TABLE 2.

Assigned classification of mutations detected during preparation of ChimeriVax-DEN viruses and the genetic stability passages shown in

Type of mutation	Mutation detected
	In uncloned P2 virus	After the first plaque purificationa	After the third plaque purificationb	In genetic stability passages
Introduced by SP6 polymerase (17 nucleotide changes in 11.5 genomes)	−	+	+	+
Introduced by YFpol (2 nucleotide changes in 10.5 genomes)	−	−	+	+
Beneficial mutationc (15 and 19 nucleotide changes in the cloned and uncloned variants, respectively)	−	−	−	+

^aP2 plaque purification step of cloned ChimeriVax-DEN2₂₀₀₀ or P3 of cloned ChimeriVax-DEN1, -DEN2₂₀₀₁, -DEN3, and -DEN4 (Table 1) (Fig. 1).

^bP6 of cloned ChimeriVax-DEN2₂₀₀₀ or P6 or P7 of cloned variants of other chimeras (Table 1).

^cBeneficial mutations that accumulated in genetic stability passages of cloned variants were introduced by YFpol, while it is unknown which polymerase (SP6 or YFpol) introduced mutations that accumulated in uncloned variants. The E-204 and E-251 mutations beneficial for ChimeriVax-DEN1 were also found after plaque purifications in some of the clones of the chimera.