Passage of virus with wt or SVpzf nsP4 and one or neither of the two SVlm21 mutations in nsP1.

Infectious clones were constructed and virus passaged as described in Materials and Methods. Virus harvested at each passage was assayed for the ability to form plaques on a monolayer of mosquito cells maintained under agarose containing 100 µM methionine (NM), and under agarose containing only 5 µM methionine (LM). Virus populations with the LMR phenotype, i.e. with both the nt 319 and the nt 321 mutations, should give rise to approximately equal numbers of plaques under the LM overlay and under the NM overlay.