Detection of Leishmania RNA virus in Leishmania parasites

Abstract

Background: Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence.

Methodology/principal findings: This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice.

Conclusions/significance: We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

Figure 1. Detection of LRV in nucleic acid extracts.

A and B. Visualization of viral genomic dsRNA by gel electrophoresis. A. Total nucleic acid from stationary phase promastigotes was treated with ssRNase then migrated in a 1% agarose gel. The sample was either kept intact (1 µg, upper panel) or digested with RQ-DNase (5 µg, lower panel). B. To quantify viral dsRNA in Lg 1398 relative to Lg M4147 LRV^high, various concentrations of nucleic acid (2, 1 and 0.5 µg) were digested with RQ-DNase and migrated as above. C. Quantification of LRV transcript by qRT-PCR. Total parasitic and viral cDNA was prepared for qRT-PCR and amplified using primers specific for LRV (SetA and SetB, see material and methods for sequences). Viral transcript was quantified as normalized to the parasitic housekeeping gene kmp11 then adjusted relative to Lg M4147 LRV^high.

Figure 2. Detection of LRV with a polyclonal anti-capsid antibody (g018d53) and epitope mapping.

A. Western blot. Total parasitic protein extract (40 µg) was separated on a 10% acrylamide denaturing gel then transferred onto a nitrocellulose membrane where the LRV capsid could be detected using the rabbit polyclonal antibody g018d53 (upper panel). A Ponceau staining of the same membrane shows total parasitic protein (lower panel). B. Immunofluorescence microscopy. Red: capsid (g018d53 Ab). Blue: DAPI integrated into kinetoplast and nuclear DNA. Capsid immunofluorescence was visualized with a standardized exposure time in all images. C. 74 overlapping peptides (20-mer) covering the complete sequence of Lg M4147 LRV1-4 capsid were spotted on a cellulose membrane (30 peptides per lane as indicated) and incubated with the g018d53 antibody to identify the recognized epitopes. D. Sequence alignment of the LRV capsids from Lg M4147, Lg M5313 and Lg 1398 in the C-terminal region covering the epitopes recognized by the g018d53 antibody (shown in C). The residues that are not identical to the Lg M5313 LRV sequence are highlighted in a black box.

Figure 3. Detection of LRV with a monocolonal anti-dsRNA (J2) antibody by immunofluorescence microscopy.

A. Reference strain analysis (protocol A, see “Material and methods”). Green: dsRNA (J2 Ab). Blue: DAPI (standardized exposure time in all images). B. Phase and immunofluorescent images of Lg M4147 LRV^high or LRV^neg cells were obtained in the presence or absence of J2 antibody (protocol B). C. Quantitative immunofluorescence (protocol B). The fluorescent intensity per cell was assessed using Image J software on Lg M4147 LRV^high or LRV^neg cells following IFM with the J2 antibody. Cells from phase images were identified and the fluorescent intensity average over the area of the cell was recorded. 108–160 cells from 2 distinct fields were measured, and histogram plots were made using Excel software. LRV^high, no primary antibody (▪, dashed line); LRV^high with J2 (▪, solid line); LRV^neg, no primary antibody (•, dashed line); LRV^neg with J2 (•, solid line).

Figure 4. Detection of LRV using slot blots and J2 antibody.

A. 5×10⁴ parasites were blotted onto nitrocellulose membranes and incubated with J2 or anti-histone H2A antibodies. B. Quantification of the signal intensity for cells in logarithmic or stationary growth phase: dsRNA signal was quantified relative to the histone H2A signal. The cut-off line was calculated as 3 standard deviations (SD) above the mean absorbance of the LRV-negative that showed the highest value (log phase).

Figure 5. Detection of LRV in total parasite lysate using J2 antibody.

A. ELISA. Total lysates from 5×10⁶ promastigotes were coated on 96 wells plates and dsRNA was quantified colorimetrically at 490 nm relative to Lg M4147 LRV^high after background subtraction (uncoated control wells). The cut-off line was calculated as 3 standard deviations (SD) above the mean absorbance of the LRV-negative that showed the highest value (Lg 1881). B. Dot blot. 10⁵ to 5×10⁵ promastigotes were spread directly onto a nitrocellulose membrane and dsRNA was detected using the J2 antibody (upper panel). A Ponceau stain of the membrane shows total protein concentration was similar across samples (lower panel). C. Dot blot sensitivity screening. A dot blot was performed in a serial dilution of 1000 to 10 parasites from LRV-positive and negative control strains (Lg M4147 LRV^high and Lg M4147 LRV^neg).

Figure 6. Screening for LRV in human isolates of Leishmania.

Parasites of 5 different L. braziliensis strains previously shown to harbor LRV were analyzed by dot blot (1 to 4 µg total protein/spot).

Figure 7. Screening for LRV in freshly-isolated human L. braziliensis.

A. Dot blot analysis of two parasite samples obtained from separate lesion biopsies in an infected patient: Lb 2169 and Lb 2192. Live parasites (1 to 4 µg total proteins) were spotted on a nitrocellulose membrane for LRV dsRNA detection by dot blot (J2 antibody). Lg M4147 LRV^high and LRV^neg were used as positive and negative controls. Upper panel: dsRNA detection by dot blot (J2). Lower panel: verification of protein quantity by Ponceau staining. B. J2 anti-dsRNA analysis of Lb 2169 by fluorescence microscopy. Green: dsRNA (J2 Ab). Blue: DAPI. C. Isolation of viral genomic dsRNA from the Lb 2169 strain. Intact and DNase-digested total nucleic acids from Lb 2169 parasites and Lg M4147 LRV^high as a control, were analyzed by gel electrophoresis (similarly to Figure 1A). Note: with high resolution gels such as presented here (in contrast to Figure 1), the viral genome often appears as a doublet.

Figure 8. Detection of LRV in mice footpad lesions.

Dot blot analysis on total RNA extracted from mice lesions infected with Lg M4147 LRV^high and Lg M4147 LRV^neg. Whole parasite (‘total’) and RNA extracts from Lg M4147 promastigotes were also loaded as a control. The amount of protein and RNA loaded is indicated on the left and right side of the figure respectively.