Stabilization of enveloped viruses by dimethyl sulfoxide.

In a recent review on the storage of animal viruses at subzero temperatures (8), it was pointed out that enveloped viruses present special problems. For example, herpesvirus even at -90 C loses infectivity after prolonged storage and at -20 C is even more labile than at 4 C. Repeated freezings and thawings of this virus result in marked loss in activity. Members of the herpesvirus group can be stabilized to some degree at freezing temperatures by suspending the viruses in serum, glycerol, sorbitol, sucrose, skim milk, or other proteins (1, 3, 11, 13, 14). These stabilizers are believed to act by preventing the formation of destructive ice crystals. The stabilizers of herpesviruses are atso recognized as preservatives for cultured cells in the frozen state. The protective effects of sugars and proteins on cells have been attributed to the action of these compounds in lowering the effective concentrations of electrolytes, and thus preventing the denaturation of the cells by the toxic effects of concentrated salts and solutes, or by preventing the formation of intracellular ice crystals (4-7, 9). Since the infectivity of enveloped viruses may depend on the integrity of the envelope derived from the host cell (2, 10, 12), their preservation during freezing may require treating the viruses as if they were cultured cells. This led us to investigate dimethyl sulfoxide (DMSO), as a preservative for enveloped viruses, because this compound is the current preservative of choice for cultured cells. All viruses used in this study were grown in green monkey kidney (GMK) cells maintained with protein-free medium B (0.5%,, lactalbumin hydrolysate in Earle's salt solution). Viruses were assayed in GMK cultures using the plaqueforming unit (PFU) method. Herpesvirus (JES strain) was used as a model agent. Other enveloped viruses used were measles (Edmonston strain), Sindbis (ar 334 strain), and vesicular stomatitis virus (Indiana strain). Nonenveloped viruses studied were vaccinia (WR strain), adenovirus (SV15 strain), and poliovirus (type 1 Mahoney strain). Since repeated freezing and thawing are deleterious to enveloped viruses, DMSO was compared with fetal calf serum, which has well known preservative properties. Figure 1 shows the results of repeated freezing and thawing on undiluted herpesvirus (control) and on virus in the presence of DMSO and of serum. Herpesvirus was inactivated twofold after a single freezing and thawing; after four cycles of freezing and thawing, a 10-fold decrease in titer was manifested. Fetal serum conferred protection, but not as well as DMSO.