Leishmania RNA Virus 1 Research Articles

Epidemiology and Genetic Characterization of Leishmania RNA Virus in Leishmania (Viannia) spp. Isolates from Cutaneous Leishmaniasis Endemic Areas in Panama.

Leishmania (Viannia) spp. can harbor a double-stranded RNA virus known as Leishmania RNA virus 1 (LRV-1), whose presence has been reported in nine countries across the Americas and seven Leishmania species. Here, we studied 100 Leishmania (Viannia) isolates from patients with cutaneous leishmaniasis collected from different endemic areas in Panama from 2016 to 2022. We identified L. (V.) panamensis, L. (V.) guyanensis, L. (V.) braziliensis/guyanensis hybrid, and L. (V.) panamensis sp.1. (genetic variant). LRV-1 was detected by RT-PCR in 9% of L. (Viannia) isolates (eight cases in L. (V.) panamensis, and one in L. (V.) guyanensis). Phylogenetic analysis based on sequencing data classified all LRV-1 isolates within genotype A, suggesting that LRV phylogenetic proximity is closely aligned with geographical distribution or to the phylogenetic proximity of the Leishmania host in the case of the L. (V.) panamensis and L. (V.) guyanensis in Panama.

Transcriptomics analysis highlights potential ways in human pathogenesis in Leishmania braziliensis infected with the viral endosymbiont LRV1.

The parasite Leishmania (Viannia) braziliensis is widely distributed in Brazil and is one of the main species associated with human cases of different forms of tegumentary leishmaniasis (TL) such as cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). The mechanisms underlying the pathogenesis of TL are still not fully understood, but it is known that factors related to the host and the parasite act in a synergistic and relevant way to direct the response to the infection. In the host, macrophages have a central connection with the parasite and play a fundamental role in the defense of the organism due to their ability to destroy intracellular parasites and present antigens. In the parasite, some intrinsic factors related to the species or even the strain analyzed are fundamental for the outcome of the disease. One of them is the presence of Leishmania RNA Virus 1 (LRV1), an endosymbiont virus that parasitizes some species of Leishmania that triggers a cascade of signals leading to a more severe TL phenotype, such as ML. One of the strategies for understanding factors associated with the immune response generated after Leishmania/host interaction is through the analysis of molecular patterns after infection. Thus, the gene expression profile in human monocyte-derived macrophages obtained from healthy donors infected in vitro with L. braziliensis positive (LbLRV1+) and negative (LbLRV1-) for LRV1 was evaluated. For this, the microarray assay was used and 162 differentially expressed genes were identified in the comparison LbLRV1+ vs. LbLRV1-, 126 upregulated genes for the type I and II interferons (IFN) signaling pathway, oligoadenylate synthase OAS/RNAse L, non-genomic actions of vitamin D3 and RIG-I type receptors, and 36 down-regulated. The top 10 downregulated genes along with the top 10 upregulated genes were considered for analysis. Type I interferon (IFNI)- and OAS-related pathways results were validated by RT-qPCR and Th1/Th2/Th17 cytokines were analyzed by Cytometric Bead Array (CBA) and enzyme-linked immunosorbent assay (ELISA). The microarray results validated by RT-qPCR showed differential expression of genes related to IFNI-mediated pathways with overexpression of different genes in cells infected with LbLRV1+ compared to LbLRV1- and to the control. No significant differences were found in cytokine levels between LbLRV1+ vs. LbLRV1- and control. The data suggest the activation of gene signaling pathways associated with the presence of LRV1 has not yet been reported so far. This study demonstrates, for the first time, the activation of the OAS/RNase L signaling pathway and the non-genomic actions of vitamin D3 when comparing infections with LbLRV1+ versus LbLRV1- and the control. This finding emphasizes the role of LRV1 in directing the host's immune response after infection, underlining the importance of identifying LRV1 in patients with TL to assess disease progression.

Extracellular vesicles in parasitic protozoa: Impact of Leishmania exosomes containing Leishmania RNA virus 1 (LRV1) on Leishmania infectivity and disease progression.

