Estimate of absolute specific infectivity of wound tumor virus purified with polyethylene glycol

Purification of wound tumor virus (WTV) was made simpler and quicker and a much greater proportion of active virions were obtained in the purified preparations of this labile virus by precipitating it with polyethylene glycol (PEG) 6000 before using density gradient centrifugation. Adjustment of clarified extracts to pH 5.7 gave the best yield of total (active plus inactive) virus and the highest specific infectivity. An optimal NaCl concentration (0.3 m) was important in obtaining good separation of the virus from host components during subsequent zonal density gradient centrifugation and maximal yields. NaCl at 0.2 m yielded purified virus of somewhat greater specific infectivity. Maximal yields of total virus were obtained with the use of 4% PEG for 2 hr although 3% PEG gave virus of about 1.5 times greater specific infeetivity. A histidine-MgCl₂ buffer solution for resuspending virus precipitated by PEG resulted in at least 50% greater total virus yields than did a glycine-MgCl₂ solution. WTV purified under the optimal conditions determined in this investigation yielded 3–5 times more total virus and at least 50 times more infective virus than did the standard method used previously. The purified virus had an absolute specific infectivity of about 25–30%, that is, it contained about 2 or 3 inactivated virions to each infective one. Purified preparations containing about 3 × 10¹¹ total virions per ml could be quick-frozen and stored at ?80 ° for at least a year without detectable loss of infectivity.