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 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.

The First Non-LRV RNA Virus in Leishmania.

In this work, we describe the first Leishmania-infecting leishbunyavirus-the first virus other than Leishmania RNA virus (LRV) found in trypanosomatid parasites. Its host is Leishmaniamartiniquensis, a human pathogen causing infections with a wide range of manifestations from asymptomatic to severe visceral disease. This virus (LmarLBV1) possesses many characteristic features of leishbunyaviruses, such as tripartite organization of its RNA genome, with ORFs encoding RNA-dependent RNA polymerase, surface glycoprotein, and nucleoprotein on L, M, and S segments, respectively. Our phylogenetic analyses suggest that LmarLBV1 originated from leishbunyaviruses of monoxenous trypanosomatids and, probably, is a result of genomic re-assortment. The LmarLBV1 facilitates parasites' infectivity in vitro in primary murine macrophages model. The discovery of a virus in L.martiniquensis poses the question of whether it influences pathogenicity of this parasite in vivo, similarly to the LRV in other Leishmania species.

Leishmania RNA virus exacerbates Leishmaniasis by subverting innate immunity via TLR3-mediated NLRP3 inflammasome inhibition.

Leishmania RNA virus (LRV) is an important virulence factor associated with the development of mucocutaneous Leishmaniasis, a severe form of the disease. LRV-mediated disease exacerbation relies on TLR3 activation, but downstream mechanisms remain largely unexplored. Here, we combine human and mouse data to demonstrate that LRV triggers TLR3 and TRIF to induce type I IFN production, which induces autophagy. This process results in ATG5-mediated degradation of NLRP3 and ASC, thereby limiting NLRP3 inflammasome activation in macrophages. Consistent with the known restricting role of NLRP3 for Leishmania replication, the signaling pathway triggered by LRV results in increased parasite survival and disease progression. In support of this data, we find that lesions in patients infected with LRV+ Leishmania are associated with reduced inflammasome activation and the development of mucocutaneous disease. Our findings reveal the mechanisms triggered by LRV that contribute to the development of the debilitating mucocutaneous form of Leishmaniasis.

Global status of synchronizing Leishmania RNA virus in Leishmania parasites: A systematic review with meta-analysis.

Leishmaniasis is one of the most neglected tropical diseases caused by protozoan parasites belonging to the genus Leishmania. There is much evidence regarding prevalence of Leishmania RNAvirus (LRV) causing Old World leishmaniasis (OWL) and New World leishmaniasis (NWL); however, a combined evidence-based knowledge on this topic is not still available. The purpose of this systematic review and meta-analysis was to address the global status of synchronizing LRV in Leishmania in the available literature. The data were systematically collected from the English electronic databases up to May 2018. Then, the studies were screened based on the inclusion and exclusion criteria. The random-effect model was used by forest plot with 95% confidence interval (CI). Overall, 877 samples from 17 articles were included in this study. Given species of Leishmania, the highest prevalence of LRV belonged to Leishmania (L.) Viannia (V.) guyanensis and L. V. braziliensis. Additionally, the virus was detected also in L. V. amazonensis, L. V. panamanensis, L. V. lainsoni, L. aethiopica, L. major and L. infantum. By random-effect model, the global prevalence of LRV was estimated to be 26.2% (95% CI: 14.4% - 40.1%). The high prevalence of LRV among causative agents of NWLisolated from the metastatic clinical forms suggests potential association of LRV with metastatic clinical forms in New World endemic regions. A comprehensive investigation on experimental and clinical aspects of LRV is needed to fully appraise the role of these viruses in pathogenicity of Leishmania parasites and their drug resistance.

First report of treatment failure in a patient with cutaneous leishmaniasis infected by Leishmania (Viannia) naiffi carrying Leishmania RNA virus: a fortuitous combination?

Abstract We report the case of a 32-year-old man from Rio de Janeiro, who was infected in the Amazon region of Brazil by Leishmania (Viannia) naiffi. Generally, patients with L. naiffi cutaneous leishmaniasis exhibit a good therapeutic response to either pentavalent antimonials or pentamidine. However, after pentamidine treatment, this patient's infection evolved to therapeutic failure. To understand this clinical outcome, we investigated the presence of the Leishmania RNA virus (LRV) in parasites isolated from the cutaneous lesion; herein, we discuss the possible association between a poor response to pentamidine therapy and the presence of the LRV.

