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.

Department of Biochemistry and Molecular Biology Institute of Biosciences Vilnius Universitygrid 6441 7 Vilnius Lithuania

Department of Pathophysiology and Allergy Research Center of Pathophysiology Infectiology and Immunology Medical University of Vienna Vienna Austria

Faculty of Science University of South Bohemia České Budějovice Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice Czech Republic

Laboratory of Genetics Institute of Botany Nature Research Centre Vilnius Lithuania

Life Science Research Centre Faculty of Science University of Ostravagrid 412684 d Ostrava Czech Republic

National Center for Biotechnology Informationgrid 419234 9 National Library of Medicine National Institutes of Health Bethesda Maryland USA

Zobrazit více v PubMed

WHO. 2020. Leishmaniasis. https://www.who.int/en/news-room/fact-sheets/detail/leishmaniasis. Accessed July 02, 2022.

Bruschi F, Gradoni L. 2018. The leishmaniases: old neglected tropical diseases. Springer, Cham, Switzerland. doi:10.1007/978-3-319-72386-0. DOI

Kaye P, Scott P. 2011. Leishmaniasis: complexity at the host-pathogen interface. Nat Rev Microbiol 9:604–615. doi:10.1038/nrmicro2608. PubMed DOI

Ghabrial SA, Castón JR, Jiang D, Nibert ML, Suzuki N. 2015. 50-plus years of fungal viruses. Virology 479-480:356–368. doi:10.1016/j.virol.2015.02.034. PubMed DOI

Poulos BT, Tang KF, Pantoja CR, Bonami JR, Lightner DV. 2006. Purification and characterization of infectious myonecrosis virus of penaeid shrimp. J Gen Virol 87:987–996. doi:10.1099/vir.0.81127-0. PubMed DOI

Zhai Y, Attoui H, Mohd Jaafar F, Wang HQ, Cao YX, Fan SP, Sun YX, Liu LD, Mertens PP, Meng WS, Wang D, Liang G. 2010. Isolation and full-length sequence analysis of Armigeres subalbatus totivirus, the first totivirus isolate from mosquitoes representing a proposed novel genus (Artivirus) of the family Totiviridae. J Gen Virol 91:2836–2845. doi:10.1099/vir.0.024794-0. PubMed DOI

Løvoll M, Wiik-Nielsen J, Grove S, Wiik-Nielsen CR, Kristoffersen AB, Faller R, Poppe T, Jung J, Pedamallu CS, Nederbragt AJ, Meyerson M, Rimstad E, Tengs T. 2010. A novel totivirus and piscine reovirus (PRV) in Atlantic salmon (Salmo salar) with cardiomyopathy syndrome (CMS). Virol J 7:309. doi:10.1186/1743-422X-7-309. PubMed DOI PMC

Xin C, Wu B, Li J, Gong P, Yang J, Li H, Cai X, Zhang X. 2016. Complete genome sequence and evolution analysis of Eimeria stiedai RNA virus 1, a novel member of the family Totiviridae. Arch Virol 161:3571–3576. doi:10.1007/s00705-016-3020-7. PubMed DOI

Janssen ME, Takagi Y, Parent KN, Cardone G, Nibert ML, Baker TS. 2015. Three-dimensional structure of a protozoal double-stranded RNA virus that infects the enteric pathogen Giardia lamblia. J Virol 89:1182–1194. doi:10.1128/JVI.02745-14. PubMed DOI PMC

Grybchuk D, Kostygov AY, Macedo DH, d'Avila-Levy CM, Yurchenko V. 2018. RNA viruses in trypanosomatid parasites: a historical overview. Mem Inst Oswaldo Cruz 113:e170487. PubMed PMC

Wang AL, Wang CC. 1986. Discovery of a specific double-stranded RNA virus in Giardia lamblia. Mol Biochem Parasitol 21:269–276. doi:10.1016/0166-6851(86)90132-5. PubMed DOI

Hartley MA, Ronet C, Zangger H, Beverley SM, Fasel N. 2012. Leishmania RNA virus: when the host pays the toll. Front Cell Infect Microbiol 2:99. doi:10.3389/fcimb.2012.00099. PubMed DOI PMC

Brettmann EA, Shaik JS, Zangger H, Lye LF, Kuhlmann FM, Akopyants NS, Oschwald DM, Owens KL, Hickerson SM, Ronet C, Fasel N, Beverley SM. 2016. Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response. Proc Natl Acad Sci USA 113:11998–12005. doi:10.1073/pnas.1615085113. PubMed DOI PMC

Cantanhêde LM, Mata-Somarribas C, Chourabi K, Pereira da Silva G, Dias das Chagas B, de Oliveira RPL, Cortes Boite M, Cupolillo E. 2021. The maze pathway of coevolution: a critical review over the Leishmania and its endosymbiotic history. Genes 12:657. doi:10.3390/genes12050657. PubMed DOI PMC

