Monitoring virus evolution.

<p>Top panel: Phylogenetic approaches are first used to characterize a viral outbreak. From left to right: Sequencing data identifies a new viral genotype by phylogenetics. This new strain is represented in red in the phylogenetic tree. By combining these data with sampling dates, a Bayesian skyline plot reveals the demographic history of the epidemic. Middle panels: First, by functional genomics, this new variant is tested in vitro and in vivo. Survival curves show the phenotype of this new strain. Second, experimental evolution is performed to a) recapitulate the evolution observed in nature using ancestral genotypes and b) predict the next mutations likely to emerge, using as a starting point the newly identified variant (in red). Bottom panel: Implementation of genotype–phenotype maps would help monitor evolution and potentially predict future trajectories towards, or away from, virulence.</p

5-FU-mediated U:C and A:G transitions are distributed across the CoV genome at low frequency.

<p>(<b>A</b>) and (<b>B</b>) The genomic distribution of low frequency statistically significant U:C and A:G variants within the SARS-ExoN+ population following treatment with 0 or 400 µM 5-FU. (<b>C</b>) and (<b>D</b>) Same as in A and B except for the SARS-ExoN− population. For all panels, SARS-ExoN+ viruses are shown in blue, and SARS-ExoN− viruses are shown in green. U:C transitions are denoted by a diamond, whereas A:G transitions are plotted as circles.</p

The increased sensitivity of MHV-ExoN− viruses to 5-FU is consistent with mutagenesis.

<p>(<b>A</b>) DBT cells in 96-well plates were incubated with DMEM alone, or DMEM containing 20% ethanol (EtOH), 4% DMSO, or the indicated concentration of 5-FU for 12 h. Cell viability was determined using CellTiter-Glo (Promega) according to manufacturer's instructions. All values were normalized to the untreated (DMEM) control. Mean values ± S.E.M. are shown, n = 2. (<b>B</b>) MHV-ExoN+ (filled circle) and MHV-ExoN− (open circle) virus sensitivity to 5-FU during single- (solid lines; MOI = 1 PFU/cell) and multi-cycle (dotted lines; MOI = 0.01 PFU/cell) replication. MHV-ExoN+ viruses are shown in blue and MHV-ExoN− viruses are shown in green. The change in virus titer was calculated by dividing virus titers following treatment by the untreated controls. Mean values ± S.E.M. are shown, n = 4. (<b>C</b>) The change in titer (filled bars) and genomic RNA levels (hatched bars) of MHV-ExoN+ (blue) and MHV-ExoN− (green) viruses following treatment with 5-FU is shown. DBT cells were infected with MHV-ExoN+ or MHV-ExoN− in the presence or absence of 5-FU, and virus titer was determined by plaque assay. Genomic RNA levels were determined using two-step real-time qRT-PCR and primers optimized to amplify a ∼120 nt region of ORF1a <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003565#ppat.1003565-Donaldson1" target="_blank">[33]</a>. The change in genomic RNA levels (2^−ΔΔCt) is shown relative to endogenous GAPDH expression and was normalized to RNA levels from untreated samples. Mean values ± S.E.M. are shown, n = 6. For all parts, statistical significance was determined using an unpaired, two-tailed Student's <i>t</i> test (*P<0.05, **P<0.01, ***P<0.0001).</p

The antiviral activity of RBV against ExoN− viruses is not primarily due to mutagenesis.