This chapter focuses on the interplay between Leishmania parasites and their host, particularly on Leishmania RNA virus (LRVs) and extracellular vesicles (EVs) in modulating host-pathogen interactions. Leishmania EVs have been shown to facilitate gene transfer, including drug-resistance genes, enhancing the parasites' survival and resistance to antileishmanial therapeutics. These EVs also play a significant role in host immune modulation by altering cytokine production in macrophages and promoting an anti-inflammatory environment that favours parasitic persistence. The presence of virulence factors such as GP63 within these EVs further underscores their role in the parasite's immunopathogenesis. Over the last few decades, LRVs have been established as drivers of the severity and persistence of leishmaniasis by exacerbating inflammatory responses and potentially influencing treatment outcomes. This chapter discusses the evolutionary origins and classification of these viruses, and explores their role in parasitic pathogenicity, highlighting their ubiquity across protozoan parasites and their impact on disease progression.

Novel detection of Leishmania RNA virus-1 (LRV-1) in clinical isolates of Leishmania Viannia panamensis.

American tegumentary leishmaniasis comprises a discrete set of clinical presentations endemic to Latin America. Leishmania RNA virus-1 (LRV-1) is a double-stranded RNA virus identified in 20–25% of the Leishmania Viannia braziliensis and L. V. guyanensis, however not in L. V. panamensis. This is the first report of LRV-1 in L. V. panamensis and its associations with clinical phenotypes of ATL. Unique surplus discard clinical isolates of L. V. panamensis were identified from the Public Health Ontario Laboratory (PHOL) and the Leishmania Clinic of the Instituto de Medicina Tropical ‘Alexander von Humboldt’ between 2012 and 2019 and screened for LRV-1 by real-time polymerase chain reaction. Patient isolates were stratified according to clinical phenotype. Of 30 patients with L. V. panamensis, 14 (47%) and 16 (53%) patients had severe and non-severe ATL, respectively. Five (36%) of 14 severe cases and 2 (12%) of 16 non-severe cases were positive for LRV-1, respectively. No differences in sex were observed for clinical phenotype and LRV-1 status. Although an association between LRV-1 status and clinical phenotype was not demonstrated, this is the first description of the novel detection of LRV-1 in L. V. panamensis, a species that has been documented predominantly in Central America.

Exploring Host-Specificity: Untangling the Relationship between Leishmania (Viannia) Species and Its Endosymbiont Leishmania RNA Virus 1.

A relevant aspect in the epidemiology of Tegumentary Leishmaniasis (TL) are the Leishmania parasites carrying a viral endosymbiont, Leishmania RNA Virus 1 (LRV1), a dsRNA virus. Leishmania parasites carrying LRV1 are prone to causing more severe TL symptoms, increasing the likelihood of unfavorable clinical outcomes. LRV1 has been observed in the cultured strains of five L. (Viannia) species, and host specificity was suggested when studying the LRV1 from L. braziliensis and L. guyanensis strains. The coevolution hypothesis of LRV1 and Leishmania was based on phylogenetic analyses, implying an association between LRV1 genotypes, Leishmania species, and their geographic origins. This study aimed to investigate LRV1 specificity relative to Leishmania (Viannia) species hosts by analyzing LRV1 from L. (Viannia) species. To this end, LRV1 was screened in L. (Viannia) species other than L. braziliensis or L. guyanensis, and it was detected in 11 out of 15 L. naiffi and two out of four L. shawi. Phylogenetic analyses based on partial LRV1 genomic sequencing supported the hypothesis of host specificity, as LRV1 clustered according to their respective Leishmania species' hosts. These findings underscore the importance of investigating Leishmania and LRV1 coevolution and its impact on Leishmania (Viannia) species dispersion and pathogenesis in the American Continent.

Detection of Leishmania RNA Virus 1 in Leishmania (Viannia) panamensis Isolates, Panama.

We detected Leishmania RNA virus 1 (LRV1) in 11 isolates of Leishmania (Viannia) panamensis collected during 2014-2019 from patients from different geographic areas in Panama. The distribution suggested a spread of LRV1 in L. (V.) panamensis parasites. We found no association between LRV1 and an increase in clinical pathology.