Grybchuk D, Kostygov AY, Macedo DH, Votypka J, Lukes J, Yurchenko V. 2018. RNA viruses in Blechomonas (Trypanosomatidae) and evolution of Leishmaniavirus. mBio 9:e01932-18. doi:10.1128/mBio.01932-18. PubMed DOI PMC

Ives A, Ronet C, Prevel F, Ruzzante G, Fuertes-Marraco S, Schutz F, Zangger H, Revaz-Breton M, Lye LF, Hickerson SM, Beverley SM, Acha-Orbea H, Launois P, Fasel N, Masina S. 2011. Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis. Science 331:775–778. doi:10.1126/science.1199326. PubMed DOI PMC

Rossi M, Castiglioni P, Hartley MA, Eren RO, Prevel F, Desponds C, Utzschneider DT, Zehn D, Cusi MG, Kuhlmann FM, Beverley SM, Ronet C, Fasel N. 2017. Type I interferons induced by endogenous or exogenous viral infections promote metastasis and relapse of leishmaniasis. Proc Natl Acad Sci USA 114:4987–4992. doi:10.1073/pnas.1621447114. PubMed DOI PMC

Hartley MA, Bourreau E, Rossi M, Castiglioni P, Eren RO, Prevel F, Couppie P, Hickerson SM, Launois P, Beverley SM, Ronet C, Fasel N. 2016. Leishmaniavirus-dependent metastatic leishmaniasis is prevented by blocking IL-17A. PLoS Pathog 12:e1005852. doi:10.1371/journal.ppat.1005852. PubMed DOI PMC

Barrow P, Dujardin JC, Fasel N, Greenwood AD, Osterrieder K, Lomonossoff G, Fiori PL, Atterbury R, Rossi M, Lalle M. 2020. Viruses of protozoan parasites and viral therapy: is the time now right? Virol J 17:142. doi:10.1186/s12985-020-01410-1. PubMed DOI PMC

Widmer G, Dooley S. 1995. Phylogenetic analysis of Leishmania RNA virus and Leishmania suggests ancient virus-parasite association. Nucleic Acids Res 23:2300–2304. doi:10.1093/nar/23.12.2300. PubMed DOI PMC

Kostygov AY, Grybchuk D, Kleschenko Y, Chistyakov DS, Lukashev AN, Gerasimov ES, Yurchenko V. 2021. Analyses of Leishmania-LRV co-phylogenetic patterns and evolutionary variability of viral proteins. Viruses 13:2305. doi:10.3390/v13112305. PubMed DOI PMC

Scheffter S, Widmer G, Patterson JL. 1994. Complete sequence of Leishmania RNA virus 1–4 and identification of conserved sequences. Virology 199:479–483. doi:10.1006/viro.1994.1149. PubMed DOI

Scheffter SM, Ro YT, Chung IK, Patterson JL. 1995. The complete sequence of Leishmania RNA virus LRV2-1, a virus of an Old World parasite strain. Virology 212:84–90. doi:10.1006/viro.1995.1456. PubMed DOI

Adaui V, Lye LF, Akopyants NS, Zimic M, Llanos-Cuentas A, Garcia L, Maes I, De Doncker S, Dobson DE, Arevalo J, Dujardin JC, Beverley SM. 2016. Association of the endobiont double-stranded RNA virus LRV1 with treatment failure for human leishmaniasis caused by Leishmania braziliensis in Peru and Bolivia. J Infect Dis 213:112–121. doi:10.1093/infdis/jiv354. PubMed DOI PMC

Abtahi M, Eslami G, Cavallero S, Vakili M, Hosseini SS, Ahmadian S, Boozhmehrani MJ, Khamesipour A. 2020. Relationship of Leishmania RNA Virus (LRV) and treatment failure in clinical isolates of Leishmania major. BMC Res Notes 13:126. doi:10.1186/s13104-020-04973-y. PubMed DOI PMC

Bourreau E, Ginouves M, Prevot G, Hartley MA, Gangneux JP, Robert-Gangneux F, Dufour J, Sainte-Marie D, Bertolotti A, Pratlong F, Martin R, Schutz F, Couppie P, Fasel N, Ronet C. 2016. Presence of Leishmania RNA Virus 1 in Leishmania guyanensis increases the risk of first-line treatment failure and symptomatic relapse. J Infect Dis 213:105–111. doi:10.1093/infdis/jiv355. PubMed DOI

Ro YT, Scheffter SM, Patterson JL. 1997. Hygromycin B resistance mediates elimination of Leishmania virus from persistently infected parasites. J Virol 71:8991–8998. doi:10.1128/JVI.71.12.8991-8998.1997. PubMed DOI PMC

