Leishmania RNA Virus 1 Research Articles

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.

Concentration of 2′C-methyladenosine triphosphate by Leishmania guyanensis enables specific inhibition of Leishmania RNA virus 1 via its RNA polymerase

Leishmania is a widespread trypanosomatid protozoan parasite causing significant morbidity and mortality in humans. The endobiont dsRNA virus Leishmania RNA virus 1 (LRV1) chronically infects some strains, where it increases parasite numbers and virulence in murine leishmaniasis models, and correlates with increased treatment failure in human disease. Previously, we reported that 2'-C-methyladenosine (2CMA) potently inhibited LRV1 in Leishmania guyanensis (Lgy) and Leishmania braziliensis, leading to viral eradication at concentrations above 10 μm Here we probed the cellular mechanisms of 2CMA inhibition, involving metabolism, accumulation, and inhibition of the viral RNA-dependent RNA polymerase (RDRP). Activation to 2CMA triphosphate (2CMA-TP) was required, as 2CMA showed no inhibition of RDRP activity from virions purified on cesium chloride gradients. In contrast, 2CMA-TP showed IC50 values ranging from 150 to 910 μm, depending on the CsCl density of the virion (empty, ssRNA-, and dsRNA-containing). Lgy parasites incubated in vitro with 10 μm 2CMA accumulated 2CMA-TP to 410 μm, greater than the most sensitive RDRP IC50 measured. Quantitative modeling showed good agreement between the degree of LRV1 RDRP inhibition and LRV1 levels. These results establish that 2CMA activity is due to its conversion to 2CMA-TP, which accumulates to levels that inhibit RDRP and cause LRV1 loss. This attests to the impact of the Leishmania purine uptake and metabolism pathways, which allow even a weak RDRP inhibitor to effectively eradicate LRV1 at micromolar concentrations. Future RDRP inhibitors with increased potency may have potential therapeutic applications for ameliorating the increased Leishmania pathogenicity conferred by LRV1.

Development of a semi-automated image-based high-throughput drug screening system.

We previously reported that the innate sensing of the endosymbiont Leishmania RNA virus 1 (LRV1) within Leishmania (Viannia) guyanensis through Toll-like receptor 3, worsens the pathogenesis of parasite infection in mice. The presence of LRV1 has been associated with the failure of first-line treatment in patients infected with LRV1 containing -L. guyanensis and -L. braziliensis parasites. Here, we established a semi-automated image-based high-throughput drug screening (HTDS) protocol to measure parasiticidal activity of the Prestwick chemical library in primary murine macrophages infected with LRV1-containing L. guyanensis. The two-independent screens generated 14 hit compounds with over sixty-nine percent reduction in parasite growth compared to control, at a single dose in both screens. Our screening strategy offers great potential in the search for new drugs and accelerates the discovery rate in the field of drug repurposing against Leishmania. Moreover, this technique allows the concomitant assessment of the effect of drug toxicity on host cell number.

Influence of Leishmania RNA Virus 1 on Proinflammatory Biomarker Expression in a Human Macrophage Model of American Tegumentary Leishmaniasis.

Species of the Leishmania Viannia (L. V.) subgenus harbor the double-stranded Leishmania RNA virus 1 (LRV-1), previously identified in isolates from Brazil and Peru. Higher levels of LRV-1 in metastasizing strains of L. V. guyanensis have been documented in both human and murine models, and correlated to disease severity. Expression of proinflammatory biomarkers, including interleukin (IL) 1β, tumor necrosis factor alpha (TNF-α), CXCL10, CCL5, IL-6, and superoxide dismutase, in human macrophages infected with 3 ATCC and 5 clinical isolates of L. V. braziliensis, L. V. guyanensis, and L. V. panamensis for 24 and 48 hours were measured by commercial enzyme immunoassay. Analyses were performed at 24 and 48 hours, stratified by LRV-1 status and species. LRV-1-positive L. V. braziliensis demonstrated significantly lower expression levels of TNF-α (P = .01), IL-1β (P = .0015), IL-6 (P = .001), and CXCL10 (P = .0004) compared with LRV-1-negative L. V. braziliensis. No differences were observed in strains of L. V. panamensis by LRV-1 status. Compared to LRV-1-negative L. V. braziliensis, LRV-1-positive strains of L. V. braziliensis produced a predominant Th2-biased immune response, correlated in humans to poorer immunologic control of infection and more severe disease, including mucosal leishmaniasis. Effects of LRV-1 on the pathogenesis of American tegumentary leishmaniasis may be species specific.

