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Quantitative Assays for Evaluation of HTLV-III Inactivation Procedures: Tri(N-butyl)phosphate:sodium cholate and ß-Propiolactone¹

Laboratory of Virology, The Lindsley F. Kimball Research Institute of The New York Blood Center, New York, New York 10021 [A. M. P.]; Blood Derivatives Program, The New York Blood Center [B. H.]; Biotest Pharma, GmbH, Frankfurt/Main, West Germany [H. D., W. S.]; and Laboratory of Tumor Cell Biology, National Cancer Institute, NIH, Bethesda, Maryland [R. C. G.]

Human T-lymphotropic retrovirus type III (HTLV-III) can be quantitatively assayed for infectivity by inoculation of serial dilutions into cultures of the H-9 cell line and testing for reverse transcriptase in the culture supernatants. Sequential harvests revealed that 14 days of incubation of cultures fed twice weekly was sufficient to reveal maximal titers. Stocks prepared from unconcentrated H9:HTLV-III_b supernatants have contained from 10^4.5 to 10^6.0 (TCID₅₀)/ml. Stocks prepared by 100-fold concentration of such fluids by pelleting or by polyethylene glycol precipitation followed by pelleting onto sucrose cushions contained 10^6.0–10^6.5 TCID₅₀/ml.

Preliminary studies are under way to utilize this system for evaluation of sterilization processes which can be applied to blood derivatives. Exposure of HTLV-III suspended in Factor VIII preparations to 0.3% tri(n-butyl)phosphate-0.2% sodium cholate resulted in inactivation of 10^4.5 TCID₅₀ in 2.5 h at 27°C. Exposure of HTLV-III suspended in 4 g of -globulin per 100 ml to 0.14% ß-propiolactone for 4 h at room temperature at pH 8.0 inactivated 10^4.5 TCID₅₀. However, exposure to -globulin alone inactivated about 99% of HTLV-III infectivity.