BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. FINDINGS: We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS: To the best of our knowledge, this is the first detection of LRV2 in the New World.

• MeSH
• Leishmania infantum * genetika   MeSH
• leishmanióza viscerální * veterinární   MeSH
• lidé MeSH
• psi MeSH
• RNA-dependentní RNA-polymerasa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Brazílie MeSH
• Názvy látek
• RNA-dependentní RNA-polymerasa MeSH

Leishmaniaviruses (LRVs) have been demonstrated to enhance progression of leishmaniasis, a vector-transmitted disease with a wide range of clinical manifestations that is caused by flagellates of the genus Leishmania. Here, we used two previously proposed strategies of the LRV ablation to shed light on the relationships of two Leishmania spp. with their respective viral species (L. guyanensis, LRV1 and L. major, LRV2) and demonstrated considerable difference between two studied systems. LRV1 could be easily eliminated by the expression of exogenous capsids regardless of their origin (the same or distantly related LRV1 strains, or even LRV2), while LRV2 was only partially depleted in the case of the native capsid overexpression. The striking differences were also observed in the effects of complete viral elimination with 2'C-methyladenosine (2-CMA) on the transcriptional profiles of these two Leishmania spp. While virtually no differentially expressed genes were detected after the LRV1 removal from L. guyanensis, the response of L. major after ablation of LRV2 involved 87 genes, the analysis of which suggested a considerable stress experienced even after several passages following the treatment. This effect on L. major was also reflected in a significant decrease of the proliferation rate, not documented in L. guyanensis and naturally virus-free strain of L. major. Our findings suggest that integration of L. major with LRV2 is deeper compared with that of L. guyanensis with LRV1. We presume this determines different effects of the viral presence on the Leishmania spp. infections. IMPORTANCE Leishmania spp. represent human pathogens that cause leishmaniasis, a widespread parasitic disease with mild to fatal clinical manifestations. Some strains of leishmaniae bear leishmaniaviruses (LRVs), and this has been shown to aggravate disease course. We investigated the relationships of two distally related Leishmania spp. with their respective LRVs using different strategies of virus removal. Our results suggest the South American L. guyanensis easily loses its virus with no important consequences for the parasite in the laboratory culture. Conversely, the Old-World L. major is refractory to virus removal and experiences a prominent stress if this removal is nonetheless completed. The drastically different levels of integration between the studied Leishmania spp. and their viruses suggest distinct effects of the viral presence on infections in these species of parasites.

• Klíčová slova
• LRV1, LRV2, Leishmania guyanensis, Leishmania major, capsid,
• MeSH
• Leishmania * genetika   MeSH
• Leishmaniavirus * genetika   MeSH
• leishmanióza * parazitologie   MeSH
• lidé MeSH
• virové plášťové proteiny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• virové plášťové proteiny MeSH

Leishmania spp. are important pathogens causing a vector-borne disease with a broad range of clinical manifestations from self-healing ulcers to the life-threatening visceral forms. Presence of Leishmania RNA virus (LRV) confers survival advantage to these parasites by suppressing anti-leishmanial immunity in the vertebrate host. The two viral species, LRV1 and LRV2 infect species of the subgenera Viannia and Leishmania, respectively. In this work we investigated co-phylogenetic patterns of leishmaniae and their viruses on a small scale (LRV2 in L. major) and demonstrated their predominant coevolution, occasionally broken by intraspecific host switches. Our analysis of the two viral genes, encoding the capsid and RNA-dependent RNA polymerase (RDRP), revealed them to be under the pressure of purifying selection, which was considerably stronger for the former gene across the whole tree. The selective pressure also differs between the LRV clades and correlates with the frequency of interspecific host switches. In addition, using experimental (capsid) and predicted (RDRP) models we demonstrated that the evolutionary variability across the structure is strikingly different in these two viral proteins.

• Klíčová slova
• Leishmaniavirus, coevolution, phylogenomics,
• MeSH
• Leishmania virologie   MeSH
• leishmanióza virologie   MeSH
• lidé MeSH
• RNA virová analýza   MeSH
• RNA-dependentní RNA-polymerasa genetika   MeSH
• RNA-viry genetika   MeSH
• virové plášťové proteiny genetika   MeSH
• virové proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA virová MeSH
• RNA-dependentní RNA-polymerasa MeSH
• virové plášťové proteiny MeSH
• virové proteiny MeSH

