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The influence of purification protocol and pH on tomato spotted wilt virions
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Two purification protocols were tested for their influence on yield of tomato spotted wilt virions. Protocol "A" was developed by Black et al (2), modified by Mohammed et al (34), and described by Gonsalves and Trujillo (16). Protocol "B" was developed by Tas et al (45), and described by Verkleij and Peters (48). Three levels of pH were tested using three separate buffers, each of which was selected according to the most useful pK value; ammonium citrate (pH 6.0), phosphate (pH 7.0), and Tris-HCI (pH 8.0). The purified virions were characterized according to spectrophotometric readings, double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA), infectivity assay, and by electron microscopy. The purified virons were spectrophotometrically scanned in the ultraviolet range from 310 to 240 nm to produce the "virus profile" from which the 280/260 ratios were calculated and analyzed for nucleic acid per cent according to the Warburg and Christian data (52) . Extracts of the tissue from which the virus was purifed were analyzed by DAS ELISA (Agdia, Inc.), as were the results from each protocol, and each pH level using protocol B. Infectivity was determined by mechanically inoculating Nicotiana tabacum L. cv. Benthamiana seedlings at the three to fiveleaf stage (approximately 21 days old) with the virus suspensions obtained from each protocol, and at each pH level. The seedlings were tested for infectivity by DAS ELISA 8 to 10 days post-inoculation. To identify virions among other particles within the final pellet, an immunosorbent electron microscopy (ISEM) assay was performed on the purified virons obtained from each of the protocols. When the pH level was varied using protocol B, the purified virions were observed without the benefit of an ISEM. The ISEM results revealed the most uniform, spherical, intact particles when the assay was performed on spotted wilt infected Benthamiana seedlings. Results from virions obtained at three pH levels from each of the protocols, revealed a mixture of particle types and sizes, with very little uniformity. Data obtained in which an ISEM was not performed was inconclusive. The preparations revealed nearly the same amorphous particle shapes from protocols, A and B, and at each pH level from protocol B. Nucleic acid content in the viral suspensions ranged from 59/o to nearly 20@o. Infectivity on the Benthamiana seedlings was correlated to nucleic acid per cent, with the greatest per cent infectivity observed as the nucleic acid content of the purified virions approached 59/0. The D.-k,@ ELISA readings were inversely correlated to nucleic acid per cent and per cent infectivity.
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Shimek, Christina Marie (1993). The influence of purification protocol and pH on tomato spotted wilt virions. Master's thesis, Texas A&M University. Available electronically from
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