Type I interferons induced by endogenous or exogenous viral infections promote metastasis and relapse of leishmaniasis

The presence of the endogenous Leishmania RNA virus 1 (LRV1) replicating stably within some parasite species has been associated with the development of more severe forms of leishmaniasis and relapses after drug treatment in humans. Here, we show that the disease-exacerbatory role of LRV1 relies on type I IFN (type I IFNs) production by macrophages and signaling in vivo. Moreover, infecting mice with the LRV1-cured Leishmania guyanensis (LgyLRV1- ) strain of parasites followed by type I IFN treatment increased lesion size and parasite burden, quantitatively reproducing the LRV1-bearing (LgyLRV1+ ) infection phenotype. This finding suggested the possibility that exogenous viral infections could likewise increase pathogenicity, which was tested by coinfecting mice with L. guyanensis and lymphocytic choriomeningitis virus (LCMV), or the sand fly-transmitted arbovirus Toscana virus (TOSV). The type I IFN antiviral response increased the pathology of L. guyanensis infection, accompanied by down-regulation of the IFN-γ receptor normally required for antileishmanial control. Further, LCMV coinfection of IFN-γ-deficient mice promoted parasite dissemination to secondary sites, reproducing the LgyLRV1+ metastatic phenotype. Remarkably, LCMV coinfection of mice that had healed from L. guyanensis infection induced reactivation of disease pathology, overriding the protective adaptive immune response. Our findings establish that type I IFN-dependent responses, arising from endogenous viral elements (dsRNA/LRV1), or exogenous coinfection with IFN-inducing viruses, are able to synergize with New World Leishmania parasites in both primary and relapse infections. Thus, viral infections likely represent a significant risk factor along with parasite and host factors, thereby contributing to the pathological spectrum of human leishmaniasis.

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.

Mammalian Innate Immune Response to a Leishmania-Resident RNA Virus Increases Macrophage Survival to Promote Parasite Persistence.

Some strains of the protozoan parasite Leishmania guyanensis (L.g) harbor a viral endosymbiont called Leishmania RNA virus 1 (LRV1). LRV1 recognition by TLR-3 increases parasite burden and lesion swelling invivo. However, the mechanisms by which anti-viral innate immune responses affect parasitic infection are largely unknown. Upon investigating the mammalian host's response to LRV1, we found that miR-155 was singularly and strongly upregulated in macrophages infected with LRV1+ L.g when compared to LRV1- L.g. LRV1-driven miR-155 expression was dependent on TLR-3/TRIF signaling. Furthermore, LRV1-induced TLR-3 activation promoted parasite persistence by enhancing macrophage survival through Akt activation in a manner partially dependent on miR-155. Pharmacological inhibition of Akt resulted in a decrease in LRV1-mediated macrophage survival and consequently decreased parasite persistence. Consistent with these data, miR-155-deficient mice showed a drastic decrease in LRV1-induced disease severity, and lesional macrophages from these mice displayed reduced levels of Akt phosphorylation.

Correlation between presence of Leishmania RNA virus 1 and clinical characteristics of nasal mucosal leishmaniosis

IntroductionMucosal leishmaniosis (ML) is a severe clinical form of leishmaniosis. Complex factors related to the parasite and the host are attributed to the development of mucosal lesions. Leishmania RNA virus 1 (LRV1) can disrupt immune response, and may be the main determinant of severity of the disease; it should be investigated. ObjectiveTo study the existence of clinical differences between patients with ML with endosymbiosis by LRV1 and. those without it. MethodsA cross-sectional cohort study with clinical evaluation, polymerase chain reaction (PCR) detection of Leishmania, species classification, and search of LRV1 was performed. Only patients with confirmed diagnosis of ML by positive PCR and with nasal mucosa injuries were included in this analysis. ResultsOut of 37 patients, 30 (81.1%) were diagnosed with Leishmania braziliensis, five (13.5%) with Leishmania guyanensis, and two (5.4%) with mixed infection of L. braziliensis and L. guyanensis. LVR1 virus was present in 26 (70.3%) of the cases. ConclusionCorrelation between clinical phenotype and presence of LRV1 was not observed, although the frequency of the virus is two-fold higher in mucosal lesions than that found in the literature on skin lesions in the same geographical area.

