Preparation of Poliovirus Labeled with Phosphorus-33

Abstract

Phosphorus-33 ( 33 P), a weak (0.25 Mev) beta-emitting isotope of phosphorus with a half-life of 25 days, has been used to label poliovirus in cell culture. HeLa cell monolayers were depleted of phosphate and then labeled by incubating at 37 C in a medium (LM) containing about 10 μCi of 33 P as orthophosphate per ml. Labeled cells were infected at a high multiplicity with poliovirus type 1 and incubated for 8 hr in LM medium. Virus from infected cells was then concentrated and purified. Virus purity was confirmed by comparison of virus infectivity and radioactivity after CsCl density gradient centrifugation and by observing purified virus preparations with electron microscopy. With the method described, yields of about 10 10 to 5 × 10 10 plaque-forming units (PFU) of highly purified poliovirus with specific activities of about 3 × 10 -4 to 10 -3 disintegrations per min per PFU have been obtained from 1.5 × 10 8 to 3.0 × 10 8 HeLa cells.