Purification of Group IV ((+)ss) RNA viruses: Nidovirales: Coronaviridae and Arteriviridae

There are now many published papers that report the use of iodixanol gradients not only to purify viruses but also to investigate their assembly. In all comparative studies between CsCl and iodixanol, the recovery of virus infectivity is much higher and the particle:infectivity ratio much lower when viruses are purified in iodixanol. 

Although sucrose is generally less deleterious to viral infectivity than CsCl, it can nevertheless also have serious effects on certain important aspects of viral function; in particular the loss of surface glycoproteins from retroviruses has been noted [1]. This may be related to its viscosity, which, in solutions of the same density, is much higher than that of iodixanol. 

Like CsCl, sucrose must be dialyzed before infectivity can be measured. In contrast both infectivity measurements using cultured cells and many add-on techniques can be performed without dialysis of iodixanol. Combined with the availability of OptiPrep as a sterile solution, this makes the use of OptiPrep for virus purification and assembly analysis much more convenient than the use of either CsCl or sucrose. 

Clarified suspensions of murine coronavirus [2] and later human coronavirus [3] were first concentrated in PEG 8000 (containing 0.5 M NaCl) and subsequently purified in Nycodenz gradients. 

Initially the virus was concentrated at the boundary of a 10% and 50% (w/v) Nycodenz discontinuous gradient (83,000 g for 3.5 h) and then it was further purified in a continuous 10-50% Nycodenz gradient (83,000 g for 16 h). The Nycodenz solutions were made up in 0.1 M NaCl, 1 mM EDTA, 100 mM Tris-maleate, pH 6.2.