Relationship between mutation and evolution rates across viruses.

<p>Symbols for each Baltimore group are the same as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002685#ppat-1002685-g001" target="_blank">Figure 1</a>. <b>a</b>: evolution rates versus mutation rates for seven viruses (HSV-1: herpes simplex virus 1; TMV: tobacco mosaic virus; AHBV: avian hepatitis B virus; FLUVA: influenza A virus; HIV-1: human immunodeficiency virus 1; PV-1: poliovirus 1; HCV: hepatitis C virus). <b>b</b>: log-scale mean ± SEM mutation and evolution rates for each Baltimore group. The dotted line indicates the prediction from a purely neutral model, whereas the dashed line corresponds to a model that incorporates deleterious mutations.</p

Mutation and evolution rate estimates for the major groups defined by the Baltimore classification of viruses.

<p><b>a</b>: mutation rates; <b>b</b>: evolution rates. Each data point corresponds to an individual estimate. Bars indicate log-scale (geometric) means.</p

Expected relationship between mutation and evolution rates according to the neutral-deleterious evolution model for four human viruses: HCV (hepatitis C virus), PV-1 (poliovirus 1), FLUVA (influenza A virus), and HIV-1 (human immunodeficiency virus 1).

<p>Curves indicate the prediction obtained using log₁₀<i>a</i> = 2.387, <i>b</i> = 3.744, and the corresponding genome size of each virus. White dots show the observed average rates. These four viruses were chosen for representation because of the relatively high number of estimates available (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002685#ppat.1002685-Sanjun1" target="_blank">[7]</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002685#ppat.1002685.s001" target="_blank">Text S1</a>). Fewer data are available for the other three viruses appearing in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002685#ppat-1002685-g002" target="_blank">Figure 2a</a>, and their predicted rates deviated more from the observed values.</p

Molecular clone sequencing of VSV from BHK-21 cells.

<p>Molecular clone sequencing of VSV from BHK-21 cells.</p

Fluctuation tests of VSV in BHK-21 cells.

<p>Fluctuation tests of VSV in BHK-21 cells.</p

Genome-Wide Estimation of the Spontaneous Mutation Rate of Human Adenovirus 5 by High-Fidelity Deep Sequencing

<div><p>Rates of spontaneous mutation determine the ability of viruses to evolve, infect new hosts, evade immunity and undergo drug resistance. Contrarily to RNA viruses, few mutation rate estimates have been obtained for DNA viruses, because their high replication fidelity implies that new mutations typically fall below the detection limits of Sanger and standard next-generation sequencing. Here, we have used a recently developed high-fidelity deep sequencing technique (Duplex Sequencing) to score spontaneous mutations in human adenovirus 5 under conditions of minimal selection. Based on >200 single-base spontaneous mutations detected throughout the entire viral genome, we infer an average mutation rate of 1.3 × 10⁻⁷ per base per cell infection cycle. This value is similar to those of other, large double-stranded DNA viruses, but an order of magnitude lower than those of single-stranded DNA viruses, consistent with the possible action of post-replicative repair. Although the mutation rate did not vary strongly along the adenovirus genome, we found several sources of mutation rate heterogeneity. First, two regions mapping to transcription units L3 and E1B-IVa2 were significantly depleted for mutations. Second, several point insertions/deletions located within low-complexity sequence contexts appeared recurrently, suggesting mutational hotspots. Third, mutation probability increased at GpC dinucleotides. Our findings suggest that host factors may influence the distribution of spontaneous mutations in human adenoviruses and potentially other nuclear DNA viruses.</p></div

Spontaneous mutational spectrum of HAdv5.

<p>Spontaneous mutational spectrum of HAdv5.</p

Correlation between genome sizes and mutation rates in viruses.

<p>Published mutation rate estimates are shown for a viroid (star), RNA viruses including retroviruses (white circles), single-stranded DNA viruses (black squares; from left to right: bacteriophage ØX174 and bacteriophage m13), double-stranded DNA viruses (black triangles; from left to right: HAdv5 from this study, bacteriophage λ, herpes simplex virus, bacteriophage T2, murine cytomegalovirus, and human cytomegalovirus). See text for DNA virus mutation rate references. Details on mutation rate data can be found in a recent review [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006013#ppat.1006013.ref026" target="_blank">26</a>]. Bacterial and viroid mutation rates were taken from a previous review [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006013#ppat.1006013.ref007" target="_blank">7</a>]. The dashed line corresponds to a mutation rate of 0.003 per site.</p

Experimental setup for HAdv5 mutation rate measurement.

<p>Three serial endpoint dilution transfers were carried out in 96-well plates (hypothetical infected wells are depicted in red) and then passaged twice in 10-cm plates at high MOI. Each endpoint dilution should remove pre-existing diversity. The estimated number of infection cycles during which mutations could accumulate are shown below: two for growth of the virus from a single PFU in the last endpoint dilution step, and one for each high-MOI transfer. Viral DNA was purified and used directly for Duplex Sequencing. The entire experiment (endpoint dilutions, amplification, and sequencing) was repeated three times (R1, R2, and R3).</p

Relationship between mutation rate and the in vivo sequence diversity.

<p><b>A</b>. Nucleotide diversity along the adenovirus genome. The protein-coding regions of different transcription units are indicated on top with arrows showing their orientation. The red line shows Nei and Li´s nucleotide diversity averaged over a 1 kpb sliding window, using alignments of adenovirus C sequences retrieved from GenBank. Some small discontinuities appear because the analysis was done of a per-gene basis to facilitate sequence alignment. <b>B</b>. Cumulative probability of nucleotide diversity values obtained from GenBank sequences, comparing sites that showed mutations in our experimental system (red) versus non-mutated sites (grey). Notice that the y-axis is broken and that most sites showed no diversity. <b>C</b>. Same figure for the two low-mutation regions (defined in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006013#ppat.1006013.g002" target="_blank">Fig 2B</a>)</b> versus the rest of the viral genome. <b>D.</b> Sequence variants found around genome site 14,073 in GenBank sequences. Accession numbers are included in sequence names. We retrieved 47 adenovirus C sequences in total, but only one example per variant is shown for clarity. Similar figures for other recurrently mutated sites (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006013#ppat.1006013.t002" target="_blank">Table 2</a></b>) are provided in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006013#ppat.1006013.s005" target="_blank">S2 Fig</a></b>.</p