Correlation between presence of Leishmania RNA virus 1 and clinical characteristics of nasal mucosal leishmaniosis / Correlação entre a presença de Leishmania RNA Vírus 1 e as características clínicas da leishmaniose de mucosa nasal

ABSTRACT INTRODUCTION: Mucosal 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. OBJECTIVE: To study the existence of clinical differences between patients with ML with endosymbiosis by LRV1 and. those without it. METHODS: A 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. RESULTS: Out 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. CONCLUSION: Correlation 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.

RESUMO Introdução: A leishmaniose de mucosa (LM) é uma forma clínica grave da leishmaniose. Fatores complexos ligados ao parasita e ao hospedeiro são atribuídos ao desenvolvimento das lesões de mucosa. Leishmania RNA Vírus 1 (LRV1) pode subverter a resposta imune, podendo ser o principal determinante da gravidade da doença e deve ser pesquisado. Objetivo: Estudar a existência de diferenças clínicas entre pacientes portadores de LM com endosimbiose por LRV1 e as que não possuem. Métodos: Foi realizado um estudo de coorte histórica com corte transversal com avaliação clínica, detecção da Leishmania por técnica de PCR, classificação da espécie e pesquisa de LRV1. Foram incluídos na análise da pesquisa somente os pacientes com diagnóstico confirmado de LM com PCR positivo, com lesão de mucosa nasal. Resultados: Dos 37 pacientes, 30 (81,1%) foram diagnosticados com L. braziliensis, 5 (13,5%) com L. guyanensis e 2 (5,4%) com infecção mista de L. braziliensis e L. guyanensis. O vírus LVR1 estava presente em 26 casos (70,3%). Conclusão: A correlação entre o fenótipo clínico e a presença do LRV1 não foi constatada, porém a frequência do vírus é duas vezes maior em lesão de mucosa do que encontrado em trabalho, da mesma região, sobre lesão cutânea.

Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response.

BACKGROUND: Infection with Leishmania parasites causes mainly cutaneous lesions at the site of the sand fly bite. Inflammatory metastatic forms have been reported with Leishmania species such as L. braziliensis, guyanensis and aethiopica. Little is known about the factors underlying such exacerbated clinical presentations. Leishmania RNA virus (LRV) is mainly found within South American Leishmania braziliensis and guyanensis. In a mouse model of L. guyanensis infection, its presence is responsible for an hyper-inflammatory response driven by the recognition of the viral dsRNA genome by the host Toll-like Receptor 3 leading to an exacerbation of the disease. In one instance, LRV was reported outside of South America, namely in the L. major ASKH strain from Turkmenistan, suggesting that LRV appeared before the divergence of Leishmania subgenera. LRV presence inside Leishmania parasites could be one of the factors implicated in disease severity, providing rationale for LRV screening in L. aethiopica. METHODOLOGY/PRINCIPAL FINDINGS: A new LRV member was identified in four L. aethiopica strains (LRV-Lae). Three LRV-Lae genomes were sequenced and compared to L. guyanensis LRV1 and L. major LRV2. LRV-Lae more closely resembled LRV2. Despite their similar genomic organization, a notable difference was observed in the region where the capsid protein and viral polymerase open reading frames overlap, with a unique -1 situation in LRV-Lae. In vitro infection of murine macrophages showed that LRV-Lae induced a TLR3-dependent inflammatory response as previously observed for LRV1. CONCLUSIONS/SIGNIFICANCE: In this study, we report the presence of an immunogenic dsRNA virus in L. aethiopica human isolates. This is the first observation of LRV in Africa, and together with the unique description of LRV2 in Turkmenistan, it confirmed that LRV was present before the divergence of the L. (Leishmania) and (Viannia) subgenera. The potential implication of LRV-Lae on disease severity due to L. aethiopica infections is discussed.

The therapeutic potential of immune cross-talk in leishmaniasis.

