LR1: a candidate RNA virus of Leishmania.

Abstract

Although viruses are important biological agents and useful molecular tools, little is known about the viruses of parasites. We report here the discovery of a candidate for an RNA virus in a kinetoplastid parasite. This potential virus, which we term LR1, is present in the promastigote form of the human pathogen Leishmania braziliensis guyanensis CUMC1-1A but not in 11 other stocks ofLeishmania that were examined nor in Trypanosoma brucei. The candidate viral RNA has a size of -6000 nucleotides, is single-stranded, and is largely, if not exclusively, located in the cytoplasm. No homologous LR1 sequences are detected in genomic DNA. The candidate viral RNA is associated with a spherical particle 32 nm in diameter that has a sedimentation coefficient of =130 S. There is as yet no evident effect of this potential virus on parasite physiology or the disease caused by the parasite.The presence of viruses in parasitic protozoa may be relevant to the diseases caused by these organisms and may be useful for molecular biological studies. DNA viruses have been reported in Amoeba (1) and virus-like particles have been observed in several Plasmodium species (1), in Nagleria (1), in Endotrypanum (2), in the cytoplasm of Leishmania hertigi (3, 4), and in the flagellum of Trypanosoma melophagium (5). Double-stranded RNA viruses have been found in Giardia (6) and Trichomonas (7). In addition, circular DNAs have been detected in Leishmania (8).We report here the discovery and preliminary characterization of a multicopy RNA in the cytoplasm ofLeishmania braziliensis guyanensis that is associated with a spherical particle 32 nm in diameter. This may be an RNA virus, to the best of our knowledge, the first virus found in a kinetoplastid parasite.MATERIALS AND METHODS Organisms. The Leishmania stocks examined in this study are shown in Table 1. They were grown as the promastigote forms at 28°C as described (9).Nucleic Acid Isolation. Total cellular RNA was prepared by the urea/phenol/cesium chloride method (10). Genomic DNA was prepared as described in Milhausen et al. (11). Gel purified LR1 RNA was prepared from 1.35 x 1010 L. braziliensis guyanensis CUMC1-1A cells by lysis in 5 ml of 1% NaDodSO4/proteinase K (1 mg/ml)/25 mM EDTA for 1 hr at 50°C. Chromosomal DNA was removed by potassium acetate precipitation and the supernatant was precipitated with 1-propanol, resuspended, and electrophoresed in a 0.7% agarose gel in TBE (89 mM Tris borate, pH 8.3/2 mM EDTA). The 6000-nucleotide band was excised from the gel, reelectrophoresed, electroeluted, and ethanol-precipitated.Electrophoresis and Hybridizations. Pulse-field gel electrophoresis was performed as described (9). RNA was electrophoresed either in 1.2% agarose/2.2 M formaldehyde gels as described (10) or in native 0.6% agarose gels in TBE at 1.8 V/cm for 12-16 hr. After electrophoresis RNA was treated with 50 mM NaOH for 30 min and transferred to a Nytran membrane (Schleicher & Schuell) (10). Hybridization to the LR1 cDNA riboprobe was carried out in 50% (vol/vol) formam...

Prevalence and Distribution of Leishmania RNA Virus 1 in Leishmania Parasites from French Guiana

“…[18][19][20]22 We here report the detection of LRV1 from different L. guyanensis and L. braziliensis isolates. 7,10,[18][19][20] None of the six L. amazonensis isolates tested were positive for LRV1. With the exception of the Leishmania panamensis species for which no LRV1 has been identified, 18 no information is currently available for the other New World Leishmania species.…”

“…Surveys of New World parasites have identified LRV1 only in isolates that originated from the Amazon basin, such as L. guyanensis and L. braziliensis. 7,10,[18][19][20] The average molecular prevalence of LRV that we report here in Leishmania spp. isolates circulating in French Guiana is equivalent to the one described by Bourreau and others, 21 but substantially higher (74%) than those reported in other South American countries, which ranged from 5.8% in Colombia, 18% in Peru, and 25.5% in the Brazilian city of Caratinga, Minas Girais.…”

“…6 The first discovery of VLPs in Leishmania was reported by Molyneux 2 in Leishmania hertigi, a nonhuman parasite species. The presence of VLPs in another Leishmania species was then highlighted by Tarr and others, 7 namely the Leishmania (braziliensis) guyanensis species. Leishmania RNA virus (LRV) is an icosahedral encapsidated double-stranded 5.3-kb RNA virus, belonging to the Totiviridae family.…”

“…8,9 New World strains are grouped into the LRV1 type, which is divided in 14 subtypes (LRV1-1 to LRV1-14) that spread over South America. 7,10,11 Old World strains belong to the LRV2 type. 9 The LRV complementary DNA (cDNA) was first completely sequenced by Stuart and others 12 for the prototype virus termed LRV1-1.…”

Successful transient introduction of leishmania RNA virus into a virally infected and an uninfected strain of Leishmania

“…Subsequently, Wang and Wang (2) reported the presence of a double-stranded (ds) RNA virus in Giardia lamblia. dsRNA viruses have been isolated in Trichomonas vaginalis (3) and most recently in Leishmania (4,5). To date, all of the viruses have shown a number of similarities, and all have a single, linear, dsRNA genome of 5-6 kb.…”