TABLE 1.

Quantification of different RNAs present in C-S8p260 and C-S8p260p3d

Amplification no.a	Position of primersb		Size of expected product (bp)	Quantified RNAsc	No. of RNA molecules/μld
	Sense	Antisense			C-S8p260	C-S8p260p3d
28	5344-5363	5699-5678	356	total RNA	2.1 ± 0.6 × 108	3.2 ± 0.8 × 108
29	1539-1560	2926-2903	1388	st	<1.7 ± 0.4 × 104e	1.7 ± 0.4 × 108
30	2744-2770	2926-2903	183	st + Δ417	1.3 ± 0.1 × 108	1.7 ± 0.4 × 108
31	1305-1330	2140-2122	836	st + Δ999	8.5 ± 3.4 × 107	2.2 ± 0.9 × 108
32	1539-1560	2983-2961	1445 + 429	st + Δ1017	8.2 ± 5.8 × 107	5.8 ± 4.8 × 108
33	1692-1710	2140-2122	449	st + Δ417 + Δ999	2.2 ± 0.6 × 108	2.6 ± 0.6 × 108
34	3175-3194	3518-3496	344	st + Δ417 + Δ1017	1.6 ± 0.2 × 108	2.6 ± 0.8 × 108
35	1305-1330	1619-1596	315	st + Δ999 + Δ1017	9.8 ± 2.4 × 107	1.9 ± 0.6 × 108

^aAmplifications were carried out with primers that could amplify specifically each class of RNA. Numbers are correlative to those of the amplifications shown in Fig. 1A.

^bPosition refers to the FMDV C-S8c1 genome, with the numbering used in reference 10.

^cClass of RNA quantified by the specific primers. In C-S8p260p3d (free of defective genomes), all the amplifications quantified only standard (st) genomes.

^dNumber of RNA molecules per microliter of supernatant of infected cells. 10⁸ RNA molecules/μl corresponds to about 10⁵ molecules per infected cell. Each value is the average of at least two independent determinations and represents the mean of triplicate amplification assays; standard deviations are indicated.

^eNo standard RNA was detected in C-S8p260 by real-time PCR (Light Cycler). To ensure that standard (st) RNA could be amplified in the presence of an excess of RNAs with deletions amplifications with primers located within the sequences deleted in ΔRNAs (amplification number 29) were carried out with a fixed amount of C-S8p260 RNA (corresponding to 1 μl [≈3.4 ng RNA] of an undiluted RNA preparation extracted from C-S8p260) and 10-fold serial dilutions of C-S8p260p3d (corresponding to RNA extract from C-S8p260p3d). Standard RNA was detected even when present at a 10⁻⁴ dilution relative to ΔRNAs; the intensity of the RT-PCR band was about 50% of the intensity observed in the absence of RNAs with deletions. Since the last dilution of C-S8p260p3d in which there was positive amplification was 10⁻⁴, in C- S8p260 the maximum amount of st RNA was 1.7×10⁴ molecules per μl of supernatant of infected cells.