3 - Propiolactone Decontamination of Simian Virus - 40 as Determined by a Rapid Fluorescent - Antibody Assay

LEVINE, S. I. (The National Drug Co., Philadelphia, Pa.), N. R. GOULET, AND 0. C. Liu. ,B-Propiolactone decontamination of Simian virus-40 as determined by a rapid fluorescent-antibody assay. Appl. Microbiol. 13:70-72. 1965.-,3-Propiolactone vapor treatment of vaccine production facilities has been shown to be approximately 90%o effective in the elimination of large quantities of Simian virus-40 (SV40). The use of a rapid fluorescent-antibody assay for the detection of SV40 was also studied. Simian virus-40 (SV40) contamination of animal quarters and of production or research laboratories is of considerable concern to manufacturers of virus vaccines. f-Propiolactone (BPL) vapor has been utilized to decontaminate enclosed spaces (Bruch, 1961; Hoffman and Warshowsky, 1958). The efficacy of BPL decontamination in these studies was based solely on results obtained with exposed bacterial spore strips. This report is concerned with BPL vapor decontamination of a specific virus (SV-40) intentionally introduced into three vaccine production areas, and the development of a rapid assay for the detection of SV40 . MATERIALS AND METHODS Virus. SV40 used was isolated from a formalinized adenovirus vaccine. The virus was propagated in African Grivet monkey kidney (AGMK) cultures. The infectious titer of the virus was approximately 10-8 . per ml. Identification was established by cross-neutralization tests employing rabbit antisera prepared against the homologous virus and the 45-54 strain of SV40 (obtained from G. W. Workman, National Institutes of Health). Decontamination Procedure. Three buildings were included in the experiment: an animal holding barn, a vaccine production building, and a research facility. A large quantity of SV40 (10-' . dilution) was swabbed on the floors, bench tops, sinks, cupboards, etc., in wax-delineated areas. As a control against inactivation of the virus by residual surface disinfectants, these areas were sampled for SV40 1 day later by reswabbing with a sterile moistened swab, and then retreated with fresh virus. Decontamination (performed by the Wilmot Castle Co., Rochester, N.Y.) of each area was effected by atomizing a quantity of BPL calculated to give a maximal concentration of 10 mg per liter of space. The temperature and relative humidity were kept to 30 C and 80%C, respectively. Since the half-life of BPL at 25 C is 210 min (Hoffman and Warshowsky, 1958), it was deemed safe to work in the treated areas after a minimum of 4 hr. However, as an added precaution, the treated areas were kept sealed overnight. Although the BPL should have been completely hydrolyzed in that time, a residual odor remained. This odor was dissipated after an aeration period of 15 min. The wax-delineated areas were then sampled for a second time. Swabs were placed in a tube containing sterile saline (Hanks and Wallace, 1949), 400 units of penicillin G, and 1,000 mg of streptomycin, and held at -40 C until assayed for SV40 . The effectiveness of the decontamination procedure per se was determined by placing duplicate spore strips of Bacillus subtilis var. niger (B. globigii) or B. stearothermophilus in open petri dishes at strategic locations in each building. The petri dishes were sealed after BPL decontamination, and the spore strips were tested (performed by the Wilmot Castle Co., Rochester, N.Y.) for the presence of viable bacteria. Detection of SV40 by the fluorescent-antibody technique (FA). Rabbit antiserum prepared against SV40 was fractionated with ammonium sulfate and conjugated with fluorescein isothiocyanate. The conjugated globulin was then dialyzed against a 0.01 M, pH 7.2 phosphate-buffered saline (PBS) at 4 C until free of visible fluorescence. The conjugate was adsorbed twice with mouse liver powder to remove nonspecific substances. Cover slip AGMK cultures were inoculated with 0.5 ml of the samples to be assayed and incubated 70 on O cber 0, 2017 by gest ht://aem .sm .rg/ D ow nladed fom DECONTAMINATION OF SIMIAN VIRUS-40 at 37 C for 7 days. The cultures were then washed four times with PBS, air-dried, and fixed in acetone for 10 min. Fluorescent staining was done in a moist chamber by covering the cover slip cultures with the conjugated globulin and incubating at 37 C for 60 min. Cultures were washed twice in PBS, once in distilled water, air-dried, and mounted on a microslide (Rodriguez and Deinhardt, 1960). The cultures were then read for SV40 nuclear fluorescence. Comparison of the FA technique and cytopathogenic effect (CPE) method. A direct comparison of the FA and CPE methods of detecting SV40 was made as follows. For the FA method, 13 AGMK slide cultures were challenged with 0.1 ml of SV40 for each half-log virus dilution through 10-9-r. The cultures were incubated at 37 C for 7 days and then stained for nuclear fluorescence as described above. For the CPE method, 15 AGMK cultures were challenged with 0.1 ml of the same virus dilutions. Cultures were read for characteristic SV40 CPE for 25 days. Those cultures not showing CPE were individually harvested, frozen and thawed once, subpassed into each of two fresh AGMK cultures, and observed for another 20 days.