Heterogeneity in evolutionary rates of host-associated bat rabies viruses.

<p>Median substitutions per site per year in the 3^rd codon position of the nucleoprotein gene for estimates generated by the HPM (filled circles) and ILMs (open circles). Colors and dashed gray lines distinguish bat genera as indicated below the x-axis. Credible intervals denote the 95% highest posterior density interval on evolutionary rate.</p

Predictors of viral evolutionary rate from the Bayesian hierarchical phylogenetic model.

<p>(A) Effect sizes (<i>β</i>) on a log scale for each predictor were conditioned on the inclusion of that term in the model (i.e., <i>β</i> | <i>δ</i> = 1). Climatic region, coloniality, seasonal inactivity and long distance migration were categorical variables. Horizontal lines are the 95% highest posterior density intervals on conditional effect sizes and squares (median effect sizes) are proportional to effect sizes. (B) Violin plot showing the effect of climatic region on viral evolutionary rate. White points, black boxes and whiskers indicate the median, inter-quartile range and the total range of values for that group, respectively. The gray shading shows the probability density of evolutionary rate at different values.</p

Evolutionary lability of viral substitution rates in the rabies virus phylogeny.

<p>(A) Bayesian phylogenetic tree of bat rabies viruses with viral lineage names and climatic regions denoted in black (TE: temperate; ST: subtropical; TR: tropical). Host-associated lineages are condensed to triangles connecting the most recent common ancestor to the sampled branches. Lineages are colored along a blue (slowest) to red (fastest) continuum according to evolutionary rate in CP₃ using estimates from the Independent Lineage Models (ILMs). Bayesian posterior support values (>0.70) are given above branches to the lineage level only. All colored lineages received Bayesian posterior support values of ≥0.91. (B) Frequency histogram of expected values of Blomberg's <i>K</i> from 5000 random assignments of substitution rates estimated from the ILM to lineages. Dashed lines indicate 95% bounds of the null distribution and diamonds denote the median values of <i>K</i> for the randomized rate model, RRM (gray), the ILMs (blue), and the HPM (red).</p

Convergent changes among branches of interest in the bat and terrestrial RABV datasets.

1<p>Branch numbers are corresponding to the number shown in the nodes (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002786#ppat.1002786.s001" target="_blank">Figure S1</a>).</p

Test results for positive selection analysis, and summary of amino acid substitutions.

1<p>Significance of test statistics (* <i>P</i><0.05, ** <i>P</i><0.01).</p>2<p>The infinite value is due to absence of synonymous change in foreground branch.</p>3<p>The information is arranged in the order: position, change, branch.</p>4<p>Amino acid changes with bold character were changes that occurred at terminal branches.</p

Reconstruction of post-host shift amino acid changes.

<p>Left: Bayesian tree of EF-W1 lineage, based on the entire coding region of G gene (1572 nuc). The branches involved in host-shift are marked with red. The amino acid changes occurred within these branches were defined “post-host shift” changes. Other branches that lead to terrestrial mammal RABV are marked with green. Right: the magnification of the three clusters involved in the Flagstaff host shifts. For clarity, the branch length did not reflect substitutions per site. The branches with non-synonymous changes are marked with pink, and numbers of changes are shown above and below. For more details see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002786#ppat-1002786-t002" target="_blank">Table 2</a>.</p

Bayesian tree of the EF-W1 lineage.

<p>Skunk viruses are colored in red, fox viruses are colored in blue, other mesocarnivoran viruses are colored in green; bat viruses are black. Posterior probabilities are indicated at key nodes.</p

A Pan-<i>Lyssavirus</i> Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable <i>Rabies virus</i> and Other Lyssaviruses

<div><p>Rabies, resulting from infection by <i>Rabies virus</i> (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the <i>Lyssavirus</i> genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the <i>Lyssavirus</i> genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional <i>Lyssavirus</i> species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.</p></div

Sequence alignment of the LN34 assay target region from isolates of 14 <i>Lyssavirus</i> species.

<p>Sequence alignment of the LN34 assay target region from isolates of 14 <i>Lyssavirus</i> species.</p

<i>Rabies</i> virus positive samples used to validate the sensitivity of the LN34 real-time RT-PCR assay.

<p><i>Rabies</i> virus positive samples used to validate the sensitivity of the LN34 real-time RT-PCR assay.</p