Table 2.

Quasispecies Analysis of the 3D-Coding Region of FMDV Populations Passaged in the Absence or Presence of 5-Fluorouracil^a

						Synonymous Mutations (%)g
Viral Populationa	Number of Nucleotides (clones)b	Mutation Frequency [min]c	Mutation Frequency [max]d	Shannon Entropye	Transition Biasf	Found (Expected) [min]h	Found (Expected) [max]i
FMDV-wt	72,610 (53)	5.9 × 10−4 (43)	6.2 × 10−4 (45)	0.7	3.3	34.9 (27.4)	35.6 (27.4)
FMDV-wt + FU	52,060 (38)	1.8 × 10−3 (96)	2.6 × 10−3 (137)	1.0	6.2	38.5* (29.5)	44.5** (29.2)
FMDV-3D(V173I)	68,500 (50)	4.8 × 10−4 (33)	5.0 × 10−4 (34)	0.5	4.7	18.2 (29.0)	17.6 (29.2)
FMDV-3D(V173I) + FU	58,910 (43)	1.7 × 10−3 (103)	2.0 × 10−3 (120)	1.0	3.3	43.7** (31.0)	47.5** (31.2)

^aWild-type FMDV or mutant FMDV-3D(V173I) were subjected to 10 serial passages in the absence or presence of FU (400 µg/ml) (+FU).

^bThe mutant spectrum analysis involved nucleotides 6,610–7,980 in the 3D-coding region; the number of individual clones analyzed is given in parenthesis.

^cMutation frequency [min] is the number of different mutations found in the mutant spectrum (relative to the sequence of the parental, unpassaged FMDV clone), divided by the total number of nucleotides sequenced (given in the second column); values in parenthesis are the total number of unique mutations. The statistical significance of the differences between values is the following: FMDV-wt, no FU versus FU: χ² = 42.7, P = 6.5 × 10⁻¹¹; FMDV-3D(V173I), no FU versus FU: χ² = 47.6, P = 5.0 × 10⁻¹²; no FU, FMDV-wt versus FMDV-3D(V173I): χ² = 0.79, P = 0.37; FU, FMDV-wt versus FMDV-3D(V173I): χ² = 0.14, P = 0.71. (In all cases, Pearson’s chi-squared test, df = 1).

^dMutation frequency [max] is the number of total mutations found in the mutant spectrum (relative to the sequence of the parental, unpassaged FMDV clone), divided by the total number of nucleotides sequenced (given in the second column); values in parenthesis are the total number of mutations. The statistical significance of the differences between values is the following: FMDV-wt, no FU versus FU: χ² = 84.1, P = 4.5 × 10⁻¹²; FMDV-3D(V173I), no FU versus FU: χ² = 62.2, P = 2.9 × 10⁻¹⁵; no FU, FMDV-wt versus FMDV-3D(V173I): χ² = 0.95, P = 0.33; FU, FMDV-wt versus FMDV-3D(V173I): χ² = 4.2, P = 3.9 × 10⁻². (In all cases, Pearson’s chi-squared test, df = 1).

^eShannon entropy (S) is calculated by the formula S =  − [S_i (p_i × ln p_i)]/ln N, in which p_i is the frequency of each sequence in the quasispecies, and N is the total number of sequences compared. It is a measure of heterogeneity of the sequences sampled in the mutant spectrum (S = 0, all sequences are identical; S = 1, each sequence is different).

^fTransition bias is defined here as the ratio between the frequency of transitions A → G plus U → C, relative to the frequency of transitions G–A plus C–U: $\left[\left(\frac{A\to G}{A}\right)+\left(\frac{U\to C}{U}\right)/\left(\frac{G\to A}{G}\right)+\left(\frac{C\to U}{C}\right)\right]$.

^gActual and expected (in parenthesis) percentage of synonymous mutations. The expected values were computed from the sequence of the 3D-coding region of FMDV, assuming the all mutations of a given type are equally probable, and considering the same relative frequencies of different types of mutations as in the corresponding mutant spectra. Asterisks indicate a significant difference between the actual and expected values (*P < 0.05; **P < 0.01; binomial test). (See also footnotes h, i.)

^hThe statistical significance of the differences between the found and expected values is the following: FMDV-wt, no FU: P = 0.17; FMDV-wt, FU: P = 0.036; FMDV-3D(V173I), no FU: P = 0.94; FMDV-3D(V173I), FU: P = 0.004. (In all cases, Exact binomial test, df =1).

ⁱThe statistical significance of the differences between the found and expected values is the following: FMDV-wt, no FU: P = 0.1451; FMDV-wt, FU: P = 0.0001; FMDV-3D(V173I), no FU: P = 0.95; FMDV-3D(V173I), FU: P = 0.0001. (In all cases, Exact binomial test, df =1).