Veterans of infection, Leishmania parasites have been plaguing mammals for centuries, causing a morbidity toll second only to that of malaria as the most devastating protozoan parasitic disease in the world. Cutaneous leishmaniasis (CL) is, by far, the most prevalent form of the disease, with symptoms ranging from a single self-healing lesion to chronic metastatic leishmaniasis (ML). In an increasingly immunocompromised population, complicated CL is becoming a more likely outcome, characterized by severely inflamed, destructive lesions that are often refractory to current treatment. This is perhaps because our ageing arsenal of variably effective antileishmanial drugs may be directly or indirectly immunomodulatory and may thus have variable effects in each type and stage of CL. Indeed, widely differing immune biases are created by the various species of Leishmania, and these immunological watersheds are further shifted by extrinsic disturbances in immune homeostasis. For example, we recently showed that a naturally occurring RNA virus (Leishmania RNA virus (LRV)) within some Leishmania parasites creates hyperinflammatory cross-talk, which can predispose to ML: a case of immunological misfire that may require a different approach to immunotherapy, whereby treatments are tailored to underlying immune biases. Understanding the intersecting immune pathways of leishmaniasis and its co-infections will enable us to identify new drug targets, and thereby design therapeutic strategies that work by untangling the immunological cross-wires of pathogenic cross-talk.

Detection of Leishmania RNA virus in Leishmania parasites.

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. METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.

Desorganização da polpa branca esplênica está associada com a apresentação clínica mais grave da leishmaniose visceral em cães naturalmente infectados / Disorganization of the splenic white pulp is associated with more severe clinical presentation of visceral leishmaniasis in naturally infected dogs

O baço é o maior órgão linfoide secundário em seres humanos e em cães. Em ambos, a ausência do baço está associada com um risco aumentado de ocorrência de infecções localizadas e disseminadas, incluindo sepse generalizada. A leishmaniose visceral e outras infecções podem alterar a estrutura histológica do baço, o que leva a uma destruição dos microambientes da polpa branca. Trabalhos anteriores mostraram que a uptura da estrutura de polpa branca é mais frequente em cães com marcadores laboratoriais de suscetibilidade à leishmaniose visceral, como a cultura esplênica positiva e LST negativo, do que nos animais em que estes marcadores de susceptibilidade estavam ausentes. Neste estudo, o nosso objetivo é examinar a relação entre a desorganização histológica da polpa branca esplênica e a gravidade da leishmaniose visceral. As amostras e os dados utilizados neste estudo foram coletados de 206 cães de rua provenientes de uma área endêmica para leishmaniose visceral, a cidade de Jequié (Bahia, Brasil). Os animais foram examinados clinicamente e foram realizados os testes ELISA e LST. Aspirados de baço foram coletados para a cultura, e fragmentos de baço foram coletadas para estudos de biologia molecular e de estudos morfológicos. Os animais foram classificados de acordo com o grau de organização estrutural da polpa branca esplênica em grupos com baço (a), bem organizado, (b) ligeiramente desorganizado, (c) a moderadamente a extensivamente desorganizado. Em relação à positividade no ELISA juntamente com a desorganização do baço, conjuntivite ( P= 0,0116), hiperproteinemia (p= 0,021) foram mais frequentes no grupo de animais com ELISA positivo e baço desorganizado, quando comparado com os outros grupos. Os animais polissintomáticos são mais frequentes no grupo com ELISA positivo e baço do tipo 3 (p= 0,004). Os scores clínicos atribuídos à intensidade da conjuntivite (P <0,05), dermatite (P <0,05) e linfadenopatia (P <0,01), alopecia (P <0,01), onicogrifose (P <0,05) foram mais elevados nos animais com ELISA positivo e baço desorganizado do que outros grupos, bem como o número de sinais clínicos atribuíveis à leishmaniose visceral canina (p= 0,0014).Em relação à análise da positividade na cultura esplênica juntamente com a desorganização do baço, a frequência de cães polissintomáticos foi maior em animais com baço ligeiramente desorganizado (P <0,05) e baço desorganizado (P <0,005), do que em animais com baço organizado. Alopecia (P <0,01), conjuntivite (P <0,05), desidratação (P <0,001), dermatite (P <0,05), onicogrifose (p <0,01), anemia (P <0,05), úlcera (P <0,001) e alto escore clínico (P <0,001) foram mais frequentes em animais com cultura esplênica positiva e baço desorganizado, do que nos animais com cultura negativa e baço organizado. Em conclusão, os cães com desorganização do baço associada com leishmaniose visceral têm mais sinais clínicos e pior estado clínico do que os animais com leishmaniose visceral, mas sem desorganização do baço.

Leishmania RNA virus: when the host pays the toll.

