REOVIRUS MULTIPLICITY REACTIVATION hanced susceptibility to thermal inactivation after chymotrypsin treatment

MCCLAN, MARY E. (California State Department of Public Health, Berkeley), AND REX S. SpENmLovE. Multiplicity reactivation of reovirus particles after exposure to ultraviolet light. J. Bacteriol. 92:1422-1429. 1966.-Exposure of reovirus suspensions to moderate doses of ultraviolet light results in essentially exponential inactivation of infectivity to survivals of 10-2 to 10-3. With suspensions of sufficiently high particle concentration, larger doses of ultraviolet light (6 to 12 min) are associated with multiplicity reactivation (MR) which is demonstrable both by immunofluorescent-cell count and by plaque assay in FL human amnion cells. Similar effects are produced by photodynamic inactivation in the presence of proflavine, but not by thermal inactivation at 50 C. All three reovirus types exhibit MR under appropriate conditions, and all three interact in mixed ultraviolet suspensions with high efficiency. Progeny from FL cells infected under conditions ofMR were as infectious as those of unirradiated inocula, with yields per cell ranging from 104 to 4 X 104 infective units. Since the original observation by Luria (11) of multiplicity reactivation (MR) among the T-even group of bacterial viruses, and the subsequent theoretical development of the phenomenon by Luria and Dulbecco (12), demonstrations of similar effects with animal viruses have been relatively few. Among the ribonucleic acid (RNA) viruses, those of poliomyelitis (5), influenza (2), fowl plaque (17), and Newcastle disease (6, 9) have been shown to exhibit MR. With the deoxyribonucleic acid (DNA) animal viruses, MR has been reported only for the poxvirus group (1, 8). It is of interest that genetic recombination has so far been demonstrated with these same viruses. It is reasonable to assume, as postulated for the bacteriophages, that, with animal viruses, the same basic mechanisms are involved in these two phenomena. The present report presents evidence for the occurrence of multiplicity reactivation with a double-stranded RNA virus after exposure to ultraviolet (UV) light. MATERIALS AND MErHODS Cells. The FL human amnion cell line was maintained and used for virus assay and propagation as previously described (20). Viruses. Reoviruses type 1 (Lang), 2 (Erwin, States), and 3 (Willis) were used. Stock suspensions were pepared from FL human amnion cells harvested 66 to 96 hr after infection, and were frozen and thawed six times. For use in these experiments, strains Lang and States were partially purified by exposure to a-chymotrypsin and nucleases, extraction with Genetron, and dialysis against 0.01 M phosphate-buffered saline (PBS). Strains Erwin and Willis were used as crude cell harvest and were clarified by centrifugation and treated with chymotrypsin for enhancement of infectivity (21). All virus suspensions were stored at -20 C. Infectivity tests. Two methods of virus assay were used: immunofluorescent-cell count (ICC; 20) and plaque assay. Titers were expressed as fluorescent cells per milliliter (FC/ml) or plaque-forming units per milliliter (PFU/ml). Exposure to ultraviolet light. Source of radiation was a 15-w General Electric germicidal lamp emitting more than 95% of its radiation at 2.537 A wavelength. Energy flux, measured with a Latarjet dosimeter, at the surface of irradiated samples was 15 erg per mm' per sec. Purified virus suspensions were diluted 5fold, and crude suspension was diluted 30-fold in PBS for ultraviolet (UV) exposure. Sample volumes of 2 to 2.5 ml in 60-mm petri dishes were exposed with magnetic stirring. Dose of UV is expressed In minutes of exposure.