Purification of human respiratory syncytial virus: superiority of sucrose gradient over percoll, renografin, and metrizamide gradients.
A method was devised for producing and purifying human respiratory syncytial virus (HRSV) preparations with high titers. Previous attempts to obtain substantial amounts of purified human respiratory syncytial virus have been unsuccessful due to the extreme lability of this virus, its close association with the host cell membrane, and its tendency to aggregate during concentration procedures. We describe a comparative study of various purification media as well as a novel approach for obtaining high titers of HRSV. Virus was produced in HEp-2 cells grown on gelatin beads and concentrated either by precipitation with polyethylene glycol or by centrifugation over a sucrose cushion, the latter yielding 100% recovery. These procedures employed EDTA to disaggregate the virus and MgSO4 as a virus stabilizing agent. Attempts to purify HRSV over percoll, renografin and metrizamide gradients lead to loss of infectivity. Sucrose was found to represent the best purification medium with a yield of up to 60%.
Mbiguino
A
A
Laboratory of Immunovirology, Department of Microbiology & Immunology, University of Montreal, Quebec, Canada.
Menezes
J
J
D002277:Carcinoma; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods*; D003470:Culture Media; D003974:Diatrizoate Meglumine; D006801:Humans; D007822:Laryngeal Neoplasms; D008793:Metrizamide; D009997:Osmotic Pressure; D011205:Povidone; D012136:Respiratory Syncytial Viruses / Q000254:growth & development / Q000302:isolation & purification*; D012822:Silicon Dioxide; D014775:Virus Activation; D014783:Viscosity
D003470:Culture Media; D003974:Diatrizoate Meglumine; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone; D008793:Metrizamide