Leishmania RNA Virus Is Not Detected in All Species of the Leishmania Viannia Subgenus: The Case of L. (V.) panamensis in Colombia.

The endosymbiotic Leishmania RNA virus 1 (LRV1) has been associated with severity and clinical manifestations of American tegumentary leishmaniasis caused by species of the Leishmania (Viannia) subgenus. Between and within Leishmania species, and among endemic countries, the prevalence of LRV is highly variable. The LRV virus has not been detected in L. (V.) panamensis, the second-most prevalent species in Central America and Colombia. However, no systematic screening of LRV has been conducted in L. (V.) panamensis, and thus it is still controversial whether this virus is truly absent from the species. We sought to determine the prevalence of LRV1 in L. (V.) panamensis clinical strains isolated from patients with cutaneous leishmaniasis (CL), from different geographic areas of Colombia. We analyzed 219 clinical strains; 78% were L. (V.) panamensis, 18% were L. (V.) braziliensis, and 4% were L. (V.) guyanensis. Screening for LRV1 was performed by quantitative reverse transcription-polymerase chain reaction. The LRV1 was detected in 18% (7 of 40) of L. (V) braziliensis strains, and was not detected in any of the L. (V.) guyanensis or L. (V.) panamensis strains. The LRV1-positive L. (V). braziliensis strains came from the Amazon Basin. Of the seven LRV1-positive strains, two were isolated from patients with mucocutaneous leishmaniasis, and the remaining from patients with CL. Our results confirm the absence of LRV1 in L. (V.) panamensis in Colombia.

In vitro miltefosine and amphotericin B susceptibility of strains and clinical isolates of Leishmania species endemic in Brazil that cause tegumentary leishmaniasis

Tegumentary leishmaniasis encompasses a spectrum of clinical manifestations caused by the parasitic protozoa of the genus Leishmania. In Brazil, there are at least seven Leishmania species that are endemic and responsible for this set of clinical manifestations of the disease. Current treatment is limited to a restricted number of drugs that in general have several drawbacks including parenteral use, toxicity, and severe side effects. Amphotericin B is considered a second-line drug for tegumentary leishmaniasis in Brazil, while miltefosine was recently approved for clinical use in the treatment of this disease. In this study, we investigated the in vitro susceptibility of Leishmania strains representative of the species endemic to Brazil, as well as a panel of thirteen clinical isolates of tegumentary leishmaniasis, to both amphotericin B and miltefosine. A moderate variation in the susceptibility to both drugs was found, where the EC50 values varied from 11.43 to 52.67 μM for miltefosine and from 12.89 to 62.36 nM for amphotericin B in promastigotes, while for the intracellular amastigotes, values ranged from 1.08 to 9.60 μM and from 1.69 to 22.71 nM for miltefosine and amphotericin B respectively. Furthermore, the clinical isolates and strains of the subgenus Viannia were evaluated for the presence of Leishmania RNA virus 1 (LRV1), as this is an important factor associated with disease severity and treatment outcome. These findings provide a preclinical dataset of the activity of these drugs against the causative species of tegumentary leishmaniasis in Brazil.

First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil.

Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. To the best of our knowledge, this is the first detection of LRV2 in the New World.

Virion structure of Leishmania RNA virus 1

The presence of Leishmania RNA virus 1 (LRV1) enables Leishmania protozoan parasites to cause more severe disease than the virus-free strains. The structure of LRV1 virus-like particles has been determined previously, however, the structure of the LRV1 virion has not been characterized. Here we used cryo-electron microscopy and single-particle reconstruction to determine the structures of the LRV1 virion and empty particle isolated from Leishmania guyanensis to resolutions of 4.0 Å and 3.6 Å, respectively. The capsid of LRV1 is built from sixty dimers of capsid proteins organized with icosahedral symmetry. RNA genomes of totiviruses are replicated inside the virions by RNA polymerases expressed as C-terminal extensions of a sub-population of capsid proteins. Most of the virions probably contain one or two copies of the RNA polymerase, however, the location of the polymerase domains in LRV1 capsid could not be identified, indicating that it varies among particles.Importance.Every year over 200 000 people contract leishmaniasis and more than five hundred people die of the disease. The mucocutaneous form of leishmaniasis produces lesions that can destroy the mucous membranes of the nose, mouth, and throat. Leishmania parasites carrying Leishmania RNA virus 1 (LRV1) are predisposed to cause aggravated symptoms in the mucocutaneous form of leishmaniasis. Here, we present the structure of the LRV1 virion determined using cryo-electron microscopy.