Lye LF, Owens K, Shi H, Murta SM, Vieira AC, Turco SJ, Tschudi C, Ullu E, Beverley SM. 2010. Retention and loss of RNA interference pathways in trypanosomatid protozoans. PLoS Pathog 6:e1001161. doi:10.1371/journal.ppat.1001161. PubMed DOI PMC

Kuhlmann FM, Robinson JI, Bluemling GR, Ronet C, Fasel N, Beverley SM. 2017. Antiviral screening identifies adenosine analogs targeting the endogenous dsRNA Leishmania RNA virus 1 (LRV1) pathogenicity factor. Proc Natl Acad Sci USA 114:E811–E819. doi:10.1073/pnas.1619114114. PubMed DOI PMC

Robinson JI, Beverley SM. 2018. Concentration of 2'C-methyladenosine triphosphate by Leishmania guyanensis enables specific inhibition of Leishmania RNA virus 1 via its RNA polymerase. J Biol Chem 293:6460–6469. doi:10.1074/jbc.RA117.001515. PubMed DOI PMC

Widmer G. 1995. Suppression of Leishmania RNA virus replication by capsid protein overexpression. J Virol 69:4122–4126. doi:10.1128/JVI.69.7.4122-4126.1995. PubMed DOI PMC

Herskowitz I. 1987. Functional inactivation of genes by dominant negative mutations. Nature 329:219–222. doi:10.1038/329219a0. PubMed DOI

Castiglioni P, Hartley MA, Rossi M, Prevel F, Desponds C, Utzschneider DT, Eren RO, Zangger H, Brunner L, Collin N, Zehn D, Kuhlmann FM, Beverley SM, Fasel N, Ronet C. 2017. Exacerbated leishmaniasis caused by a viral endosymbiont can be prevented by immunization with its viral capsid. PLoS Negl Trop Dis 11:e0005240. doi:10.1371/journal.pntd.0005240. PubMed DOI PMC

Lukša J, Ravoitytė B, Konovalovas A, Aitmanaitė L, Butenko A, Yurchenko V, Serva S, Servienė E. 2017. Different metabolic pathways are involved in response of Saccharomyces cerevisiae to L-A and M viruses. Toxins 9:e233. doi:10.3390/toxins9080233. PubMed DOI PMC

Aitmanaitė L, Konovalovas A, Medvedevas P, Servienė E, Serva S. 2021. Specificity determination in Saccharomyces cerevisiae killer virus systems. Microorganisms 9:236. doi:10.3390/microorganisms9020236. PubMed DOI PMC

Yao W, Bruenn JA. 1995. Interference with replication of two double-stranded RNA viruses by production of N-terminal fragments of capsid polypeptides. Virology 214:215–221. doi:10.1006/viro.1995.9938. PubMed DOI

Zakharova A, Albanaz ATS, Opperdoes FR, Škodová-Sveráková I, Zagirova D, Saura A, Chmelová L, Gerasimov ES, Leštinová T, Bečvář T, Sádlová J, Volf P, Lukeš J, Horváth A, Butenko A, Yurchenko V. 2022. Leishmania guyanensis M4147 as a new LRV1-bearing model parasite: phosphatidate phosphatase 2-like protein controls cell cycle progression and intracellular lipid content. PLoS Negl Trop Dis 16:e0010510. doi:10.1371/journal.pntd.0010510. PubMed DOI PMC

Ishemgulova A, Kraeva N, Hlaváčová J, Zimmer SL, Butenko A, Podešvová L, Leštinová T, Lukeš J, Kostygov A, Votýpka J, Volf P, Yurchenko V. 2017. A putative ATP/GTP binding protein affects Leishmania mexicana growth in insect vectors and vertebrate hosts. PLoS Negl Trop Dis 11:e0005782. doi:10.1371/journal.pntd.0005782. PubMed DOI PMC

Eren RO, Reverte M, Rossi M, Hartley MA, Castiglioni P, Prevel F, Martin R, Desponds C, Lye LF, Drexler SK, Reith W, Beverley SM, Ronet C, Fasel N. 2016. Mammalian innate immune response to a Leishmania-resident RNA virus increases macrophage survival to promote parasite persistence. Cell Host Microbe 20:318–328. doi:10.1016/j.chom.2016.08.001. PubMed DOI PMC

Zangger H, Hailu A, Desponds C, Lye LF, Akopyants NS, Dobson DE, Ronet C, Ghalib H, Beverley SM, Fasel N. 2014. Leishmania aethiopica field isolates bearing an endosymbiontic dsRNA virus induce pro-inflammatory cytokine response. PLoS Negl Trop Dis 8:e2836. doi:10.1371/journal.pntd.0002836. PubMed DOI PMC

Dinman JD, Wickner RB. 1992. Ribosomal frameshifting efficiency and gag/gag-pol ratio are critical for yeast M1 double-stranded RNA virus propagation. J Virol 66:3669–3676. doi:10.1128/JVI.66.6.3669-3676.1992. PubMed DOI PMC