Leishmania parasites cause a variety of symptoms, including mucocutaneous leishmaniasis, which results in the destruction of the mucous membranes of the nose, mouth, and throat. The species of Leishmania carrying Leishmania RNA virus 1 (LRV1), from the family Totiviridae, are more likely to cause severe disease and are less sensitive to treatment than those that do not contain the virus. Although the importance of LRV1 for the severity of leishmaniasis was discovered a long time ago, the structure of the virus remained unknown. Here, we present a cryo-electron microscopy reconstruction of the virus-like particle of LRV1 determined to a resolution of 3.65 Å. The capsid has icosahedral symmetry and is formed by 120 copies of a capsid protein assembled in asymmetric dimers. RNA genomes of viruses from the family Totiviridae are synthetized, but not capped at the 5' end, by virus RNA polymerases. To protect viral RNAs from degradation, capsid proteins of the L-A totivirus cleave the 5' caps of host mRNAs, creating decoys to overload the cellular RNA quality control system. Capsid proteins of LRV1 form positively charged clefts, which may be the cleavage sites for the 5' cap of Leishmania mRNAs. The putative RNA binding site of LRV1 is distinct from that of the related L-A virus. The structure of the LRV1 capsid enables the rational design of compounds targeting the putative decapping site. Such inhibitors may be developed into a treatment for mucocutaneous leishmaniasis caused by LRV1-positive species of LeishmaniaIMPORTANCE Twelve million people worldwide suffer from leishmaniasis, resulting in more than 30 thousand deaths annually. The disease has several variants that differ in their symptoms. The mucocutaneous form, which leads to disintegration of the nasal septum, lips, and palate, is caused predominantly by Leishmania parasites carrying Leishmania RNA virus 1 (LRV1). Here, we present the structure of the LRV1 capsid determined using cryo-electron microscopy. Capsid proteins of a related totivirus, L-A virus, protect viral RNAs from degradation by cleaving the 5' caps of host mRNAs. Capsid proteins of LRV1 may have the same function. We show that the LRV1 capsid contains positively charged clefts that may be sites for the cleavage of mRNAs of Leishmania cells. The structure of the LRV1 capsid enables the rational design of compounds targeting the putative mRNA cleavage site. Such inhibitors may be used as treatments for mucocutaneous leishmaniasis.

• Klíčová slova
• CAP-4, LRV1, Leishmania, RNA, Totiviridae, Viannia, capsid, cryo-electron microscopy, decapping, genome, leishmaniasis, mRNA, parasite, structure, uncoating, virion, virus,
• MeSH
• elektronová kryomikroskopie MeSH
• genom virový MeSH
• kapsida chemie   metabolismus   MeSH
• Leishmaniavirus chemie   genetika   metabolismus   MeSH
• RNA virová genetika   metabolismus   MeSH
• virové plášťové proteiny chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA virová MeSH
• virové plášťové proteiny MeSH

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.

• Klíčová slova
• Bunyavirales, Leishmania martiniquensis, leishbunyavirus,
• MeSH
• fylogeneze * MeSH
• genom virový * MeSH
• Leishmania patogenita   virologie   MeSH
• makrofágy parazitologie   MeSH
• myši MeSH
• otevřené čtecí rámce MeSH
• reassortantní viry MeSH
• RNA-dependentní RNA-polymerasa MeSH
• RNA-viry klasifikace   genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA-dependentní RNA-polymerasa MeSH

Here we report sequence and phylogenetic analysis of two new isolates of Leishmania RNA virus 2 (LRV2) found in Leishmania major isolated from human patients with cutaneous leishmaniasis in south Uzbekistan. These new virus-infected flagellates were isolated in the same region of Uzbekistan and the viral sequences differed by only nineteen SNPs, all except one being silent mutations. Therefore, we concluded that they belong to a single LRV2 species. New viruses are closely related to the LRV2-Lmj-ASKH documented in Turkmenistan in 1995, which is congruent with their shared host (L. major) and common geographical origin.

• Klíčová slova
• LRV2, Leishmania RNA virus, next-generation sequencing,
• MeSH
• fylogeneze MeSH
• genom virový * MeSH
• jednonukleotidový polymorfismus MeSH
• Leishmania major virologie   MeSH
• Leishmaniavirus klasifikace   genetika   patogenita   MeSH
• mutace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Uzbekistán MeSH

In this work, we analyzed viral prevalence in trypanosomatid parasites (Blechomonas spp.) infecting Siphonaptera and discovered nine species of viruses from three different groups (leishbunyaviruses, narnaviruses, and leishmaniaviruses). Most of the flagellate isolates bore two or three viral types (mixed infections). Although no new viral groups were documented in Blechomonas spp., our findings are important for the comprehension of viral evolution. The discovery of bunyaviruses in blechomonads was anticipated, since these viruses have envelopes facilitating their interspecific transmission and have already been found in various trypanosomatids and metatranscriptomes with trypanosomatid signatures. In this work, we also provided evidence that even representatives of the family Narnaviridae are capable of host switching and evidently have accomplished switches multiple times in the course of their evolution. The most unexpected finding was the presence of leishmaniaviruses, a group previously solely confined to the human pathogens Leishmania spp. From phylogenetic inferences and analyses of the life cycles of Leishmania and Blechomonas, we concluded that a common ancestor of leishmaniaviruses most likely infected Leishmania first and was acquired by Blechomonas by horizontal transfer. Our findings demonstrate that evolution of leishmaniaviruses is more complex than previously thought and includes occasional host switching.IMPORTANCE Flagellates belonging to the genus Leishmania are important human parasites. Some strains of different Leishmania species harbor viruses (leishmaniaviruses), which facilitate metastatic spread of the parasites, thus aggravating the disease. Up until now, these viruses were known to be hosted only by Leishmania Here, we analyzed viral distribution in Blechomonas, a related group of flagellates parasitizing fleas, and revealed that they also bear leishmaniaviruses. Our findings shed light on the entangled evolution of these viruses. In addition, we documented that Blechomonas can be also infected by leishbunyaviruses and narnaviruses, viral groups known from other insects' flagellates.