Leishmania guyanensis suppressed inducible nitric oxide synthase provoked by its viral endosymbiont.

Inducible nitric oxide synthase (iNOS) is essential to the production of nitric oxide (NO), an efficient effector molecule against intracellular human pathogens such as Leishmania protozoan parasites. Some strains of Leishmania are known to bear a viral endosymbiont termed Leishmania RNA virus 1 (LRV1). Recognition of LRV1 by the innate immune sensor Toll-like receptor-3 (TLR3) leads to conditions worsening the disease severity in mice. This process is governed by type I interferon (type I IFNs) arising downstream of TLR3 stimulation and favoring the formation of secondary metastatic lesions. The formation of these lesions is mediated by the inflammatory cytokine IL-17A and occurs in the absence, or low level of, protective cytokine IFN-γ. Here, we described that the presence of LRV1 led to the initial expression of iNOS and low production of NO that failed to control infection. We subsequently showed that LRV1-triggered type I IFN was essential but insufficient to induce robust iNOS induction, which requires strong activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Leishmania guyanensis carrying LRV1 (LgyLRV1+) parasites mitigated strong iNOS production by limiting NF-kB activation via the induction of tumor necrosis factor-alpha-induced protein 3 (TNFAIP3), also known as A20. Moreover, our data suggested that production of LRV1-induced iNOS could be correlated with parasite dissemination and metastasis via elevated secretion of IL-17A in the draining lymph nodes. Our findings support an additional strategy by which LRV1-bearing Leishmania guyanensis evaded killing by nitric oxide and suggest that low levels of LRV1-induced NO might contribute to parasite metastasis.

Dissection of the macrophage response towards infection by the Leishmania-viral endosymbiont duo and dynamics of the type I interferon response.

Leishmania RNA virus 1 (LRV1) is a double-stranded RNA virus found in some strains of the human protozoan parasite Leishmania, the causative agent of leishmaniasis, a neglected tropical disease. Interestingly, the presence of LRV1 inside Leishmania constitutes an important virulence factor that worsens the leishmaniasis outcome in a type I interferon (IFN)–dependent manner and contributes to treatment failure. Understanding how macrophages respond toward Leishmania alone or in combination with LRV1 as well as the role that type I IFNs may play during infection is fundamental to oversee new therapeutic strategies. To dissect the macrophage response toward infection, RNA sequencing was performed on murine wild-type and Ifnar-deficient bone marrow–derived macrophages infected with Leishmania guyanensis (Lgy) devoid or not of LRV1. Additionally, macrophages were treated with poly I:C (mimetic virus) or with type I IFNs. By implementing a weighted gene correlation network analysis, the groups of genes (modules) with similar expression patterns, for example, functionally related, coregulated, or the members of the same functional pathway, were identified. These modules followed patterns dependent on Leishmania, LRV1, or Leishmania exacerbated by the presence of LRV1. Not only the visualization of how individual genes were embedded to form modules but also how different modules were related to each other were observed. Thus, in the context of the observed hyperinflammatory phenotype associated to the presence of LRV1, it was noted that the biomarkers tumor-necrosis factor α (TNF-α) and the interleukin 6 (IL-6) belonged to different modules and that their regulating specific Src-family kinases were segregated oppositely. In addition, this network approach revealed the strong and sustained effect of LRV1 on the macrophage response and genes that had an early, late, or sustained impact during infection, uncovering the dynamics of the IFN response. Overall, this study contributed to shed light and dissect the intricate macrophage response toward infection by the Leishmania-LRV1 duo and revealed the crosstalk between modules made of coregulated genes and provided a new resource that can be further explored to study the impact of Leishmania on the macrophage response.

