- Volume 66, Issue 10, 1985
Volume 66, Issue 10, 1985
- Animal
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Phosphorylation of the N and M1 Proteins of Rabies Virus
More LessSUMMARYPhosphorylation of rabies virus proteins was followed in vivo and in vitro. The N and M1 proteins were both found to be phosphorylated. The M1 protein was present in the virion in two phosphorylated states, but only the hypophosphorylated form of M1 was found in infected cells. The hypothesis that some of the M1 molecules become hyperphosphorylated during the maturation process by a membrane-bound kinase was examined. The phosphorylation of the viral proteins by the kinase present in purified rabies virions was studied using an in vitro transcriptase assay: under the conditions of the assay, additional phosphate groups were rapidly attached to the N protein. The M1 protein was similarly hyperphosphorylated although more slowly. Whether the hyperphosphorylation of the N protein is responsible for the poor efficiency of the in vitro transcriptase reaction is not clear. No detectable change in the phosphorylation of cellular proteins was observed in the course of rabies virus infection.
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Conditions for Haemolysis by Flaviviruses and Characterization of the Haemolysin
More LessSUMMARYThe 17D vaccine strain of yellow fever virus (YF 17D) was used to establish the optimal conditions for lysis of chick erythrocytes. Tissue culture-grown, polyethylene glycol-concentrated virus showed peak activity at pH 5.4 in citrate buffer when incubated at 37 °C. A further two- to fourfold increase in titre was obtained by pretreatment of the chick erythrocytes with 250 µg/ml trypsin. These conditions were also shown to be optimal for Japanese encephalitis (JE), West Nile (WN) and dengue-2 (den2) viruses. The ratio of haemagglutination (HA) titre to haemolysis (HL) titre approximated to unity, suggesting that the two functions are associated with the same molecule although as separable entities since selective inactivation of the HL activity of the virus was accomplished using 60 µg/ml trypsin. HL could be demonstrated at neutral pH if the chick erythrocytes were first subjected to treatment with acidic pH buffer. The effect on the virus envelope is thus not the sole contribution of a low pH environment to optimal HL. Hyperimmune rabbit antiserum prepared against purified YF 17D virions inhibited HA and HL if added before agglutination had occurred by the virus but when added after agglutination had taken place it showed specific anti-HL activity. Monoclonal antibodies that inhibited HA (HAI) by YF 17D did not inhibit HL (HLI) activity when applied after agglutination had taken place. Moreover, monoclonal antibodies specific for the 54K glycoprotein of YF virus but without HAI activity also had no effect on HL when added either before or after agglutination. As yet, we have been unable to identify a monoclonal antibody displaying specific anti-HL activity but all those directed against the 54K envelope glycoprotein possessing HAI activity showed HA to be a prerequisite for HL.
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- Plant
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Cell-free Translation of Beet Necrotic Yellow Vein Virus: Readthrough of the Coat Protein Cistron
More LessSUMMARYRNA from isolate F13 of beet necrotic yellow vein virus, an isolate that lacks RNA-4, has been translated in a rabbit reticulocyte lysate. Abundant translation products of RNA-1 had molecular weights of 150 000 (150K) and 50K. RNA-2 directed synthesis of the 22K viral coat protein and an 85K polypeptide. Both RNA-2 translation products were precipitated by antiserum against virus. The time course of appearance of the 22K and 85K polypeptides, comparative peptide mapping, and translation experiments with added suppressor tRNA indicate that the cistron for the 22K viral coat protein is situated near the 5′ terminus of RNA-2 and that the 85K polypeptide arises by partial readthrough of the coat protein cistron.
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Monoclonal Antibody-based Biotin-Avidin ELISA for the Detection of Soybean Mosaic Virus in Soybean Seeds
More LessSUMMARYTwo monoclonal antibodies (M-Ab) specific for different epitopes on particles of soybean mosaic virus (SMV) were used in a double-antibody sandwich ELISA (M-Ab ELISA). The non-isotopic immunoassay, which used a biotinylated second antibody and an avidin-alkaline phosphatase detection system to detect SMV in soybean seed extracts, was compared with a polyclonal antibody-based solid-phase radioimmunoassay (SPRIA). m-Ab ELISA detected less than 10 ng of SMV/ml and was more sensitive than the SPRIA which detected 25 ng SMV/ml. Furthermore, m-Ab ELISA required less than 36 h for seed sample assays and only 5.5 h for assays involving purified virus, whereas SPRIA required 3 or 4 days. When seeds from 33 field plots, in which 0%, 30% and 50% of the soybean plants had been inoculated with SMV, were assayed by both systems, results of the two tests correlated for 31 of 33 (94%) seed samples. This suggests that dual-site biotin-avidin m-Ab ELISA systems have potential utility for routine screening of seed samples.
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Detection of Viroid and Viroid-like RNAs from Grapevine
More LessSUMMARYAnalysis by polyacrylamide gel electrophoresis of nucleic acid preparations, obtained from several varieties of grapevine by a procedure designed to isolate and purify viroids, revealed the presence of RNA species with some of the characteristic physical properties of viroids. Under non-denaturing conditions, a band with a mobility faster than that of citrus exocortis viroid (CEV) was detected, and under fully denaturing conditions two bands were observed, one co-migrating with the circular forms of CEV and a second migrating faster than the linear forms of this viroid. This RNA species did not hybridize with a cDNA probe to CEV. Some of the grapevine preparations were infective for Gynura aurantiaca, inducing symptoms similar to those caused by CEV, and the appearance of an RNA which had the same mobility as CEV in denaturing and non-denaturing electrophoretic systems and hybridized with cDNA to CEV. These results suggest that viroid-like and viroid RNAs can be recovered from grapevine, the former (with no detectable sequence homology to CEV) at a concentration sufficient to be observed as a physical entity in gels, and the latter (with close sequence homology to CEV) whose presence could only be revealed by bioassay. The possible involvement of these RNAs in some grapevine diseases of unknown aetiology is discussed.
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