First report of Leishmania RNA virus 1 in Leishmania (Viannia) braziliensis clinical isolates from Rio de Janeiro State - Brazil.

BACKGROUND: Leishmania parasites carry a double-stranded RNA virus (Leishmania RNA virus - LRV) that has been divided in LRV1 and LRV2. OBJECTIVES: Leishmania (Viannia) braziliensis clinical isolates were assessed in order to determine LRV presence. METHODS: Two-round polymerase chain reaction (PCR and nested PCR) was performed to detect LRV1 or LRV2 in L. (V.) braziliensis clinical isolates (n = 12). FINDINGS: LRV1 was detected in three clinical isolates which was phylogenetically related to other sequences reported from other American tegumentary leishmaniasis (ATL) endemic areas of Brazil. Patients infected with L. (V.) braziliensis LRV-negative showed only cutaneous lesions while LRV-positive reported different manifestations. MAIN CONCLUSION: Data presented here show for the first time that LRV1 is circulating in L. (V.) braziliensis clinical isolates from Rio de Janeiro State in Brazil.

Leishmania RNA Virus 1 (LRV-1) in Leishmania (Viannia) braziliensis Isolates from Peru: A Description of Demographic and Clinical Correlates.

RNA virus 1-1 (LRV-1-1) is a dsRNA virus identified in isolates of Leishmania (Viannia) braziliensis and thought to advance localized cutaneous leishmaniasis (LCL) to mucocutaneous or mucosal leishmaniasis (MCL/ML). We examined the prevalence of LRV-1 and its correlation to phenotypes of American tegumentary leishmaniasis caused by L. (V.) braziliensis from Peru to better understand its epidemiology. Clinical isolates of L. (V.) braziliensis were screened for LRV-1 by real-time polymerase chain reaction (PCR) and stratified according to the phenotype: LCL (< 4 ulcers in number) MCL/ML; inflammatory ulcers (erythematous, purulent, painful ulcers with or without lymphatic involvement) or multifocal ulcers (≥ 4 in ≥ 2 anatomic sites). Proportionate LRV-1 positivity was compared across phenotypes. Of 78 L. (V.) braziliensis isolates, 26 (54.2%) had an inflammatory phenotype, 22 (28%) had the MCL/ML phenotype, whereas 30 (38.5%) had LCL. Mucocutaneous or mucosal leishmaniasis was found exclusively in adult male enrollees. Leishmania RNA virus 1 positivity by phenotype was as follows: 9/22 (41%) with MCL/ML; 5/26 (19%) with an inflammatory/multifocal cutaneous leishmaniasis phenotype; and 7/30 (23%) with LCL (P = 0.19). Leishmania RNA virus 1 positivity was not associated with age (P = 0.55) or gender (P = 0.49). Relative LRV-1 copy number was greater in those with MCL/ML than those with inflammatory/multifocal CL (P = 0.02). A direct association between LRV-1 status and clinical phenotype was not demonstrated; however, relative LRV-1 copy number was highest in those with MCL/ML. Future analyses to understand the relationship between viral burden and pathogenesis are required to determine if LRV-1 is truly a contributor to the MCL/ML phenotype.

New insights into the genetic diversity of Leishmania RNA Virus 1 and its species-specific relationship with Leishmania parasites.

Cutaneous leishmaniasis is a neglected parasitic disease that manifests in infected individuals under different phenotypes, with a range of factors contributing to its broad clinical spectrum. One factor, Leishmania RNA Virus 1 (LRV1), has been described as an endosymbiont present in different species of Leishmania. LRV1 significantly worsens the lesion, exacerbating the immune response in both experimentally infected animals and infected individuals. Little is known about the composition and genetic diversity of these viruses. Here, we investigated the relationship between the genetic composition of LRV1 detected in strains of Leishmania (Viannia) braziliensis and L. (V.) guyanensis and the interaction between the endosymbiont and the parasitic species, analyzing an approximately 850 base pair region of the viral genome. We also included one LRV1 sequence detected in L. (V.) shawi, representing the first report of LRV1 in a species other than L. braziliensis and L. guyanensis. The results illustrate the genetic diversity of the LRV1 strains analyzed here, with smaller divergences detected among viral sequences from the same parasite species. Phylogenetic analyses showed that the LRV1 sequences are grouped according to the parasite species and possibly according to the population of the parasite in which the virus was detected, corroborating the hypothesis of joint evolution of the viruses with the speciation of Leishmania parasites.

