Purification of Rabies Virus Grown in Tissue Culture

Abstract

Extracellular rabies virus, grown in monolayer cultures of BHK21 cells in the presence of medium supplemented with bovine serum albumin, was purified by the following procedure. Virus was precipitated from infectious tissue culture fluid by zinc acetate and was resuspended in a solution of ethylenediaminetetraacetate. The suspension was filtered through a Sephadex column and was treated with ribonuclease and deoxyribonuclease. The virions were then pelleted by centrifugation at high speed and were resuspended in buffer solution. Banding of the virus by centrifugation in a sucrose density gradient was the final step in the purification procedure. Purified preparations contained bullet-shaped virus particles of variable length and little (up to 5%) contaminating host-cell material. Most of the virions were “complete”, i.e., 180 nm long, but some virus particles were shorter. The length distribution of the virions was nonrandom. Shorter virions seemed to be noninfectious and showed markedly decreased hemagglutinating activity. The complement-fixing activity and the ribonucleic acid to protein ratio of the virions were not related to the length of the virus particles. Although the properties of extracellular and intracellular viruses were similar, the procedure was not suitable for purification of intracellular rabies virus.

A human cytomegalovirus function inhibits replication of herpes simplex virus

“…), cells were labeled with [3H]thymidine (10 1xCi/ml), and after labeling, infected cells were frozen and thawed three times to release virus. 3H-labeled HSV was then precipitated with 0.05 M zinc-acetate by the procedure of Sokol et al (38). 3H-labeled HSV was suspended in EDTA, and subjected to cesium chloride density gradient centrifugation, and the infectious virus band was collected (M. K. Howett, personal communication).…”

“…Controls including pACYC184 (6) vector DNA and HEL cell DNA (provided by Brian Wigdahl) as well as a reconstruction mixture (20 p.l) composed of HEL cell DNA (10 jig) in the presence of HSV-1 virion DNA in decreasing quantities (1 to 0 ,ug) were blotted onto nitrocellulose. The HSV-1 virion DNA was extracted from HEL cells infected with HSV-1 (1.0 PFU per cell) as described by Sokol et al (38) and adapted by M. K. Howett (personal communication). The visible virus band collected from a cesium chloride step gradient (1.2 to 1.4 g/ml) was digested for 1 h at 37°C with proteinase K (2 mglml)-0.…”

Characterization of guinea pig cytomegalovirus DNA

“…Intracellular virus DNA was purified by treatment of the pellet with 0.1% Nonidet P-40 and 1% sodium deoxycholate. Extracellular virions were precipitated with 1.0 M zinc acetate, and virus DNA was extracted as described previously for purification of rabies virus DNA (58). Virus DNA was separated from cell DNA by CsCl buoyant density gradient centrifugation as previously described for purification of HSV DNA (10,69,70).…”

In vivo interference in vesicular stomatitis virus infection

“…Virus and DI particle concentrates were prepared as described by Crick and Brown (6). Virus harvests were clarified by low-speed centrifugation and centrifuged for 1 h at 20,000 rpm in the SW25 rotor of a Spinco ultracentrifuge, and the resuspended pellet was centrifuged for 2 h at 20,000 rpm in a 15 to 45% linear gradient of sucrose in NTE buffer [0.13 M NaCl, 0.001 M sodium ethylenediamine-tetraacetate, and 0.05 M tris(hydroxymethyl)aminomethane, pH 7.8 (28). The virion or DI particle bands were withdrawn with a syringe after piercing the tube wall at the appropriate position.…”