Table 4.

Stability of the assignments at L protein amino acid positions 1321 and 1313 in the 1321K(AAA)/1313(TCA) mutant during passage at restrictive temperature^a

Virus descriptionb	% of cultures with codon 1321 revertantsc	Codon 1321		Codon 1313
		Revertant codon(s) observedd	Amino acid(s)e	Codon observedd	Amino acide
1321E(GAA)/1313C(TGC)f	50	G[A/T]A	E:V	TGC	C
	20	[G/A]AA	E:K	TGC	C
	10	G[A/T/C]A	E:V:A	TGC	C
1321K(AAA)/1313S(TCA)	0			TCA	S

^aTen replicate 25-cm² flasks of HEp-2 cells were infected with the indicated 831L mutant at a multiplicity of infection of 0.1 PFU/cell at 37°C. Virus was harvested between 5 and 7 days postinfection, serially passaged again at 37°C, and serially passaged twice at 38°C, for a total of four passages, each by transferring 1 ml (of a total of 5 ml) of supernatant to a fresh 25-cm² flask of HEp-2 cells. In parallel, two control flasks per mutant were passaged four times at the permissive temperature of 32°C. For each passage, aliquots were frozen for titration and genotype analysis. Genotype analysis was done after the fourth passage by sequencing of a 249-nt region of the RSV L gene (RSV nucleotides 12261 to 12511; GenBank accession number M74568). No mutations were detected in the 32°C controls (not shown).

^bAmino acid assignments of codons 1321 and 1313 are indicated in single-letter code. The codon sequence is shown in parentheses.

^cPercentage of cultures with detectable revertants.

^dObserved codon sequences; mixtures are indicated in brackets. Changed nucleotides are underlined.

^eAmino acid(s) coded; colon indicates a mixed population of the specified amino acids. Changed amino acids are underlined.

^fControl to show that the abbreviated stress test sensitively detects instability.