• Klíčová slova
• Blechomonas, Leishbunyaviridae, Leishmaniavirus, Narnaviridae,
• MeSH
• fylogeneze MeSH
• genetická variace MeSH
• genom virový MeSH
• Leishmaniavirus genetika   MeSH
• molekulární evoluce * MeSH
• RNA-viry klasifikace   izolace a purifikace   MeSH
• Siphonaptera parazitologie   MeSH
• Trypanosomatina virologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Viruses of trypanosomatids are now being extensively studied because of their diversity and the roles they play in flagellates' biology. Among the most prominent examples are leishmaniaviruses implicated in pathogenesis of Leishmania parasites. Here, we present a historical overview of this field, starting with early reports of virus-like particles on electron microphotographs, and culminating in detailed molecular descriptions of viruses obtained using modern next generation sequencing-based techniques. Because of their diversity, different life cycle strategies and host specificity, we believe that trypanosomatids are a fertile ground for further explorations to better understand viral evolution, routes of transitions, and molecular mechanisms of adaptation to different hosts.

• MeSH
• hostitelská specificita MeSH
• Leishmaniavirus fyziologie   MeSH
• RNA-viry fyziologie   MeSH
• transmisní elektronová mikroskopie MeSH
• Trypanosomatina virologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Knowledge of viral diversity is expanding greatly, but many lineages remain underexplored. We surveyed RNA viruses in 52 cultured monoxenous relatives of the human parasite Leishmania (Crithidia and Leptomonas), as well as plant-infecting PhytomonasLeptomonas pyrrhocoris was a hotbed for viral discovery, carrying a virus (Leptomonas pyrrhocoris ostravirus 1) with a highly divergent RNA-dependent RNA polymerase missed by conventional BLAST searches, an emergent clade of tombus-like viruses, and an example of viral endogenization. A deep-branching clade of trypanosomatid narnaviruses was found, notable as Leptomonas seymouri bearing Narna-like virus 1 (LepseyNLV1) have been reported in cultures recovered from patients with visceral leishmaniasis. A deep-branching trypanosomatid viral lineage showing strong affinities to bunyaviruses was termed "Leishbunyavirus" (LBV) and judged sufficiently distinct to warrant assignment within a proposed family termed "Leishbunyaviridae" Numerous relatives of trypanosomatid viruses were found in insect metatranscriptomic surveys, which likely arise from trypanosomatid microbiota. Despite extensive sampling we found no relatives of the totivirus Leishmaniavirus (LRV1/2), implying that it was acquired at about the same time the Leishmania became able to parasitize vertebrates. As viruses were found in over a quarter of isolates tested, many more are likely to be found in the >600 unsurveyed trypanosomatid species. Viral loss was occasionally observed in culture, providing potentially isogenic virus-free lines enabling studies probing the biological role of trypanosomatid viruses. These data shed important insights on the emergence of viruses within an important trypanosomatid clade relevant to human disease.

• Klíčová slova
• Bunyavirales, Trypanosomatidae, coevolution, coinfection, persistent virus infection,
• MeSH
• fylogeneze MeSH
• genetická variace MeSH
• hostitelská specificita MeSH
• infekce prvoky kmene Euglenozoa parazitologie   veterinární   MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• RNA-viry genetika   izolace a purifikace   MeSH
• Trypanosomatina virologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH

The co-infection cases involving dixenous Leishmania spp. (mostly of the L. donovani complex) and presumably monoxenous trypanosomatids in immunocompromised mammalian hosts including humans are well documented. The main opportunistic parasite has been identified as Leptomonas seymouri of the sub-family Leishmaniinae. The molecular mechanisms allowing a parasite of insects to withstand elevated temperature and substantially different conditions of vertebrate tissues are not understood. Here we demonstrate that L. seymouri is well adapted for the environment of the warm-blooded host. We sequenced the genome and compared the whole transcriptome profiles of this species cultivated at low and high temperatures (mimicking the vector and the vertebrate host, respectively) and identified genes and pathways differentially expressed under these experimental conditions. Moreover, Leptomonas seymouri was found to persist for several days in two species of Phlebotomus spp. implicated in Leishmania donovani transmission. Despite of all these adaptations, L. seymouri remains a predominantly monoxenous species not capable of infecting vertebrate cells under normal conditions.

• MeSH
• fyziologická adaptace fyziologie   MeSH
• infekce prvoky kmene Euglenozoa genetika   MeSH
• koinfekce mikrobiologie   MeSH
• Leishmania donovani MeSH
• leishmanióza viscerální parazitologie   MeSH
• modely nemocí na zvířatech MeSH
• polymerázová řetězová reakce MeSH
• protozoální geny MeSH
• Psychodidae mikrobiologie   MeSH
• stadia vývoje MeSH
• stanovení celkové genové exprese MeSH
• transkriptom MeSH
• Trypanosomatina genetika   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH