal turbinate cells (OFTu) based on the appearance of abundant double- and single-membrane vesicles, the accumulation of LC3 fluorescent puncta, the enhancement of LC3-I/-II conversion, and autophagic flux. Moreover, modulation of ORFV-induced autophagy by rapamycin (RAPA), Earle's balanced salts solution (EBSS), chloroquine (CQ) or 3-methyladenime (3-MA) does not affect virus production. In conclusion, these results suggest that autophagy can be induced in host cells by ORFV infection, but which maybe not essential for ORFV replication.

Development of a lateral flow immunochromatographic assay for the rapid diagnosis of Orf virus infections.

A rapid and simple lateral-flow immunochromatographic assay (LFIA) was developed for the specific detection of Orf virus (ORFV) using two distinct monoclonal antibodies (MAbs: 5A5 and 6F2) against the ORFV ORF011 protein. The MAb 5A5 was conjugated with colloidal gold, and the MAb 6F2 and goat anti-mouse IgG were sprayed onto a nitrocellulose membrane in strips at positions designated test (T) and control (C), respectively. The results showed that samples of ORFV complexed with colloidal gold-conjugated MAb 5A5, were captured by MAb 6F2 at the T line resulting in the appearance of a purple band. When samples did not contain ORFV or when they contained a quantity of ORFV below the detection limit of the test, only the C line was visible. The analysis of sensitivity of the test demonstrated that the lowest detected quantity of ORFV was 2.03×10(3.0) TCID50/ml. Storage at room temperature for 6 months did not result in the loss of performance of the LFIA test. Using loop-mediated isothermal amplification (LAMP) as a reference test, the relative specificity and sensitivity of the LFIA test were determined to be 100% and 92.1%, respectively. Based on these results, the LFIA test developed may be a suitable tool for rapid on-site testing for ORFV infection.

Optimization of acquisition trajectories for 3D rotational coronary venography.

OBJECTIVE: Rotational coronary X-ray imaging on C-arm systems provides a multitude of diagnostic projections from the vascular tree with a single contrast agent bolus. The acquisition trajectory is typically limited to a circular arc with a fixed caudo-cranial angulation. This may cause sub- optimal projection directions for specific vessel segments for all acquired views, e.g., those segments orthogonal to the axis of rotation. In this paper, a method is presented to calculate a patient-independent acquisition trajectory with respect to vessel foreshortening and overlap for multiple vessel segments of the coronary tree. This method can be applied to artery as well as vein anatomy. METHODS: Rotational coronary venograms of 14 patients have been used to generate three-dimensional mesh representations with a semi-automatic two view modeling algorithm. The venous tree is divided into seven different vessel segments. Foreshortening and overlap of every segment are calculated and combined for all patients in a measure called obstruction value. The weighted obstruction values of all vessel segments define a cost function for the entire two-dimensional angular range of the C-arm system. Viterbi's algorithm is used to calculate an optimal trajectory with respect to this cost function. The method is validated by leave-one-out cross-validation on the 14 rotational venography data sets and on simulated venograms of a segmented computed tomography (CT) data set. Projection images with a foreshortening value below 10% and overlap below 20% are rated 'optimal'. RESULTS: In 12 (85.7%) data sets, 43% more optimal images were acquired using the presented method compared to the standard circular arc trajectory. As well, in 13 (92.8%) data sets 38% more vessel segments can be optimally visualized in the acquired images. The test on the CT data set showed that the resulting average root-mean-square error of the extracted centerline points of the segmented CT data set compared to the error based on the views from the circular arc was reduced from 2.52 to 1.55 mm. CONCLUSION: In a first test, the method proved to deliver improved image quality by reducing foreshortening and overlap of vessel segments and may therefore also improve the centerline extraction accuracy of the semi-automatic two view modeling method.