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Characterization of potato leafroll virus

University of British Columbia

Potato leafroll virus (PLRV) was purified from infected potato (Splanum tuberosum L.) by using a mixture of chloroform and butanol for clarification and polyethylene glycol for concentration. The virus sedimented as a single band in a sucrose density gradient. Yield of purified virus varied from 0.4-0.6 mg/kg fresh weight of potato foliage. Virus yield was higher from tissue immediately processed after harvest than from tissue stored at 4 C or -20 C for a day or longer. The A 260/A 280 varied from 1.74-1.82. Antiserum prepared against purified virus had a maximum titre of 1024 in agar gel double diffusion tests. PLRV in unconcentrated potato sap could not be detected by agar gel tests, but could be detected by enzyme-linked immunosorbent assay. A pure line of the mild strain of PLRV was isolated, and an antiserum with a maximum titre of 2560 by agar gel tests was produced. PLRV had a sedimentation coefficient of 127 S by linear log sucrose density gradient centrifugation and 117 S by analytical ultracentrifugai-tion. A buoyant density of 1.38-1.39 g/cm³ was obtained from isopycnic centrifugation in cesium chloride and analytical ultracentrifugation in cesium sulphate. The nucleic acid content of PLRV was estimated from the particle densities to be 26-28%.The PLRV nucleic acid had:, a moi wt of 2.0 x 10⁶; was degraded by RNase but not by DNase; reacted with orcinol but not with diphenylamine; and had a broad range of melting temperatures from 35 to 85 C in 1 x SSC buffer with hyper-chromicity of 20-21%. These properties indicated that PLRV nucleic acid is a single-stranded RNA. The sedimentation coefficient of the RNA molecule before and after treatment with formaldehyde was 34.5 S and 20.7 S, respectively. Dissociated coat protein migrated as a single band in polyacrylamide gel electrophoresis and the average subunit molwt was 26 300. PLRV should be considered a member of the luteovirus group.

Text

2010-03-23

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http://hdl.handle.net/2429/22365

Plant Science

University of British Columbia

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