Purification of Tomato Spotted Wilt Virus Isolated from Tobacco Plants in Morioka, Japan

A virus isolated from tobacco plants in Morioka was confirmed as tomato spotted wilt virus (TSWV) and designated as TSWV-M isolate. The virus shows severe symptoms on several plants including tobacco, tomato and Nicotiana benthamiana. TSWV-M was used for the purification study. Out of seven kinds of buffers for extraction of TSWV, 0.1M phosphate buffer containing 0.01M Na2SO3 and 0.01M EDTA⋅2Na was suitable with high infectivity in crude sap checked by half-leaf method using N. tabacum cv. Xanthi nc. In a purification method with isopycnic linear sucrose density gradient with concentration from 15.0% to 60.0% (w/w), the peak fraction of TSWV infectivity was indicated at 42.0% (w/w) sucrose fraction. By this method, however, the host components could not be eliminated from the peak fraction. In isopycnic step-wise sucrose density gradient method with trap of 40.0% (w/w), host components could be excluded from 42.0% (w/w) sucrose fraction with high infectivity. Intact TSWV particles were observed in the fraction by the electron microscopy. With the passage of the TSWV purification process, specific infectivities in the final step were calculated as 44 times at OD260 and 46 times higher at OD280 in the ratio to the first step preparation.