Properties of viruses of the potyvirus group. 1. A simple method to purify bean yellow mosaic virus, pea mosaic virus, lettuce mosaic virus and potato virus YN

Abstract

Bean yellow mosaic virus, pea mosaic virus, lettuce mosaic virus, and potato virus yN were purified by homogenizing and clarifying infected leaves in a mixture of 0.1 M tris-thioglycollic acid buffer pH 9, carbon tetrachloride and chloroform, followed by differential centrifuging applying moderate centrifugal forces.

Purification of tulip breaking virus and production of anti-sera for use in ELISA

“…This hypothesis is supported by the finding that centrifugation at 8000 g after treatment with Triton X-100, which dissolves cell membranes and chloroplasts (Van Oosten, 1972), did not have a negative effect. The fragmentation of TBV we observed was caused by freezing of the leaf tissue and treatment of the sap with carbon tetrachloride/chloroform and not by the high centrifugal forces as reported for other potyviruses (Huttinga, 1973). In the purification of plum pox virus high centrifugal forces were not harmful either (Kerlan et al, 1975).…”

“…Aggregation between TBV particles could be avoided by using 0.1 M trisbuffer (pH 9.0) except when PEG precipitation was applied, as found for other potyviruses (Huttinga, 1973).…”

“…After being pressed through cheese cloth, the extracts were either shaken with an equal volume of butanol/chloroform (v/v:l/1;Wetter, 1960), stirred with 80/o butanol (Damirdagh and Shepherd, 1970), or subjected to low-speed centrifugation (10 min at 1500 g or 8000 g) and treated with 5% Triton X-100 ( Van Oosten, 1972). When carbon tetrachloride/chloroform was used, the organic solvents were mixed together with leaves and buffer (Huttinga, 1973).…”

“…Leaves (fresh, frozen at --20 ~ or freeze-dried) were homogenized with a blender in one of the following buffers (w/v:2/5): 0.1 M tris-thioglycollic acid (pH 9.0; Huttinga, 1973), 0.033 M phosphate buffer (pH 7.0 or 8.0; Van Slogteren and De Vos, 1966), or 0.5 M borate buffer (pH 7.2, 8.2, or 9.0;Tomlinson, 1963), all containing 1% sodium sulfite. After being pressed through cheese cloth, the extracts were either shaken with an equal volume of butanol/chloroform (v/v:l/1;Wetter, 1960), stirred with 80/o butanol (Damirdagh and Shepherd, 1970), or subjected to low-speed centrifugation (10 min at 1500 g or 8000 g) and treated with 5% Triton X-100 ( Van Oosten, 1972).…”

“…Therefore, we compared various methods for the purification of other potyviruses (Tomlinson, 1963;Damirdagh and Shepherd, 1970;De Zoeten and Fribourg, 1971;Van Oosten, 1972;Huttinga, 1973) and combined some of them for the purification of TBV and the preparation of antisera for use in ELISA.…”