Purification and stability of epizootic hemorrhagic disease virus

Purification of epizootic hemorrhagic disease virus (EHDV) from sonicated cell culture supernatant was effected using polyethylene glycol (PEG) precipitation and two CsCl density gradient centrifugations. During purification procedures the pH was maintained at values close to pH 8.0 as pH stability for EHDV was maximal between pH 7.2 and pH 9.0. During CsCl isopycnic centrifugation at pH 8.0, a 0.1 M or greater buffer was necessary to preserve maximum viral infectivity. This method of purification resulted in the recovery of 75% or more of the infectious virus contained in infected cell culture supernatants. For the extraction of infected cell-associated virus, sonication effectively liberated infectious virus, while freeze-thawing resulted in a significant loss of viral infectivity. The use of Freon allowed for complete recovery of infectious virus while ether or chloroform resulted in lower recoveries of infectious virus.