Leishmania guyanensis M4147 as a new LRV1-bearing model parasite: Phosphatidate phosphatase 2-like protein controls cell cycle progression and intracellular lipid content.

Leishmaniasis is a parasitic vector-borne disease caused by the protistan flagellates of the genus Leishmania. Leishmania (Viannia) guyanensis is one of the most common causative agents of the American tegumentary leishmaniasis. It has previously been shown that L. guyanensis strains that carry the endosymbiotic Leishmania RNA virus 1 (LRV1) cause more severe form of the disease in a mouse model than those that do not. The presence of the virus was implicated into the parasite’s replication and spreading. In this respect, studying the molecular mechanisms of cellular control of viral infection is of great medical importance. Here, we report ~30.5 Mb high-quality genome assembly of the LRV1-positive L. guyanensis M4147. This strain was turned into a model by establishing the CRISPR-Cas9 system and ablating the gene encoding phosphatidate phosphatase 2-like (PAP2L) protein. The orthologue of this gene is conspicuously absent from the genome of an unusual member of the family Trypanosomatidae, Vickermania ingenoplastis, a species with mostly bi-flagellated cells. Our analysis of the PAP2L-null L. guyanensis showed an increase in the number of cells strikingly resembling the bi-flagellated V. ingenoplastis, likely as a result of the disruption of the cell cycle, significant accumulation of phosphatidic acid, and increased virulence compared to the wild type cells.

Leishmania RNA virus-1 is similarly detected among metastatic and non-metastatic phenotypes in a prospective cohort of American Tegumentary Leishmaniasis

American Tegumentary Leishmaniasis (ATL) is an endemic and neglected disease of South America. Here, mucosal leishmaniasis (ML) disproportionately affects up to 20% of subjects with current or previous localised cutaneous leishmaniasis (LCL). Preclinical and clinical reports have implicated the Leishmania RNA virus-1 (LRV1) as a possible determinant of progression to ML and other severe manifestations such as extensive cutaneous and mucosal disease and treatment failure and relapse. However, these associations were not consistently found in other observational studies and are exclusively based on cross-sectional designs. In the present study, 56 subjects with confirmed ATL were assessed and followed out for 24-months post-treatment. Lesion biopsy specimens were processed for molecular detection and quantification of Leishmania parasites, species identification, and LRV1 detection. Among individuals presenting LRV1 positive lesions, 40% harboured metastatic phenotypes; comparatively 58.1% of patients with LRV1 negative lesions harboured metastatic phenotypes (p = 0.299). We found treatment failure (p = 0.575) and frequency of severe metastatic phenotypes (p = 0.667) to be similarly independent of the LRV1. Parasite loads did not differ according to the LRV1 status (p = 0.330), nor did Leishmanin skin induration size (p = 0.907) or histopathologic patterns (p = 0.780). This study did not find clinical, parasitological, or immunological evidence supporting the hypothesis that LRV1 is a significant determinant of the pathobiology of ATL.

Overcoming the Negligence in Laboratory Diagnosis of Mucosal Leishmaniasis.

The northern region of Brazil, which has the largest number of cases of tegumentary leishmaniasis (TL) in the country, is also the region that has the highest diversity of species of vectors and Leishmania parasites. In this region, cases of mucosal leishmaniasis (ML), a clinical form of TL, exceed the national average of cases, reaching up to 12% of the total annual TL notifications. ML is associated with multiple factors, such as the parasite species and the viral endosymbiont Leishmania RNA virus 1 (LRV1). Being a chronic parasitological disease, laboratory diagnosis of ML poses a challenge for health services. Here, we evaluated more than 700 clinical samples from patients with clinical suspicion of TL, including patients with cutaneous leishmaniasis (CL) and mucosal leishmaniasis, comparing the results of parasitological tests—direct parasitological examination by microscopy (DP) and conventional PCR (cPCR) targeting of both kDNA and hsp70. The DP was performed by collecting material from lesions through biopsies (mucosal lesions) or scarification (cutaneous lesions); for PCR, a cervical brush was used for sample collection. Blood samples were tested employing standardized real-time PCR (qPCR) protocol targeting the HSP70 gene. PCR tests showed higher sensitivity than DP for both CL and ML samples. Considering ML samples only (N = 89), DP showed a sensitivity of 49.4% (N = 44) against 98.8% (N = 88) for kDNA PCR. The qPCR hsp70 for blood samples from patients with ML (N = 14) resulted in superior sensitivity (50%; N = 7) compared to DP (21.4%; N = 3) for samples from the same patients. Our results reinforced the need to implement a molecular test for the diagnosis of ML, in addition to proposing methods less invasive for collecting material from TL patients. Sample collection using a cervical brush in lesions observed in CL and ML patients is easy to perform and less invasive, compared to scarification and biopsies. Blood samples could be a good source for qPCR diagnosis for ML patients. Thus, we propose here a standardized method for collection and for performing of molecular diagnosis of clinical samples from suspicious ML patients that can be applied in reference services for improving ML diagnosis.