Antiviral screening identifies adenosine analogs targeting the endogenous dsRNA Leishmania RNA virus 1 (LRV1) pathogenicity factor.

The endogenous double-stranded RNA (dsRNA) virus Leishmaniavirus (LRV1) has been implicated as a pathogenicity factor for leishmaniasis in rodent models and human disease, and associated with drug-treatment failures in Leishmania braziliensis and Leishmania guyanensis infections. Thus, methods targeting LRV1 could have therapeutic benefit. Here we screened a panel of antivirals for parasite and LRV1 inhibition, focusing on nucleoside analogs to capitalize on the highly active salvage pathways of Leishmania, which are purine auxotrophs. Applying a capsid flow cytometry assay, we identified two 2'-C-methyladenosine analogs showing selective inhibition of LRV1. Treatment resulted in loss of LRV1 with first-order kinetics, as expected for random virus segregation, and elimination within six cell doublings, consistent with a measured LRV1 copy number of about 15. Viral loss was specific to antiviral nucleoside treatment and not induced by growth inhibitors, in contrast to fungal dsRNA viruses. Comparisons of drug-treated LRV1+ and LRV1- lines recapitulated LRV1-dependent pathology and parasite replication in mouse infections, and cytokine secretion in macrophage infections. Agents targeting Totiviridae have not been described previously, nor are there many examples of inhibitors acting against dsRNA viruses more generally. The compounds identified here provide a key proof-of-principle in support of further studies identifying efficacious antivirals for use in in vivo studies of LRV1-mediated virulence.

Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response.

Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.

Severe Cutaneous Leishmaniasis in a Human Immunodeficiency Virus Patient Coinfected with Leishmania braziliensis and Its Endosymbiotic Virus.

Leishmania parasites cause a broad range of disease, with cutaneous afflictions being, by far, the most prevalent. Variations in disease severity and symptomatic spectrum are mostly associated to parasite species. One risk factor for the severity and emergence of leishmaniasis is immunosuppression, usually arising by coinfection of the patient with human immunodeficiency virus (HIV). Interestingly, several species of Leishmania have been shown to bear an endogenous cytoplasmic dsRNA virus (LRV) of the Totiviridae family, and recently we correlated the presence of LRV1 within Leishmania parasites to an exacerbation murine leishmaniasis and with an elevated frequency of drug treatment failures in humans. This raises the possibility of further exacerbation of leishmaniasis in the presence of both viruses, and here we report a case of cutaneous leishmaniasis caused by Leishmania braziliensis bearing LRV1 with aggressive pathogenesis in an HIV patient. LRV1 was isolated and partially sequenced from skin and nasal lesions. Genetic identity of both sequences reinforced the assumption that nasal parasites originate from primary skin lesions. Surprisingly, combined antiretroviral therapy did not impact the devolution of Leishmania infection. The Leishmania infection was successfully treated through administration of liposomal amphotericin B.

Association of the Endobiont Double-Stranded RNA Virus LRV1 With Treatment Failure for Human Leishmaniasis Caused by Leishmania braziliensis in Peru and Bolivia.

Cutaneous and mucosal leishmaniasis, caused in South America by Leishmania braziliensis, is difficult to cure by chemotherapy (primarily pentavalent antimonials [Sb(V)]). Treatment failure does not correlate well with resistance in vitro, and the factors responsible for treatment failure in patients are not well understood. Many isolates of L. braziliensis (>25%) contain a double-stranded RNA virus named Leishmaniavirus 1 (LRV1), which has also been reported in Leishmania guyanensis, for which an association with increased pathology, metastasis, and parasite replication was found in murine models. Here we probed the relationship of LRV1 to drug treatment success and disease in 97 L. braziliensis-infected patients from Peru and Bolivia. In vitro cultures were established, parasites were typed as L. braziliensis, and the presence of LRV1 was determined by reverse transcription-polymerase chain reaction, followed by sequence analysis. LRV1 was associated significantly with an increased risk of treatment failure (odds ratio, 3.99; P = .04). There was no significant association with intrinsic Sb(V) resistance among parasites, suggesting that treatment failure arises from LRV1-mediated effects on host metabolism and/or parasite survival. The association of LRV1 with clinical drug treatment failure could serve to guide more-effective treatment of tegumentary disease caused by L. braziliensis.

