Identification of diverse RNA viruses in Obscuromonas flagellates (Euglenozoa: Trypanosomatidae: Blastocrithidiinae).

Trypanosomatids (Euglenozoa) are a diverse group of unicellular flagellates predominately infecting insects (monoxenous species) or circulating between insects and vertebrates or plants (dixenous species). Monoxenous trypanosomatids harbor a wide range of RNA viruses belonging to the families Narnaviridae, Totiviridae, Qinviridae, Leishbuviridae, and a putative group of tombus-like viruses. Here, we focus on the subfamily Blastocrithidiinae, a previously unexplored divergent group of monoxenous trypanosomatids comprising two related genera: Obscuromonas and Blastocrithidia. Members of the genus Blastocrithidia employ a unique genetic code, in which all three stop codons are repurposed to encode amino acids, with TAA also used to terminate translation. Obscuromonas isolates studied here bear viruses of three families: Narnaviridae, Qinviridae, and Mitoviridae. The latter viral group is documented in trypanosomatid flagellates for the first time. While other known mitoviruses replicate in the mitochondria, those of trypanosomatids appear to reside in the cytoplasm. Although no RNA viruses were detected in Blastocrithidia spp., we identified an endogenous viral element in the genome of B. triatomae indicating its past encounter(s) with tombus-like viruses.

RNA viruses of Crithidia bombi, a parasite of bumblebees.

Leishbuviridae (Bunyavirales) are a diverse monophyletic group of negative-sense single-stranded RNA virus infecting parasitic flagellates of the family Trypanosomatidae. The presence of RNA viruses in trypanosomatids can influence the virulence of the latter. Here, we performed a screening for viruses in Crithidia bombi - a common parasite of important pollinators Bombus spp. (bumblebees) that negatively affects its host in stressful conditions. The majority (8/10) of C. bombi isolates collected in Europe and North America were positive for a virus that we named Crithidia bombi leishbuvirus 1 with high conservation of amino acid sequences between isolates. The results of our comparative phylogenetic analyses of the trypanosomatids and their viruses suggest that the high mobility of bumblebees and frequent coinfections by different strains of C. bombi determine an extensive viral exchange between the latter.

Diversity of RNA viruses in the cosmopolitan monoxenous trypanosomatid Leptomonas pyrrhocoris.

BACKGROUND: Trypanosomatids are parasitic flagellates well known because of some representatives infecting humans, domestic animals, and cultural plants. Many trypanosomatid species bear RNA viruses, which, in the case of human pathogens Leishmania spp., influence the course of the disease. One of the close relatives of leishmaniae, Leptomonas pyrrhocoris, has been previously shown to harbor viruses of the groups not documented in other trypanosomatids. At the same time, this species has a worldwide distribution and high prevalence in the natural populations of its cosmopolitan firebug host. It therefore represents an attractive model to study the diversity of RNA viruses. RESULTS: We surveyed 106 axenic cultures of L. pyrrhocoris and found that 64 (60%) of these displayed 2-12 double-stranded RNA fragments. The analysis of next-generation sequencing data revealed four viral groups with seven species, of which up to five were simultaneously detected in a single trypanosomatid isolate. Only two of these species, a tombus-like virus and an Ostravirus, were earlier documented in L. pyrrhocoris. In addition, there were four new species of Leishbuviridae, the family encompassing trypanosomatid-specific viruses, and a new species of Qinviridae, the family previously known only from metatranscriptomes of invertebrates. Currently, this is the only qinvirus with an unambiguously determined host. Our phylogenetic inferences suggest reassortment in the tombus-like virus owing to the interaction of different trypanosomatid strains. Two of the new Leishbuviridae members branch early on the phylogenetic tree of this family and display intermediate stages of genomic segment reduction between insect Phenuiviridae and crown Leishbuviridae. CONCLUSIONS: The unprecedented wide range of viruses in one protist species and the simultaneous presence of up to five viral species in a single Leptomonas pyrrhocoris isolate indicate the uniqueness of this flagellate. This is likely determined by the peculiarity of its firebug host, a highly abundant cosmopolitan species with several habits ensuring wide distribution and profuseness of L. pyrrhocoris, as well as its exposure to a wider spectrum of viruses compared to other trypanosomatids combined with a limited ability to transmit these viruses to its relatives. Thus, L. pyrrhocoris represents a suitable model to study the adoption of new viruses and their relationships with a protist host.

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.

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.

Evolution of RNA viruses in trypanosomatids: new insights from the analysis of Sauroleishmania.

RNA viruses play an important role in Leishmania biology and virulence. Their presence was documented in three (out of four) Leishmania subgenera. Sauroleishmania of reptiles remained the only underinvestigated group. In this work, we analyzed the viral occurrence in Sauroleishmania spp. and detected RNA viruses in three out of seven isolates under study. These viruses were of two families-Narnaviridae and Totiviridae. Phylogenetic inferences demonstrated that totiviruses from L. adleri and L. tarentolae group together within a larger cluster of LRV2s, while a narnavirus of L. gymnodactyli appeared as a phylogenetic relative of narnaviruses of Blechomonas spp. Taken together, our work not only expanded the range of trypanosomatids that can host RNA viruses but also shed new light on the evolution and potential routes of viral transmission in these flagellates.

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

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.

Elimination of LRVs Elicits Different Responses in Leishmania spp.

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.