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.

Leishmania Viannia guyanensis

Leishmania of the Viannia subgenus, including Leishmania Viannia guyanensis, is the agent responsible for cutaneous and mucocutaneous leishmaniasis (CL and MCL) in the Americas from the US to Argentina. 48,000 new cases of Cl and MCL are reported yearly, among which 1/10 are associated with L. V. guyanensis infection transmitted by female Lutzomyia sandflies during the blood meal. Inoculated metacyclic promastigotes, coupled with Leishmania exosomes, will infect various inflammatory cells at inoculation sites, where they rapidly transform into amastigotes. Parasites divide and progress in the intra-macrophage form, leading to an initial CL skin ulceration. Depending on the inoculation site and host health condition, parasites may metastasize to the nasopharyngeal tissues within a few months. L. V. guyanensis is occasionally infected with Leishmania RNA virus 1 (LRV1) that can be enveloped by exosomes and is believed to accelerate MCL development.

Exploitation of the Leishmania exosomal pathway by Leishmania RNA virus 1.

Leishmania are ancient eukaryotes that have retained the exosome pathway through evolution. Leishmania RNA virus 1 (LRV1)-infected Leishmania species are associated with a particularly aggressive mucocutaneous disease triggered in response to the double-stranded RNA (dsRNA) virus. However, it is unclear how LRV1 is exposed to the mammalian host cells. In higher eukaryotes, some viruses are known to utilize the host exosome pathway for their formation and cell-to-cell spread. As a result, exosomes derived from infected cells contain viral material or particles. Herein, we investigated whether LRV1 exploits the Leishmania exosome pathway to reach the extracellular environment. Biochemical and electron microscopy analyses of exosomes derived from LRV1-infected Leishmania revealed that most dsRNA LRV1 co-fractionated with exosomes, and that a portion of viral particles was surrounded by these vesicles. Transfer assays of LRV1-containing exosome preparations showed that a significant amount of parasites were rapidly and transiently infected by LRV1. Remarkably, these freshly infected parasites generated more severe lesions in mice than non-infected ones. Moreover, mice co-infected with parasites and LRV1-containing exosomes also developed a more severe disease. Overall, this work provides evidence that Leishmania exosomes function as viral envelopes, thereby facilitating LRV1 transmission and increasing infectivity in the mammalian host.

Prevalence and Association of Leishmania RNA Virus-1 (LRV-1) in Severe and Non-Severe Phenotypes of American Tegumentary Leishmaniasis from Peru

Background: American tegumentary leishmaniasis (ATL) comprises a discrete set of clinical presentations of leishmaniasis endemic to Latin America and particularly Peru. Leishmania RNA virus-1 (LRV-1) is a double stranded RNA virus identified in 20-25% of the Leishmania Viannia complex, and is believed to be a predictive biomarker of severe ATL. Our objective was to determine the baseline prevalence of LRV-1 and associations with clinical phenotypes of ATL in patients from Peru. Methods & Materials: Banked clinical isolates of Leishmania Viannia spp. from patients residing in or traveling to Peru between 2012 and 2016 were species identified, by PCR, RFLP analysis, and Sanger sequencing, and screened for LRV-1 by real-time PCR. Patient isolates were stratified according to clinical phenotype: localized cutaneous leishmaniasis (LCL) was defined as “non-severe” ATL, whereas “severe ATL” was defined as mucosal or mucocutaneous leishmaniasis (ML/MCL); erythematous, purulent, or painful ulcers and/or lymphatic involvement (inflammatory ulcers); or multifocal/disseminated ulcers (≥4 in ≥2 anatomic sites). Proportions of LRV-1-positivity were calculated and compared across phenotypes. Results: Of 81 patients enrolled, 45(56%) and 36(44%) of ATL cases had the severe and non-severe phenotypes, respectively. Seventeen (38%) of 45 severe ATL cases and 13(36%) of 36 non-severe ATL cases were positive for LRV-1, respectively (p = 1.00). The severe phenotype was over-represented among older patients (median age 42 years vs. 30.5 years in non-severe ATL, p = 0.01), while LRV-1 was over-represented among younger patients (median age 28 years vs. 39 years in LRV-1 negative, p = 0.02). No difference in disease severity by sex (p = 0.12) or causative species (p = 0.057) was identified. Similarly, no difference in LRV-1 status was observed by sex (p = 1.00) or causative species (p = 0.45). LRV-1 copy number was greater in those with ML/MCL compared to those with all forms of CL (p = 0.04). Conclusion: A direct association between LRV-1 status and clinical phenotype was not demonstrated, however LRV-1 copy number was higher in patients with ML/MCL. Our findings suggest age as a contributing factor to disease severity. Given the likelihood of relapse in patients with severe ATL and associated risk of treatment failure, further elucidation of LRV-1's possible contribution to disease severity in ATL warrants further exploration.

Influence of Leishmania RNA Virus-1 (LRV-1) on Virulence Factor RNA Transcript Expression of Leishmania Viannia spp

Background: Leishmania RNA Virus-1 (LRV-1) is a double stranded RNA virus identified in strains of the Viannia subgenus in Latin America. Virulence factors (VFs) are gene products that augment pathogenicity of an organism, and are mostly constitutively expressed. Both LRV-1 and VFs are known to modulate the host immune response to Leishmania infection. We aimed to quantify RNA transcripts of known VFs in Leishmania to understand the influence, if any, of LRV-1. Methods & Materials: Five clinical cultures of Leishmania Viannia spp. housed in our biobank were species identified by PCR, RFLP analysis, and Sanger sequencing. Cultures were inoculated into a macrophage model and supernatants were collected at 24- and 48-hrs. Total cellular RNA was extracted at baseline and post-macrophage infection, and cDNA was reverse transcribed. LRV-1 status was confirmed by qPCR and quantified using the 2-ΔΔ CT method. qPCR assays were performed using the 18S gene as a reference for the following RNA transcripts: zinc-metalloproteainse (gp63), mannose phosphate isomerase (mpi), cysteine proteinase B (cpb), and heat shock proteins 23, 70, 83, and 100 (hsp23, hsp70, hsp83 and hsp100, respectively). Results: Cultured isolates were derived from male patients with a median age of 22 years (range 9-80 years) and were species identified as: L. V. braziliensis (n = 1, 20%) and L. V. panamensis(n = 4, 80%). Of 5 cultured isolates, 3 (60%) were positive for LRV-1. RNA transcript expression between LRV-1 positive strains did not differ from LRV-1 negative strains for the following VFs at baseline: pooled VFs (p = 0.20), cpb (p = 0.40), gp63 (p = 0.20), mpi (p = 0.20), hsp23 (p = 0.20), hsp70 (p = 0.20), hsp83 (p = 0.20), and hsp100 (p = 0.20). Similarly, transcript expression did not differ by LRV-1 status post macrophage infection for the following: pooled VF at 24- and 48-hrs, respectively (p = 0.80 and p = 0.40), CPB at 48 hrs (p = 0.20) and MPI at 48 hrs (p > 0.99). Conclusion: VF RNA transcript expression did not differ significantly between LRV-1 positive and negative clinical strains of Leishmania at baseline and post-macrophage infection. Our findings suggest that LRV-1 does not contribute to enhanced virulence in our analyzed VFs, however further exploration of additional VFs, particularly those specific to New World or the Viannia subgenus is warranted.