Presence ofLeishmaniaRNA Virus 1 inLeishmania guyanensisIncreases the Risk of First-Line Treatment Failure and Symptomatic Relapse

Treatment failure and symptomatic relapse are major concerns in American tegumentary leishmaniasis (TL). Such complications are seen frequently in Leishmania guyanensis infections, in which patients respond variously to first-line antileishmanials and are more prone to develop chronic cutaneous leishmaniasis. The factors underlying this pathology, however, are unknown. Recently, we reported that a double-stranded RNA virus, Leishmania RNA virus 1 (LRV1), nested within L. guyanensis parasites is able to exacerbate experimental murine leishmaniasis by inducing a hyperinflammatory response. This report investigates the prevalence of LRV1 in human L. guyanensis infection and its effect on treatment efficacy, as well as its correlation to symptomatic relapses after the completion of first-line treatment. In our cohort of 75 patients with a diagnosis of primary localized American TL, the prevalence of LRV1-positive L. guyanensis infection was elevated to 58%. All patients infected with LRV1-negative L. guyanensis were cured after 1 dose (22 of 31 [71%]) or 2 doses (31 of 31 [100%]) of pentamidine. In contrast, 12 of 44 LRV1-positive patients (27%) presented with persistent infection and symptomatic relapse that required extended therapy and the use of second-line drugs. Finally, LRV1 presence was associated with a significant increase in levels of intra-lesional inflammatory markers. In conclusion, LRV1 status in L. guyanensis infection is significantly predictive (P = .0009) of first-line treatment failure and symptomatic relapse and has the potential to guide therapeutic choices in American TL.

Validation of a leishmania RNA virus-1 detection assay in a non-endemic setting

Background: Tegumentary leishmaniasis is an emerging disease among travelers, and case reports suggest that TL among travelersmaybeparticularly severe. LeishmaniaRNAvirus-1 (LRV1) has been implicated in the pathogenesis of severe TL in murine models, though whether this extends to humans is unknown. We sought to validate a LRV-1 detection assay. Methods & Materials: ATCC reference and clinical strains of Leishmania identified to species level were screened for LRV1. Two real time PCR assays for detection of LRV1 were performed with LRV1 set A and set B primers. Amplification of Leishmania kinetoplastid membrane protein 11 (kmp11) served as a quantification and extraction control. ATCC L. (V.) guyanensis strain MHOM/BR/75/M4147 was used as a positive control. Results: We screened 5 New World ATCC strains of Leishmania (L. amazonensis, L. mexicana, L. (V.) braziliensis, L. (V.) panamensis, and L. (V.) guyanensis) and 12 clinical strains from primary clinical samples including: bone marrow (N=1), whole blood (N=2), skin scraping (N=2), skin biopsy (N=2), cytology brush (N=4) and unspecified (N=1). Causative Leishmania species in clinical specimens were as follows: L. chagasi/infantum (N=3), L. (V.) braziliensis (N=3), L. tropica (N=2), L. (V.) panamensis (N=1), L. major (N=1), and L. (V.) guyanensis (N=1). Amplification of LRV1 primer sets A and B along with kmp11 only occurred with the ATCC strain of L. (V.) guyanensis known to harbour LRV1. Kmp11 alone was amplified in the remaining ATCC strains, and in 6 of 12 (50%) clinical strains, thus 80% of ATCC strains and 50% of clinical strains could be deemed negative for LRV1. Neither LRV1 setA or B primers nor kmp11 were amplified in 6 of 12 (50%) clinical strains, leading to an indeterminant result regarding LRV1 co-infection status. Conclusion: Detection of LRV1 from clinical strains of Leishmaniamaybecome relevant to clinicians in the future. Performance of the LRV1 assay was superior in cultured ATCC strains, which reflects high parasite concentrations in culture compared to primary clinical specimens. In 50% of clinical isolates, we were able to assign an LRV1 result, thus, further optimization of assay performance in primary clinical specimens is warranted.