The presence of an RNA virus in a South American subgenus of the Leishmania parasite, L. (Viannia), was detected several decades ago but its role in leishmanial virulence and metastasis was only recently described. In Leishmania guyanensis, the nucleic acid of Leishmania RNA virus (LRV1) acts as a potent innate immunogen, eliciting a hyper-inflammatory immune response through toll-like receptor 3 (TLR3). The resultant inflammatory cascade has been shown to increase disease severity, parasite persistence, and perhaps even resistance to anti-leishmanial drugs. Curiously, LRVs were found mostly in clinical isolates prone to infectious metastasis in both their human source and experimental animal model, suggesting an association between the viral hyperpathogen and metastatic complications such as mucocutaneous leishmaniasis (MCL). MCL presents as chronic secondary lesions in the mucosa of the mouth and nose, debilitatingly inflamed and notoriously refractory to treatment. Immunologically, this outcome has many of the same hallmarks associated with the reaction to LRV: production of type 1 interferons, bias toward a chronic Th1 inflammatory state and an impaired ability of host cells to eliminate parasites through oxidative stress. More intriguing, is that the risk of developing MCL is found almost exclusively in infections of the L. (Viannia) subtype, further indication that leishmanial metastasis is caused, at least in part, by a parasitic component. LRV present in this subgenus may contribute to the destructive inflammation of metastatic disease either by acting in concert with other intrinsic "metastatic factors" or by independently preying on host TLR3 hypersensitivity. Because LRV amplifies parasite virulence, its presence may provide a unique target for diagnostic and clinical intervention of metastatic leishmaniasis. Taking examples from other members of the Totiviridae virus family, this paper reviews the benefits and costs of endosymbiosis, specifically for the maintenance of LRV infection in Leishmania parasites, which is often at the expense of its human host.

Presença de formas amastigotas de leishmania chagasi e perfil leucocitário no aparelho reprodutivo de cães / Presence of amastigotes forms the leishmania chagasi and profile the leucocytes cells in the reproductive tract of dogs

A leishmaniose visceral (LV) é uma zoonose causada pelo protozoário Leishmania (Leishmania) chagasi. A leishmaniose visceral canina (LVC) é a doença de maior relevância zoonótica. Usualmente, a infecção ocorre entre um hospedeiro invertebrado para um hospedeiro vertebrado, entretanto, a transmissão na ausência do vetor já é conhecida. O objetivo principal deste estudo foi identificar a presença de formas amastigotas, quantificar as células leucocitárias, estimar o risco relativo da presença de formas amastigotas no aparelho reprodutivo de cães sorologicamente positivos com e sem sinais clínicos. Para isso, foram utilizados cães sem raça definida, sexualmente maduros e testados sorologicamente para LVC (com sinais clínicos, n=25; sem sinais clínicos, n=25), que após eutanásia, tiveram fragmentos de testículo, epidídimo (cabeça, corpo e cauda) e glândula prostática (selecionados ao acaso) impressos em lâminas. Um grupo de 20 cãs sorologicamente negativos e sem sinais clínicos foi usado como controle. Amostras do baço foram incluídas com controle parasitológico positivo. O percentual de linfócitos foi superior (P<0,05) no corpo e cauda do epidídimo, assim como no testículo. Macrófagos foram superiores (P<0,05) apenas nas regiões do corpo e cauda epididimais. A presença de amastigotas correlacionou-se entre as distintas regiões do aparelho reprodutivo. Nos sintomáticos variaram entre 0,50 a 0,80 e entre 0,79 a 0,95 nos assintomáticos. A presença de amastigotas no testículo dos cães sintomáticos foi 6,5 vezes superior aos cães assintomáticos. Os resultados obtidos demonstram o potencial epidemiológico da transmissão venérea da doença, principalmente em áreas onde os programas de controle da LVC não consideram esta forma de transmissão, que pode ser importante em populações caninas não esterilizadas.