Severity of tegumentary leishmaniasis is not exclusively associated with Leishmania RNA virus 1 infection in Brazil.

Leishmania RNA virus (LRV) has been shown to be a symbiotic component of Leishmania parasites in South America. Nested retro-transcription polymerase chain reaction was employed to investigate LRV1 presence in leishmaniasis lesions from Brazil. In endemic areas of Rio de Janeiro (RJ), no LRV1 infection was observed even with mucosal involvement. LRV1 was only detected in Leishmania (V.) guyanensis cutaneous lesions from the northern region, which were obtained from patients presenting with disease reactivation after clinical cure of their primary lesions. Our results indicated that the severity of leishmaniasis in some areas of RJ, where Leishmania (V.) brazi-liensis is the primary etiological agent, was not associated with Leishmania LRV1 infection.

Severity of tegumentary leishmaniasis is not exclusively associated with Leishmania RNA virus 1 infection in Brazil

Leishmania RNA virus (LRV) has been shown to be a symbiotic component of Leishmania parasites in South America. Nested retro-transcription polymerase chain reaction was employed to investigate LRV1 presence in leishmaniasis lesions from Brazil. In endemic areas of Rio de Janeiro (RJ), no LRV1 infection was observed even with mucosal involvement. LRV1 was only detected in Leishmania (V.) guyanensis cutaneous lesions from the northern region, which were obtained from patients presenting with disease reactivation after clinical cure of their primary lesions. Our results indicated that the severity of leishmaniasis in some areas of RJ, where Leishmania (V.) brazi-liensis is the primary etiological agent, was not associated with Leishmania LRV1 infection.

Short report: quantification of leishmaniavirus RNA in clinical samples and its possible role in pathogenesis.

Leishmaniavirus (LRV) is a double-stranded RNA virus that infects the protozoa Leishmania and has been identified in numerous strains of Leishmania braziliensis and L. braziliensis guyanensis. In general, the species of Leishmania dictates disease manifestation except in the case of L. braziliensis, which is capable of causing either cutaneous or mucocutaneous leishmaniasis. We wanted to determine 1) the quantity of LRV RNA present in a clinical sample and 2) if infection with LRV was associated with a specific disease manifestation. A real-time reverse transcriptase-polymerase chain reaction assay was used to assay clinical samples for the presence of LRV. Of 47 samples tested, 12 positive samples were obtained from patients with cutaneous lesions, lesions in the process of scarring, and cutaneous scars. This is the first study to examine the prevalence of LRV RNA within a small cohort from Brazil.

Phylogenetic analysis of the 5' subterminal region of isolates of Leishmania RNA virus-1.

Leishmania RNA virus-1 (LRV1) is a double-stranded RNA virus present in some Leishmania species. The virus genome consists of a 450-nucleotide, 5' untranslated region (UTR) followed by the coat gene and the RNA-dependent RNA polymerase (RDRP). It has been shown that the 5' end UTR of the genome promotes internal initiation of translation in an in-vitro assay, indicating the presence of an internal ribosomal entry site (IRES) element upstream of the coat gene. The nucleotide sequences of the 5' subterminal regions of six new isolates of LRV1, of different geographical origins, have now been determined. The RNA folding of the 5' subterminal region of LRV1 has been predicted, using a combination of thermodynamic parameters and folding constraints based on nucleotide substitutions. Furthermore, a putative pyrimidine-rich region (a feature unique to all IRES elements), which is complementary to the Leishmania 18S rRNA, has been identified. The significance and relevance of these findings in the context of the function of the 5' UTR of LRV1 as an IRES element are discussed.

