<i>Leishmania aethiopica</i> lines used in this work.

<p>LRV status was determined by a dot blot assay (dsRNA detection) for the eight fresh isolates, and by PCR using universal LRV-specific primers on cDNA obtained from the three cryobank lines (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002836#s2" target="_blank">Methods</a>). dsRNA was weakly detected in the <i>Lae</i> 315 strain (+). The four isolates selected for further analysis are highlighted in bold, as well as the L494 strain that was used for LRV sequencing. Parasites were isolated from patients suffering from cutaneous (CL) or diffuse cutaneous leishmaniasis (DCL) as indicated in the “pathology” column.</p

<i>L. aethiopica</i> LRV genomes analysis in comparison to LRV1 and LRV2.

<p>The three <i>L. aethiopica</i> LRV genome sequences (303, 327 and L494) were aligned and compared to each other (LRV-<i>Lae</i>), as well as to LRV2 from <i>L. major</i> ASKH and LRV1 from <i>L. guyanensis</i> CUMC1 and M4147 using LALIGN software from Expasy. Total nucleotide (Genome column) and deduced amino acid sequences of the capsid protein (CP column) and RNA-dependent RNA polymerase (RdRp column) were analyzed. The percentage of identical residues is indicated. Average percentage of identity for each LRV group was also included and highlighted in bold (‘average’ lines).</p

A unique LRV2-<i>Lae</i> genomic organization in the capsid protein/RdRp open reading frames switching region.

<p>The LRV genomic region coding for the end of the capsid protein (CP) and the beginning of the RdRp is shown for <i>L. guyanensis</i> M4147 (LRV1-<i>Lg</i>), <i>L. major</i> ASKH (LRV2-<i>Lmj</i>) and <i>L. aethiopica</i> 303 (LRV2-<i>Lae</i>). The corresponding amino acids of CP and RdRp are above and below the cDNA sequence respectively. CP stop codon is indicated by *. The overlapping region is shown in grey.</p

LRV detection in the <i>L. aethiopica</i> L494 strain.

<p>In addition to the three <i>L. aethiopica</i> cryobank lines tested, <i>Lg</i> M4147 LRV1+ was added as a positive control. <b>A. PCR amplification.</b> A portion of the LRV capsid protein open reading frame (489 and 486 bp for LRV1 and LRV2 respectively) was amplified from total cDNA using LRV universal primers (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002836#pntd.0002836.s005" target="_blank">Table S1</a>). As a cDNA quality control, a 372 bp fragment of the beta-tubulin gene was also amplified. <b>B. LRV dsRNA visualization.</b> Total RNA was analyzed on agarose gel. Ribosomal RNA (rRNA) and the complete 5.3 kb LRV genomic dsRNA are indicated.</p

LRV-<i>Lae</i> dsRNA localization by immunofluorescence microscopy.

<p>Promastigotes were fixed with formaldehyde and spread on poly-lysine coated slides before visualization of viral dsRNA with the J2 antibody (standardized exposure time in all images: 200 ms). Scale bars: 10 µm.</p

LRV2-<i>Lae</i> genome comparison to previously sequenced LRVs.

<p><b>A. Comparison of LRV1-<i>Lg</i>, LRV2-<i>Lmj</i> and LRV2-<i>Lae</i> open reading frames (ORFs).</b> The two major ORFs encoding the capsid protein (CP) and the RNA-dependent RNA polymerase (RdRp) are represented by gray and black boxes respectively. The short ORFs upstream of the CP genes are represented by open boxes with nucleotide positions indicated in italics. The two short ORFs (15–233) and (117–299) upstream of LRV1-<i>Lg</i> capsid are deduced from LRV1-<i>Lg</i> CUMC1 and LRV1-<i>Lg</i> M4147 viral genomes respectively. <b>B–D. Phylogenetic analysis of </b><b><i>L. aethiopica</i></b><b> LRVs and previously sequenced LRV genomes.</b> Total RNA genome sequence (<b>B</b>), and the deduced capsid (<b>C</b>) and RdRp amino acid sequences (<b>D</b>) of LRVs isolated from the indicated strains were analyzed by Jalview. The average distances (using BLOSUM62) are indicated on the trees.</p

LRV is found in <i>L. aethiopica</i> field isolates.

<p><b>A. LRV detection by dot blot using an anti-dsRNA antibody (J2).</b> Promastigotes were directly spotted onto a nitrocellulose membrane (2 µg of total protein/spot) before dsRNA detection by J2. A ponceau red staining of the membrane before J2 addition is added to demonstrate that similar amounts of parasites were loaded. <b>B. Viral genomic dsRNA visualization from nucleic acid extracts.</b> Total promastigote nucleic acids (5 µg for <i>Lg</i> and 20 µg for <i>Lae</i>) were digested with ssRNase and DNase I then migrated in a 0.8% agarose gel (upper panel). An undigested control of each sample acts as a loading control (lower panel).</p

The presence of LRV2-<i>Lae</i> leads to TLR3-dependent production of pro-inflammatory cytokines by <i>in vitro</i> infected macrophages.

<p>C57BL/6 (in black) and TLR3 knock-out (in grey) murine bone marrow derived macrophages were infected by <i>Leishmania</i> promastigotes (parasite/macrophage ratio 10∶1), and the level of IL-6 (<b>A</b>) and TNF-α (<b>B</b>) in culture supernatants was measured by ELISA 24 hours post-infection. Non inf.: non-infected macrophages. The cut-off line was calculated as 3 standard deviations (SD) above the mean absorbance of the uninfected macrophage control. Average values presented were obtained from two independent experiments performed in duplicates.</p

Summary of Fn/Sat×Sd/Hyg hybrid genotypes generated in <i>P. duboscqi</i>.

<p>H, hybrid; ND, not determined.</p

Summary of hybrid genotypes generated in <i>Lu. Longipalpis</i>.

<p>H, hybrid; ND, not determined.</p