title: 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.
  
  
  RESULTS
  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
  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.
authors: Department of Biochemistry, University of Lausanne, Epalinges, Vaud, Switzerland.
authors: Haroun
authors: H
authors: Zangger
authors: Catherine
authors: C
authors: Ronet
authors: Chantal
authors: C
authors: Desponds
authors: F Matthew
authors: FM
authors: Kuhlmann
authors: John
authors: J
authors: Robinson
authors: Mary-Anne
authors: MA
authors: Hartley
authors: Florence
authors: F
authors: Prevel
authors: Patrik
authors: P
authors: Castiglioni
authors: Francine
authors: F
authors: Pratlong
authors: Patrick
authors: P
authors: Bastien
authors: Norbert
authors: N
authors: Müller
authors: Laurent
authors: L
authors: Parmentier
authors: Nancy Gore
authors: NG
authors: Saravia
authors: Stephen M
authors: SM
authors: Beverley
authors: Nicolas
authors: N
authors: Fasel
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005455:Fluorescent Antibody Technique / Q000379:methods; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007891:Leishmania / Q000821:virology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D012328:RNA Viruses / Q000302:isolation & purification*; D012330:RNA, Double-Stranded / Q000276:immunology / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA; D014773:Virology / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D012330:RNA, Double-Stranded; D012367:RNA, Viral