Specific in vitro cleavage of a Leishmania virus capsid-RNA-dependent RNA polymerase polyprotein by a host cysteine-like protease.

Antibodies raised against baculovirus-expressed RNA-dependent RNA polymerase (RDRP) recognized a 95-kDa antigen and two smaller proteins in sucrose-purified Leishmania virus particles isolated from infected parasites. The 95-kDa antigen is similar in size to one predicted by translation of the RDRP open reading frame (ORF) alone. In an effort to reconcile in vitro observations of translational frameshifting on Leishmania RNA virus 1-4 transcripts, we have developed an in vitro cleavage assay system to explore the possibility that the fusion polyprotein is proteolytically processed. We show that coincubation a synthetic Cap-Pol fusion protein with lysates from Leishmania parasites yields major cleavage products similar in size to those encoded by the individual capsid and RDRP genes as well as the antigens detected in vivo. The major 82- and 95-kDa major cleavage products are specifically immunoprecipitated by capsid- or polymerase-specific antibodies, respectively, showing that cleavage occurs at or near the junction of the two functional domains. Protease inhibitor studies suggest that a cysteine-like protease is responsible for cleavage in the in vitro assay system developed here. From these results, we suggest that failure to detect a capsid-polymerase fusion protein produced by translational frameshifting in vivo may be due to specific proteolytic processing.

Intracellular localization of LRV1 in infected Leishmania cells and epitope conservation of the coat protein.

Polyclonal antibodies were raised against a recombinant fragment of the coat protein of LRV1-1 to determine the epitope conservation of the coat protein among LRV1 isolates, and the intracellular localization of LRV1 particles in promastigote cells of Leishmania braziliensis. Western blot analysis showed that specific epitopes of the coat protein are highly conserved among isolates from different geographic areas. Using indirect immunofluorescence assays LRV1 viral particles were observed as fluorescent granules, limited to the cytoplasm and with no apparent association to the host organelles or the cell membrane, characteristic of a persistent, non-infectious virus.

Phylogenetic analysis of Leishmania RNA virus and Leishmania suggests ancient virus-parasite association.

Some strains of the protozoan parasite Leishmania belonging to the new world species guyanensis and braziliensis are infected with persistent, single-segmented, non-enveloped dsRNA viruses termed LRV1. A single old world strain classified as L. major was recently found to harbor a similar virus, designated LRV2-1. The genomic nucleotide sequences of two LRV1 types (1-1 and 1-4) isolated from two L. guyanensis strains have been determined and found to be highly conserved. In contrast, LRV1-specific cDNA probes derived from the conserved genomic 5' region failed to recognize LRV2 RNA on Northern blots, suggesting a greater degree of divergence between LRV1 and LRV2 than among LRV1 types. This observation suggests a long-term association and coevolution of LRV within each parasite strain. We tested this concept by comparing nucleotide sequences of seven LRV types and PCR fingerprints of the parasite strains from which these viruses were derived. In support of the idea of virus-parasite co-evolution, we find that genetic distances between LRV types mirror the heterogeneity between parasite fingerprints and are clustered according to the geographical origin of the strains. In agreement with the postulated common origin of persistent dsRNA viruses of protozoa and fungi, we conclude that the infection of Leishmania with LRV pre-dates the divergence of Leishmania into different lineages.

Small extrachromosomal nucleic acid segments in protozoan parasites.

Viruses have been described in the following protozoa: Babesia spp., Trichomonas vaginalis, Giardia lamblia, Leishmania braziliensis and Eimeria spp. In order to study the Babesia bovis virus, merozoites have been prepared from the blood of infected cattle. Agarose gel electrophoresis of nucleic extracts from the bovine protozoa B. bovis and Babesia bigemina were separated into genomic DNA and at least two additional nucleic acids. One molecule with a relative mobility of 5.5 kilobase pairs (kbp) was identified as a double-stranded RNA virus-like particle. Another 6.2 kbp DNA molecule had sequences related to mitochondrial genome.