Table 1. Mutations detected in examples of viruses showing large plaques and/or loss of RSV F expression.

Virus clone	Construct	Plaque size	RSV F expression detected	RSV F Mutation*	HPIV3 Vector mutation**
D3-F12	F/preN	Normal	no	Y342C	ND
D11-H5	F/preN	Mixed- normal and large	no	E110 → Stop	None found
E1-B4	F/preN	Normal	no	E110 → Stop	ND
G9-E10	F/preN	Normal	no	“A” insertion at nt 5 caused frame shift and E30 → Stop	ND
C5-E3	F-H3TMCT/preN	Large	yes	No	Y3H (HN protein); V242G, N248K, C252W, F277V, I279M (L protein)
A3-D8	F/N-P	Normal	no	None found	ND
C6-F4	F/N-P	Large	no	L321P, Y342H, F352P	None found
G6-E8	F-H3TMCT/N-P	Normal	no	K132 → Stop	ND

One stock of each virus construct, at P1 stage after rescue, was subjected to terminal dilutions in LLC-MK2 cells. Terminally diluted enriched virus clones that showed large plaque phenotype and/or had lost expression of RSV F were sequenced.

* RSV F insert and flanking transcription signals were sequenced for viruses with loss of RSV F expression.

** HPIV3 backbone was sequenced for clones with large plaques.

ND, not determined.