A rapid method to produce high yields of purified rotavirus particles.
A rapid purification method of rotavirus particles to high yield retaining the double shelled structure of infectious virus is described. Group A rotavirus (UK strain) was concentrated through a cushion of colloidal silica (rho=1.10 g/cm(3)) or by precipitating with polyethylene glycol 8000. After concentration, infectious rotavirus was cleared from host cell proteins by density equilibrium centrifugation in gradients of colloidal silica using near vertical rotors. Characterisation of purified virus assessed by electron microscopy and poliacrylamide gel electrophoresis (PAGE) revealed the typical wheel shape structure of rotavirus particles and the presence of the 11 segments of dsRNA arranged in the 4-2-3-2 pattern. Presence of rotavirus structural proteins including VP6, VP4 and VP7 from the outer shell, was demonstrated by SDS-PAGE and Western blot using polyclonal and VP6-specific monoclonal antibodies. This method achieved a approximately 1500 fold purification, which retained approximately 80% infectivity depending on the concentration protocol used, while yielding 160 microg of viral protein per each litre of infected cell culture medium. The time required for the isopycnic centrifugation was only 25 min and the entire completion of the method required 3.5 h. The method is simple technically and applicable to the purification of large as well as minute amounts of virus.
Villegas
Guillermo Adolfo
GA
Centro de Virología Animal (CEVAN), Serrano 669, (1414) Buenos Aires, Argentina.
Argüelles
Marcelo Horacio
MH
Castello
Alejandro Andrés
AA
Mas
Neus Jiménez
NJ
Glikmann
Graciela
G
D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods; D005260:Female; D000882:Haplorhini; D011817:Rabbits; D012401:Rotavirus / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology; D013997:Time Factors; D015678:Viral Structural Proteins / Q000032:analysis; D014771:Virion
D015678:Viral Structural Proteins