FIG. 1.

RT-PCR amplifications used to detect defective genomes in FMDV population C-S8p260. (A) Map of the 8,115 residues of the FMDV C-S8c1 genome, excluding the internal polyribocytidylate [poly(C)] and the 3′end poly(A) (4, 10, 42). Lines indicate noncoding regions, including the 5′ untranslated region (5′ UTR) containing the poly(C) tract and the 3′ untranslated region (3′ UTR) with the terminal poly(A) tract; boxes delimit protein-coding regions. Two functional initiation AUG codons give rise to two forms of the L protease, Lab and Lb. Filled rectangles below the map indicate the position of deletions Δ417, Δ999, and Δ1017. The thin lines below the map show the RT-PCR amplification products, covering the C-S8p260 genome, used to map RNA deletions (the major class of deletion detected is indicated on the right). The location in the FMDV C-S8c1 genome of the primers used will be given upon request. RT-PCR amplifications that resulted only in DNA fragments of standard length are depicted as thick lines at the bottom of the scheme. The same RT-PCR amplifications, covering the C-S8p260p3d genome, did not detect any deletion. (B) Agarose gel showing one representative RT-PCR amplification (panel A, number 21) obtained from C-S8p260. The DNA products corresponding to standard (st) size RNA and RNAs with deletions are indicated by an arrow on the right. The DNA products obtained from C-S8p260p3d are indistinguishable from that obtained from C-S8c1. Lane M, molecular size markers (HindIII-digested φ29 DNA; the corresponding sizes [base pairs] are indicated on the left); lane −, negative control without RNA. (C) Plaque size produced by virus C-S8p260 and C-S8p260p3d. Confluent BHK-21 cell monolayers (2 × 10⁶ to 4 × 10⁶ cells per 20 cm²) were infected with serial dilutions of FMDV C-S8p260 or C-S8p260p3d, and at 24 h postinfection cell monolayers were fixed with 2% formaldehyde and stained with 2% crystal violet in 2% formaldehyde. The average diameter of the plaques produced by C-S8p260 was approximately three times smaller than that of plaques produced by C-S8p260p3d, as described in the text. Procedures for plaque assay in semisolid agar medium are described in Materials and Methods.