Measles virus: conditions for the propagation and purification of infectious virus in high yield.
Tissue culture conditions for the efficient propagation of cell-free measles virus, and a novel method for the purification of infectious virus in milligram quantities are described in this report. Infected suspension cultures of HeLa cells incubated at 32.5 degrees C yielded virus titers approaching 10(8) PFU/ml, 30-50% of which was cell-free. After concentration by ultrafiltration and sedimentation, infectious virus was separated from host cell membranes and proteins by density equilibrium centrifugation in gradients of colloidal silica. Residual contaminants and silica particles were removed by chromatographic elution through agarose gel. This protocol achieved a approximately equal to 1400 fold purification of virus which retained approximately equal to 75% infectivity while yielding approximately equal to 1.5 mg of viral protein from each liter of infected cell culture medium. Electron microscopy of the purified virus revealed only intact particles having the morphological characteristics of the paramyxoviruses. Serological studies confirmed the purified material to be antigenically reactive measles virus. SDS-PAGE analysis of the virus preparation identified eight polypeptide species as described by others. Seven of these are virus-specified structural proteins corresponding to the L, H, P, NP, F1, M, and F2 polypeptides. The eighth major structural protein was defined as host cell derived actin.
D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002500:Centrifugation, Isopycnic; D002850:Chromatography, Gel; D003102:Colloids; D006367:HeLa Cells; D006801:Humans; D008459:Measles virus / Q000032:analysis / Q000254:growth & development / Q000302:isolation & purification*; D012822:Silicon Dioxide; D013696:Temperature; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000032:analysis; D015678:Viral Structural Proteins; D014776:Virus Cultivation
D003102:Colloids; D014764:Viral Proteins; D015678:Viral Structural Proteins; D012822:Silicon Dioxide