Sequence alignment of fusion loop sequences of various strains of Ebola virus.

<p>Several conserved hydrophilic residues that might serve as potential pH sensors are highlighted: dark green—histidine, light green—threonine or glutamine, blue—lysine or arginine, red glutamate, orange—aspartates (not highly conserved). The sequence of Zaire EBOV (strain Mayinga-76) was used in this study and its residue numbers and a disulfide link that defines the fusion loop are indicated at the bottom. Basic residues are shown in blue, acidic residues in red, histidines in green, hydrophilic residues in light green, and non-conserved acidic/hydrophilic residues in orange.</p

Historic <i>Treponema pallidum</i> genomes from Colonial Mexico retrieved from archaeological remains

<div><p><i>Treponema pallidum</i> infections occur worldwide causing, among other diseases, syphilis and yaws. In particular sexually transmitted syphilis is regarded as a re-emerging infectious disease with millions of new infections annually. Here we present three historic <i>T</i>. <i>pallidum</i> genomes (two from <i>T</i>. <i>pallidum</i> ssp. <i>pallidum</i> and one from <i>T</i>. <i>pallidum</i> ssp. <i>pertenue</i>) that have been reconstructed from skeletons recovered from the Convent of Santa Isabel in Mexico City, operational between the 17^th and 19^th century. Our analyses indicate that different <i>T</i>. <i>pallidum</i> subspecies caused similar diagnostic presentations that are normally associated with syphilis in infants, and potential evidence of a congenital infection of <i>T</i>. <i>pallidum</i> ssp. <i>pertenue</i>, the causative agent of yaws. This first reconstruction of <i>T</i>. <i>pallidum</i> genomes from archaeological material opens the possibility of studying its evolutionary history at a resolution previously assumed to be out of reach.</p></div

Summary statistics of the three positive historic samples.

<p>Summary statistics of the three positive historic samples.</p

Examples for bone lesions for the three positive individuals.

<p>(A) The right tibia of individual 94A displays reactive periosteal bone on the anterior aspect of the diaphysis accompanied by progressive layering of the reactive bone. (B) The <i>pars basilaris</i> portion of the cranium of individual 94A showing pathological reactive bone in the endocranial surface, active at time of death. (C) An unidentified long bone from individual 94B displays fulminating periosteal reaction involving the whole of the diaphysis fragment. (D) The left femur of individual 133 presenting periosteal bone formation and expansion with cortical resorption characteristic of treponemal diseases. Source of the pictures: skeletal collection from Santa Isabel Convent, Mexico City, in custody of the Laboratory of Osteology, Post Graduate Studies Division, National School of Anthropology and History (ENAH), Mexico.</p

Phylogenetic trees and Circos plot [90] of the three ancient strains in comparison to modern strains.

<p>(A) Maximum Likelihood tree with bootstrap support for 39 modern strains and the three ancient strains. The strains 94A (magenta) and 94B (orange) branch with the syphilis SS14 clade while strain 133 (brown) branches with Fribourg-Blanc and other yaws strains. The scale represents the mean number of substitutions per site according to the GTR+GAMMA. Colored bars highlight the three subspecies <i>Treponema pallidum</i> ssp <i>pallidum</i> (TPA), <i>pertenue</i> (TPE) and <i>endemicum</i> (TEN). Strains of subspecies <i>pallidum</i> cause syphilis, subspecies <i>pertenue</i> cause yaws and subspecies <i>endemicum</i> causes bejel. (B) Bayesian trees visualized in Densitree overlaying phylogenetic trees based on the most probable topologies. Blue colored trees represent the most probable topology followed by red colored trees. For the ancient strains 94A and 94B two conflicting topologies are visible. The bars represent the 95% highest probability density intervals of the heights of the clades. The support value given at each clade is the fraction of trees in the tree-set that contain the clade. (C) Circos plot showing the shared SNP positions with specific clades and the coverage of the three ancient strains. From outer circle to the inner circle regions of possible recombination detected by ClonalFrameML are denoted on the outermost circle (purple). ‘ORI’ refers to the origin of replication. The genome coverages of the ancient strains 94B, 94A and 133 are represented in orange magenta and brown respectively from outward to inwards. Based on the SNPs that are specifically shared with different clades, colored bars are shown for strains 94B and 94A respectively in the innermost circles. Red bars highlight the SNP positions specifically shared with Fribourg-Blanc (supporting a phylogenetic position ancestral to the two syphilis clades). The green bars highlight the SNP positions shared with the SS14 clade while the blue bars highlight the SNP positions shared with the Nichols clade.</p

The phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection

<div><p>Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) is a lipid kinase involved in endosome maturation that emerged from a haploid genetic screen as being required for Ebola virus (EBOV) infection. Here we analyzed the effects of apilimod, a PIKfyve inhibitor that was reported to be well tolerated in humans in phase 2 clinical trials, for its effects on entry and infection of EBOV and Marburg virus (MARV). We first found that apilimod blocks infections by EBOV and MARV in Huh 7, Vero E6 and primary human macrophage cells, with notable potency in the macrophages (IC₅₀, 10 nM). We next observed that similar doses of apilimod block EBOV-glycoprotein-virus like particle (VLP) entry and transcription-replication competent VLP infection, suggesting that the primary mode of action of apilimod is as an entry inhibitor, preventing release of the viral genome into the cytoplasm to initiate replication. After providing evidence that the anti-EBOV action of apilimod is via PIKfyve, we showed that it blocks trafficking of EBOV VLPs to endolysosomes containing Niemann-Pick C1 (NPC1), the intracellular receptor for EBOV. Concurrently apilimod caused VLPs to accumulate in early endosome antigen 1-positive endosomes. We did not detect any effects of apilimod on bulk endosome acidification, on the activity of cathepsins B and L, or on cholesterol export from endolysosomes. Hence by antagonizing PIKfyve, apilimod appears to block EBOV trafficking to its site of fusion and entry into the cytoplasm. Given the drug’s observed anti-filoviral activity, relatively unexplored mechanism of entry inhibition, and reported tolerability in humans, we propose that apilimod be further explored as part of a therapeutic regimen to treat filoviral infections.</p></div

Apilimod has no effect on endosome acidification.

<p>BSC-1 cells grown overnight in 35 mm glass bottom dishes (MatTek) were treated for 3 h at 37°C with (A) DMSO (mock), (B) 25 nM bafilomycin (Baf), or (C) 0.2 μM apilimod. Acridine Orange (6.6 μg/mL) was added, and the cells were further incubated at 37°C for 20 min. The cells were then washed and imaged. Scale bars, 10 μm. Images are representative of all observed fields (30 for Baf and 35 for mock- and apilimod-treated cells, respectively).</p

Apilimod does not induce cholesterol accumulation.

<p>BSC-1 cells were grown overnight on 35 mm glass bottom dishes (MatTek). The medium on the cells was replaced with serum-free medium containing 0.05 μM TopFluor Cholesterol (Avanti) plus (A) DMSO (mock), (B) 5 μM U18666A, or (C) 0.2 μM apilimod. The cells were incubated for 18 h at 37°C, washed once with PBS, and imaged in cell imaging medium. Images are representative of all observed fields (25 per treatment).</p

Effects of Apilimod on Ebola Virus and Marburg Virus Replication

<p>Effects of Apilimod on Ebola Virus and Marburg Virus Replication</p

Apilimod has no effect on cathepsin B+L activity.

<p>Confluent dishes of BSC-1 cells were treated with the indicated concentration of apilimod, DMSO (mock), or 10 μM EST for 1 h at 37°C. The cells were then washed, lysed, and the pH adjusted to 5.0 in reaction buffer. Total cathepsin B+L activity was then assayed using the substrate Z-Phe-Arg-7-amido-4-methylcoumarin. Values represent average fluorescence units ± SD of triplicate samples.</p