Mutant spectrum complexity of P9 R and C-S8c1.

a<p>The viral populations are those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039941#pone-0039941-g001" target="_blank">Figure 1</a>.</p>b<p>The numbering of FMDV genomic residues is according to reference (Escarmís et al. 1999).</p>c<p>Number of nucleotides sequenced in each viral population.</p>d<p>The minimum mutation frequency is the number of different mutations divided by the total number of nucleotides sequenced for each viral population.</p>e<p>The maximum mutation frequency is the total number of mutations divided by the total number of nucleotides sequenced for each viral population</p>f<p><i>S_n</i>, indicates normalised Shannon entropy, that is a measure of the proportion of different sequences in a distribution. It was calculated as described in Materials and Methods.</p

Lethality of biological clones isolated from population P9 R.

<p>Groups of six mice were inoculated into the footpad with 10³ PFU of P9 R or of each of the 5 biological clones (c1 to c5) obtained as plaque-isolates from population P9 R. The nomenclature of the virus corresponds to that described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039941#pone-0039941-g001" target="_blank">Figure 1</a>. Percentage of survival of the inoculated mice during a follow-up period of 168 hours is indicated. Procedures are detailed in Materials and Methods.</p

Types of mutations found in the mutant spectra of P9 R and C-S8c1.

a<p>The viral populations are according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039941#pone-0039941-g001" target="_blank">Figure 1</a>.</p>b<p>The mutations are those found in the analysis of the mutant spectra of C-S8c1 and P9 R described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039941#pone-0039941-t001" target="_blank">Table 1</a>. The percentage of each type of mutation in relation to the total number of mutations found in the same population is shown in brackets. Transversions C→G, G→C, U→A, A→U, G→U and C→A were not observed in the analysis.</p

Mutations identified in the 3D coding region in the mutant spectrum of the population passaged in the presence of ribavirin P9 R and the parental FMDV population C-S8c1.

a<p>Population P9 R is that derived from passaging FMDV C-S8c1 nine times in the presence of 5 mM ribavirin, as detailed in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039941#pone-0039941-g001" target="_blank">Figure 1</a> and in Materials and Methods. C-S8c1 is the parental population used to initiate the passages.</p>b<p>The mutations observed in the mutant spectrum of P9 R and C-S8c1 are indicated. The total number of nucleotides sequenced, mutation frequency and Shannon entropy are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039941#pone-0039941-t002" target="_blank">Table 2</a>.</p>c<p>Amino acid substitutions deduced from the mutations listed in the second column. A dash line means a synonymous mutation.</p>d<p>The molecular clones have been numbered to identify linked and repeated mutations.</p

Complete genomic consensus nucleotide sequence of population P9 R and partial sequence of biological clones.

<p>A schematic representation of FMDV genome is displayed at the top of the figure [based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039941#pone.0039941-Mahy1" target="_blank">[64]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039941#pone.0039941-Sobrino2" target="_blank">[66]</a>. The entire genomic sequence of FMDV population P9 R and partial sequences, comprising residues 4945 to 5677 of FMDV populations P7 R, P4 R and P1 R, and P9 R-derived clones c1, c2, c3, c4 and c5 were determined. Virus nomenclature is that of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039941#pone-0039941-g001" target="_blank">Figure 1</a>. Nucleotide substitutions relative to the parental virus C-S8c1 are represented inside the boxes below the corresponding genomic region in which they are found. For non-synonymous mutations the corresponding amino acid replacement is given in parenthesis. An equivalent mixture of C (parental) and U (mutant) nucleotides in the genomic position 5087 is represented by C/U; a higher proportion of C than U (about 75% C) is indicated as C/u. Substitution C5087U leads to amino acid replacement I248T in viral protein 2C. Residue numbering is accordingly to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039941#pone.0039941-Escarmis2" target="_blank">[61]</a>.</p

Scheme of passages of FMDV C-S8c1 in BHK-21 cells in the presence of ribavirin.

<p>FMDV C-S8c1 was subjected to nine passages in BHK-21 cells in the presence of 5 mM of ribavirin. The viral population isolated from each passage is identified as a P followed by the passage number and R. The initial viral infection was performed at a moi of 0.1 PFU/cell, and subsequent infections were carried out with 1/10 of the volume of the previous passage (moi in the range of 0.01–0.1 PFU/cell). Five biological clones, represented as black squares, were isolated from viral population P9 R by dilution and plating on BHK-21 cell monolayers, as indicated with a dotted line on the right.</p

FMDV-specific T cell responses in vaccinated pigs.

<p>PBMCs were purified from blood of animals at day 30 (D30) post-immunization (pre-challenge) (white bars), and D11 post-challenge (grey bars). A. Lymphoproliferative responses of pigs 1 to 17 (groups 1, 2, 3, 4 and 5) to FMDV C-S8c1. Data are shown as stimulation index (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010414#s4" target="_blank">Material and Methods</a>) and standard deviations are indicated. B. IFN-γ release by PBMC stimulated with FMDV C-S8c1. Values were determined at 72 h of in vitro stimulation. The detection level in control cultures (medium alone) were below the sensitivity of the assay (5 pg/ml).</p

Serum IgG1, IgG2 and IgM-specific responses in pigs following immunization with C-S8p260.

<p>Each column corresponds to the antibody titer for the indicated animal (numbers 1 to 14) at different times (D, day) post-immunization and post-challenge (white bars: D15 pi; light gray: D30 pi; dark grey: D32 pi (that corresponds to D2 post-challenge), and black: D37 pi (that corresponds to D7 post-challenge). Titers are expressed as the reciprocal of the highest dilution of serum (log₁₀) that gives an OD_A460 of twice the value obtained with the pre-immune serum of the corresponding animal.</p

Immunogenicity of C-S8p260 in mice. C57BL/6 mice were inoculated in the FP with either 10³ or 10⁷ PFUs of C-S8p260, or PBS.

<p>At 90 days post-immunization, mice were challenged with 10⁴ PFUs of FMDV C-S8c1 (procedures are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010414#s4" target="_blank">Materials and Methods</a>). A. Survival curves after immunization and after challenge (indicated in the abscissa, with arrow in the bottom panel). Continuous line: animals immunized with C-S8p260; discontinuous line: animals inoculated with PBS. B. IgG titers determined by ELISA at different days post-immunization and after challenge (abscissa and bottom panel). C. IgG titers in serum of mice immunized with BEI-inactivated C-S8p260. The day of the challenge with 10⁴ PFUs of C-S8c1 is indicated with an arrow; 16 animals were inoculated in each group. D. Survival of mice inoculated with C-S8p260p3d and C-S8c1; 21 mice were inoculated with 10⁷ PFUs C-S8p260p3d (squares), and 10 mice with 10³ PFUs of C-S8c1 (diamonds).</p

Neutralizing antibody response against FMDV C-S8c1, in mice, after vaccination with C-S8p260.

a<p>Mice were vaccinated with 10⁷ PFUs or 10³ PFUs of C-S8p260, indicated for each group.</p>b<p>Neutralization titers are expressed as PRN70 (plaque-reduction neutralization titer 70): the reciprocal of the dilution of serum that causes neutralization of 70% of PFU.</p>c<p>30 days post-immunization.</p>d<p>3 days post-challenge with 10⁴ PFUs of C-S8c1.</p