A comparative analysis of freon substitutes in the purification of reovirus and calicivirus

Freon 113 (Freon) is an essential component used in some viral purification methods to separate virus from infected cell debris. With its environmental and toxic hazards, Freon's availability is limited and more tightly regulated. Several organic solvent substitutes were selected to identify a suitable Freon replacement for the purification of both cultivable reovirus and fastidious calicivirus. Reovirus was extracted from tissue cultured cells with each solvent tested and purified in cesium chloride gradients by standard techniques. Purified virions were analyzed for conservation of physical and biological properties by morphological examination and infectivity studies. The purification of calicivirus nucleic acid from stool samples using selected solvents was also examined. Solvent-extracted calicivirus RNA was reverse transcribed and quantified by polymerase chain reaction amplification of a standard diagnostic 117 bp amplicon. These studies indicated that Vertrel XF (a newly developed environmentally friendly Freon substitute) and a 7:3 mixture of isopentane/1-chlorobutane are suitable replacements. Considerations of flammability and ease of use suggest that Vertrel XF is the preferred choice as a Freon substitute for the purification of these non-enveloped viruses.