Visceral leishmaniasis (VL) is a zoonosis caused by Leishmania (Leishmania) chagasi. Canine visceral leishmaniasis (VLC) is most important. The infection occurs usually between the invertebrate host and vertebrate host; however, transmission in the absence of the vector has been reported. The aim of this study was to identify the presence of amastigote forms, quantify the leucocyte cells and to estimate the presence (odds ratio) of the amastigotes in the reproductive tract of dogs serologically positive with and without clinical signs. Sexually mature Mongrel dogs, serologically tested to VLC (symptomatic, n=25; asymptomatic, n=25), were used. After euthanasia, testes, epidydimal (caput, corpus and cauda) and prostate gland fragments (randomized) were recovered and impressed on slides. Twenty animals serologically negative and asymptomatic were used as control group. Samples of spleen were included as parasitological positive controls. Lymphocyte percentages were higher (P<0.05) in the corpus and caudal region of epididymis, similar to the testes in the symptomatic group. Macrophage percentage was higher (P<0.05) in the corpus and caudal epididymis regions. The presence of amastigote forms was associated with different regions of the reproductive tract. In the symptomatic group, the variation was between 0.50 and 0.80, and in the symptomatic between 0.79 and 0.95. The odds ratio for amastigote forms in the testicle of the symptomatic dogs was 6.5 in relation to asymptomatic dogs. The results demonstrate the epidemic potential of venereal transmission of the disease, specifically in areas where control programs of VLC do not consider this transmission route.

Identification of a ribosomal frameshift in Leishmania RNA virus 1-4.

Double-stranded Leishmania RNA virus 1-4 (LRV 1-4) has at least four open reading frames (ORFs). The two small ORFs located near its 5' terminus, ORF1 and ORFx, could encode 34- and 60-amino acid polypeptides, respectively. ORF2 encodes an 82-kDa major capsid protein, and ORF3 encodes a 98-kDa polypeptide which contains the consensus sequence for RNA-dependent RNA polymerases of plus-strand and double-stranded RNA viruses. The complete sequence of LRV 1-4 shows that ORF2 and ORF3 overlap by 71 nucleotides, and that ORF3 lacks a potential translation initiation site, suggesting that the viral polymerase may be synthesized as a 180-kDa fusion protein with the virus capsid. In this report, we present evidence for the synthesis of a fusion protein through a ribosomal frameshift. In vitro-translation experiments and immunostudies involving antiserum against the viral capsid protein demonstrated that the overlapping 71 nucleotides of ORF2 and ORF3 are contained in a region which promotes translational frameshifting. Computer analysis of the putative frameshift region revealed a potential pseudoknot structure located within the overlapping 71 nucleotide sequence.

Leishmania RNA viruses in Leishmania of the Viannia subgenus.

Karyotype analysis of 69 strains of Leishmania belonging to three species of the Viannia subgenus originating from the southeastern and southwestern regions of Colombia revealed approximately 5.3-kb RNAs in four strains of L. braziliensis and also in the World Health Organization reference strain L. guyanensis IWHI/BR/78/M5313. The RNA element in this reference strain and in L. braziliensis strains isolated from cutaneous and mucosal lesions of four patients hybridized with RNA probes prepared from cDNA of the RNA virus present in L. guyanensis strain CUMC-1-1A (LRV1-1). These strains also contained an 80-kD protein that reacted with polyclonal antibody prepared against a recombinant fragment of the coat (capsid) protein of LRV1-1. In addition, another Colombian strain of L. braziliensis was found to contain an approximately 3.5-kb RNA that did not hybridize with LRV1-1 probes. Contrasting with the strains containing the 5.3-kb RNA, a total lysate of this strain did not contain material reactive with antiserum to the capsid protein fragment. All Leishmania containing LRV1-related viruses identified to date have originated in the Amazon River basin. Karyotype analyses and biological characterization of 17 clones obtained from the highly metastatic L. guyanensis strain 5313 revealed retention of the approximately 5.3 kb RNA in all clones and no segregation of the virus with the metastatic trait. The restricted distribution of LRV1-related viruses among some strains of L. braziliensis and L. guyanensis circulating in the Amazon River basin makes these elements potential epidemiologic markers.

Monoclonal antibodies for the identification of new world Leishmania species

Monoclonal antibodies specific for selected species complexes of Leishmania have been employed for the characterization of several representative strains of Leishmania isolated from different hosts and localities in the Americas. In the past 15 years, data have been accumulated concerning (i) the specificities of a number of these monoclonal antibodies and (ii) the antigenic variation (level of the expressed antigenic determinants) ocurring among New World Leishmania species or strain variants as recognized by the monoclonal antibodies. This report is an attempt to summarize in brief the data accumulated to date on these points and to indicate the directions for future applications of these specific monoclonal antibodies for identification of leishmanial isolates.