Validation of a molecular diagnostic algorithm for leishmania detection in a non-endemic setting

Background: Tegumentary leishmaniasis is an emerging disease among travelers, and case reports suggest that TL among travelersmaybeparticularly severe. LeishmaniaRNAvirus-1 (LRV1) has been implicated in the pathogenesis of severe TL in murine models, though whether this extends to humans is unknown. We sought to validate a LRV-1 detection assay. Methods & Materials: ATCC reference and clinical strains of Leishmania identified to species level were screened for LRV1. Two real time PCR assays for detection of LRV1 were performed with LRV1 set A and set B primers. Amplification of Leishmania kinetoplastid membrane protein 11 (kmp11) served as a quantification and extraction control. ATCC L. (V.) guyanensis strain MHOM/BR/75/M4147 was used as a positive control. Results: We screened 5 New World ATCC strains of Leishmania (L. amazonensis, L. mexicana, L. (V.) braziliensis, L. (V.) panamensis, and L. (V.) guyanensis) and 12 clinical strains from primary clinical samples including: bone marrow (N=1), whole blood (N=2), skin scraping (N=2), skin biopsy (N=2), cytology brush (N=4) and unspecified (N=1). Causative Leishmania species in clinical specimens were as follows: L. chagasi/infantum (N=3), L. (V.) braziliensis (N=3), L. tropica (N=2), L. (V.) panamensis (N=1), L. major (N=1), and L. (V.) guyanensis (N=1). Amplification of LRV1 primer sets A and B along with kmp11 only occurred with the ATCC strain of L. (V.) guyanensis known to harbour LRV1. Kmp11 alone was amplified in the remaining ATCC strains, and in 6 of 12 (50%) clinical strains, thus 80% of ATCC strains and 50% of clinical strains could be deemed negative for LRV1. Neither LRV1 setA or B primers nor kmp11 were amplified in 6 of 12 (50%) clinical strains, leading to an indeterminant result regarding LRV1 co-infection status. Conclusion: Detection of LRV1 from clinical strains of Leishmaniamaybecome relevant to clinicians in the future. Performance of the LRV1 assay was superior in cultured ATCC strains, which reflects high parasite concentrations in culture compared to primary clinical specimens. In 50% of clinical isolates, we were able to assign an LRV1 result, thus, further optimization of assay performance in primary clinical specimens is warranted.

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.

Phylogenetic analysis of the 5′ subterminal region of isolates ofLeishmaniaRNA 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.

Complete Sequence of Leishmania RNA Virus 1-4 and Identification of Conserved Sequences

In order to understand the coding strategies and identify potential cis -acting sequences in Leishmania RNA virus 1 (LRV1), a complete cDNA sequence was obtained for LRV1-4 and compared to the sequence reported for LRV1-1. The results show that the 5′ end of LRV1 is conserved at the nucleotide level while open reading frames (ORFs) 2 and 3 are conserved at the amino acid level. A simple translation initiation consensus sequence is conserved at the 5′ end of ORF2 but absent from ORF3, consistent with a possibility that ORF3 is expressed as a gag-pol fusion protein. Comparison of secondary structure predictions obtained for both isolates identified nucleotide sequences capable of forming conserved stem-loops at the virus termini and in the putative frameshift region between ORF2 and ORF3. Although direct evidence is lacking, the appearance of compensatory nucleotide substitutions suggests that the structures may form in vivo. Possible functions for the conserved structures are discussed.

Molecular organization of Leishmania RNA virus 1.

The complete 5284-nucleotide sequence of the double-stranded RNA genome of Leishmania RNA virus 1 (LRV1) was determined and contains three open reading frames (ORFs) on the plus (+) (mRNA) strand. The predicted amino acid sequence of ORF3 has motifs characteristic of viral RNA-dependent RNA polymerases. ORF2, which may encode the major viral coat protein, overlaps ORF3 by 71 nucleotides, suggesting a +1 translational frameshift to produce a gag-pol type of fusion protein. Two alternative models for the frameshift are presented. The 5' splice leader sequence of kinetoplastid mRNAs is not in LRV1 RNA. This suggests that the 450-base region at the 5' end of the LRV1 (+)-strand, which contains ORF1 and is highly conserved among viral strains, does not encode protein but has a role in initiation of translation and/or RNA stability. The similarity of LRV1 genomic organization, replication cycle, and RNA-dependent RNA polymerase sequence to those of the yeast virus ScV L-A suggests a common ancestral origin. The possibility that LRV1 affects pathogenesis in leishmaniasis is intriguing.