Purification of two potyvirus isolates infecting Phaseolus vulgaris L. in Lebanon

Se desarrollo un procedimiento de purificacion para los 2 aislamientos de potivirus (52-82 y 53-82) recientemente registrados en Libano. Los aislamientos de virus se propagaron en Phaseolus vulgaris cv. Sutter Pink y/o Black Turtle Soup y se cosecharon alrededor de 3 semanas despues de la inoculacion. El tejido infectado se extrajo en 0.5 molar K2HPO4 que contenia 0.005 molar EDTA, 0.02 molar Na2SO3, 0.01 molar DIECA y 3 por ciento de Triton X-100 pH 8.5, se aclaro con 25 por ciento de cloroformo + 25 por ciento de tetracloruro de carbono y se concentro por centrifugacion de alta velocidad. El pelet obtenido se suspendio en 0.5 molar de solucion tampon de citrato sodico pH 7.5, y se purifico aun mas por centrifugacion en gradientes de densidad de sucrosa preparados con el mismo tampon. Se controlo la concn. virica en las diferentes etapas del procedimiento de purificacion mediante ELISA, y el material final purificado se evaluo por pureza y recuperacion de particulas no agregadas de virus mediante centrifugacion en gradientes de densidad de sucrosa. El rendimiento virico se estimo entre 20-30 mg/kg de tejido. El virus purificado (53-82) se inyecto en un conejo. El antisuero producido se uso para detectar virus en hojas infectadas de frijol mediante ELISA. Los valores A405 obtenidos para tejido infectado fueron altos (1.5), mientras que aquellos para frijol sano fueron de 0.1 despues de una incubacion en sustrato durante 90 min; esto indica que el procedimiento seguido produjo una preparacion virica bastante purificada. (RA- CIAT) spa

A purification procedure was developed for the 2 potyvirus isolates (52- 82 and 53-82) recently reported from Lebanon. Virus isolates were propagated in Phaseolus vulgaris cv. Sutter Pink and/or Black Turtle Soup and harvested around 3 wk. after inoculation. Infected tissue was extracted in 0.5 molar K2HPO4 containing 0.005 molar EDTA, 0.02 molar Na2SO3, 0.01 molar DIECA, and 3 percent Triton X-100 pH 8.5, clarified with 25 percent chloroform + 25 percent carbon tetrachloride, and concentrated by high speed centrifugation. The pellet obtained was suspended in 0.5 molar sodium citrate buffer pH 7.5, and further purified by centrifugation on sucrose density gradients prepared with the same buffer. Virus concn. at the different steps of the purification procedure was monitored by ELISA, and the final purified material was assessed for purity and recovery of nonaggregated virus particles by sucrose density gradient centrifugation. Virus yield was estimated to range between 20-30 mg/kg tissue. The purified virus (53-82) was injected into a rabbit. The antiserum produced was used for virus detection in infected bean leaves by ELISA. The A405 values obtained for infected tissue were high (1.5), whereas those for healthy bean were 0.1 after a 90 min substrate incubation, an indication that the procedure followed produced a fairly purified virus preparation. (AS) eng