<p>(<b>A</b>) DBT cells in 96-well plates were incubated with DMEM alone, or DMEM containing 20% ethanol (EtOH), 4% DMSO, or the indicated concentration of RBV for 12 h. Cell viability was determined using CellTiter-Glo (Promega) according to manufacturer's instructions. All values were normalized to the untreated (DMEM) control. No significant differences were found when RBV-treated values were compared to DMEM samples containing DMSO (+DMSO) using an unpaired, two-tailed Student's <i>t</i> test. Mean values ± S.E.M. are shown, n = 2. (<b>B</b>) MHV-ExoN+ (filled circle) and MHV-ExoN− (open circle) virus sensitivity to RBV during single- (solid lines; MOI = 1 PFU/cell) and multi-cycle (dotted lines; MOI = 0.01 PFU/cell) replication. MHV-ExoN+ viruses are shown in blue and MHV-ExoN− viruses are shown in green. The change in virus titer was calculated by dividing virus titers following treatment by the untreated controls. Mean values ± S.E.M. are shown, n = 4. (<b>C</b>) The change in titer (filled bars) and genomic RNA levels (hatched bars) of MHV-ExoN+ (blue) and MHV-ExoN− (green) viruses following treatment with RBV is shown. DBT cells were infected with MHV-ExoN+ or MHV-ExoN− in the presence or absence of RBV, and virus titer was determined by plaque assay. Genomic RNA levels were determined using two-step real-time qRT-PCR and primers optimized to amplify a ∼120 nt region of ORF1a <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003565#ppat.1003565-Donaldson1" target="_blank">[33]</a>. The change in genomic RNA levels (2^−ΔΔCt) is shown relative to endogenous GAPDH expression and was normalized to RNA levels from untreated samples. Mean values ± S.E.M. are shown, n = 6. (<b>D</b>) MHV-ExoN+ (filled circle) and MHV-ExoN− (open circle) virus sensitivity to mycophenolic acid (MPA) during single- (solid lines; MOI = 1 PFU/cell) and multi-cycle (dotted lines; MOI = 0.01 PFU/cell) replication. Mean values ± S.E.M. are shown, n = 2–4. RBV- or MPA-treated MHV-ExoN+ (<b>E</b>) and MHV-ExoN− (<b>F</b>) viruses with or without the addition of 100 µM guanosine (GUA) during single-cycle replication (MOI = 1 PFU/cell). Mean values ± S.E.M. are shown, n = 2. For all parts, statistical significance was determined using an unpaired, two-tailed Student's <i>t</i> test (*P<0.05, **P<0.01, ***P<0.0001).</p

Incorporation of FUMP results in increased U:C and A:G transitions.

<p>All possible base changes are shown for SARS-ExoN+ and SARS-ExoN− viruses in panels (<b>A</b>) and (<b>B</b>), respectively. Transitions (A↔G and U↔C) are shaded in grey, and 5-FU specific transitions (U:C and A:G) are marked with an asterisk. Transversions (A↔T, A↔C, C↔G, G↔T) are shown in white boxes. All values represent the number of unique statistically significant minority variants following 5-FU treatment. (<b>C</b>) The percent of all unique statistically significant minority variants represented by transversions (filled dark grey bars), C:U and G:A transitions (filled light grey bars), and the 5-FU specific transitions A:G (hatched bars) and U:C (checkered bars) are shown following 0 or 400 µM 5-FU treatment. SARS-ExoN+ viruses are shown in blue, and SARS-ExoN− viruses are shown in green.</p

SARS-ExoN− viruses have increased sensitivity to 5-FU.

<p>(<b>A</b>) Vero cells in 96-well plates were incubated with DMEM alone, or DMEM containing 20% ethanol (EtOH), 4% DMSO, or the indicated concentration of RBV or 5-FU for 24 h. Cell viability was determined using CellTiter-Glo (Promega) according to manufacturer's instructions. All values were normalized to the untreated (DMEM) control. Mean values ± S.E.M. are shown, n = 3. The change in SARS-ExoN+ (filled blue circles) and SARS-ExoN− (empty green circles) titers following treatment with RBV (<b>B</b>) or 5-FU (<b>C</b>) during single-cycle replication. Vero cells were infected with either virus at an MOI of 0.1 PFU/cell, and virus supernatant was harvest 24 h post-infection following replication in the presence or absence of RBV or 5-FU. Virus titer was determined by plaque assay on Vero cells. Mean values ± S.E.M. are shown, n = 2 (RBV) and n = 4 (5-FU). (<b>D</b>) Comparison of unique statistically significant (P<0.05) minority variants present between untreated and 5-FU treated samples for both SARS-ExoN+ and ExoN− populations. SARS-ExoN+ viruses are shown in blue, and SARS-ExoN− viruses are shown in green. For panels A–C statistical significance was determined using an unpaired, two-tailed Student's <i>t</i> test (*P<0.05, **P<0.01, ***P<0.0001).</p

Seroprevalence studies associating nutrition with infection susceptibility and arbovirus infection in humans.

<p>Seroprevalence studies associating nutrition with infection susceptibility and arbovirus infection in humans.</p

Studies observing the effect of nutrition on vector competence.

<p>Studies observing the effect of nutrition on vector competence.</p

Vectors, hosts, symptomology and estimated numbers of cases and deaths of selected arboviruses.

<p>Vectors, hosts, symptomology and estimated numbers of cases and deaths of selected arboviruses.</p

Relationships between arboviral disease severity and nutritional factors in humans.

<p>Relationships between arboviral disease severity and nutritional factors in humans.</p