Table 5.

Stability of L protein codons 831 and 1321 in rA2cp248/404/1030/ΔSH and cps2 during passage at restrictive temperatures^a

Virus	% of cultures with revertant codonsb	Codon 831L					Codon 1321
		Originally present in virus:		Revertant codon observedc	Amino acid(s)d	Reversion rate (estimated avg % of population ± SD)e	Originally present in virus:		Revertant codon observedc	Amino acidd	Reversion rate (estimated avg % of population)e
		Codon	Amino acid				Codon	Amino acid
rA2cp248/404/1030ΔSHf	70	CTG	L	C[T/A]G	L:Q	47 ± 15	AAT	N	[A/T]AT	Y	64 ± 22
	10			CAG	Q	100			[A/T]AT	Y	10
	10			CAG	Q	100					0
	10					0			TAT	Y	100
cps2	40	TTG	L	TCG	S	100	AAA	Kg			0
	10			TCG	S	100			A[A/G]A	Rh	30
	10			T[T/C]G	L:S	70					0

^aTen replicate 25-cm² flasks of HEp-2 cells were infected with the indicated virus at a multiplicity of infection of 0.1 PFU/cell at 33°C. Virus was harvested between 5 and 7 days postinfection, serially passaged again at 33°C, and serially passaged twice at 34°C, 35°C, 36°C, and 37°C, for a total of 10 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 10 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 10th passage from a 2,921-bp PCR fragment of the RSV genome (nucleotides 12271 to 15191; GenBank accession number M74568) which was partially sequenced. No mutations were detected in the 32°C controls (not shown).

^bPercentage of cultures with detectable revertants.

^cObserved codon sequence(s); mixtures are indicated in brackets. Changed nucleotides are underlined.

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

^eIn cultures with mixed populations, percentages of subpopulations with reversions were estimated from sequencing chromatograms. Values from cultures with mixed populations are shown.

^fAs noted in the text, two cDNA versions, rA2cp248/404/1030ΔSH and MEDI-559, have been constructed that share the same attenuating mutations (except for a silent codon difference at the 248 mutation) and differ by a number of incidental mutations. The virus used here is version rA2cp248/404/1030ΔSH that had previously been analyzed in clinical studies by Karron et al. (18).

^gThe stabilized codon 1321K(AAA) was used together with codon S1313(TCA); the latter site was completely stable (not shown).

^hThis assignment yields nonviable virus, as shown in Table S1 in the supplemental material, and its presence here presumably depends on complementation by nondefective virus during this in vitro infection.