Procházková M, Füzik T, Grybchuk D, Falginella FL, Podešvová L, Yurchenko V, Vácha R, Plevka P. 2021. Capsid structure of Leishmania RNA Virus 1. J Virol 95:e01957-20. doi:10.1128/JVI.01957-20. PubMed DOI PMC

de Carvalho RVH, Lima-Junior DS, da Silva MVG, Dilucca M, Rodrigues TS, Horta CV, Silva ALN, da Silva PF, Frantz FG, Lorenzon LB, Souza MM, Almeida F, Cantanhede LM, Ferreira RGM, Cruz AK, Zamboni DS. 2019. Leishmania RNA virus exacerbates leishmaniasis by subverting innate immunity via TLR3-mediated NLRP3 inflammasome inhibition. Nat Commun 10:5273. doi:10.1038/s41467-019-13356-2. PubMed DOI PMC

Saberi R, Fakhar M, Hajjaran H, Ataei-Pirkooh A, Mohebali M, Taghipour N, Ziaei Hezarjaribi H, Moghadam Y, Bagheri A. 2020. Presence and diversity of Leishmania RNA virus in an old zoonotic cutaneous leishmaniasis focus, northeastern Iran: haplotype and phylogenetic based approach. Int J Infect Dis 101:6–13. doi:10.1016/j.ijid.2020.08.033. PubMed DOI

Widmer G, Comeau AM, Furlong DB, Wirth DF, Patterson JL. 1989. Characterization of a RNA virus from the parasite Leishmania. Proc Natl Acad Sci USA 86:5979–5982. doi:10.1073/pnas.86.15.5979. PubMed DOI PMC

Yurchenko V, Lukeš J, Xu X, Maslov DA. 2006. An integrated morphological and molecular approach to a new species description in the Trypanosomatidae: the case of Leptomonas podlipaevi n. sp., a parasite of Boisea rubrolineata (Hemiptera: Rhopalidae). J Eukaryotic Microbiology 53:103–111. doi:10.1111/j.1550-7408.2005.00078.x. PubMed DOI

Ishemgulova A, Hlaváčová J, Majerová K, Butenko A, Lukeš J, Votýpka J, Volf P, Yurchenko V. 2018. CRISPR/Cas9 in Leishmania mexicana: a case study of LmxBTN1. PLoS One 13:e0192723. doi:10.1371/journal.pone.0192723. PubMed DOI PMC

Lee SE, Suh JM, Scheffter S, Patterson JL, Chung IK. 1996. Identification of a ribosomal frameshift in Leishmania RNA virus 1–4. J Biochem 120:22–25. doi:10.1093/oxfordjournals.jbchem.a021387. PubMed DOI

Záhonová K, Hadariová L, Vacula R, Yurchenko V, Eliáš M, Krajčovič J, Vesteg M. 2014. A small portion of plastid transcripts is polyadenylated in the flagellate Euglena gracilis. FEBS Lett 588:783–788. doi:10.1016/j.febslet.2014.01.034. PubMed DOI

Basu R, Bhaumik S, Basu JM, Naskar K, De T, Roy S. 2005. Kinetoplastid membrane protein-11 DNA vaccination induces complete protection against both pentavalent antimonial-sensitive and -resistant strains of Leishmania donovani that correlates with inducible nitric oxide synthase activity and IL-4 generation: evidence for mixed Th1- and Th2-like responses in visceral leishmaniasis. J Immunol 174:7160–7171. doi:10.4049/jimmunol.174.11.7160. PubMed DOI

Kraeva N, Ishemgulova A, Lukeš J, Yurchenko V. 2014. Tetracycline-inducible gene expression system in Leishmania mexicana. Mol Biochem Parasitol 198:11–13. doi:10.1016/j.molbiopara.2014.11.002. PubMed DOI

Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. doi:10.1093/bioinformatics/btu170. PubMed DOI PMC

Langmead B, Salzberg SL. 2012. Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359. doi:10.1038/nmeth.1923. PubMed DOI PMC

Aslett M, Aurrecoechea C, Berriman M, Brestelli J, Brunk BP, Carrington M, Depledge DP, Fischer S, Gajria B, Gao X, Gardner MJ, Gingle A, Grant G, Harb OS, Heiges M, Hertz-Fowler C, Houston R, Innamorato F, Iodice J, Kissinger JC, Kraemer E, Li W, Logan FJ, Miller JA, Mitra S, Myler PJ, Nayak V, Pennington C, Phan I, Pinney DF, Ramasamy G, Rogers MB, Roos DS, Ross C, Sivam D, Smith DF, Srinivasamoorthy G, Stoeckert CJ, Jr., Subramanian S, Thibodeau R, Tivey A, Treatman C, Velarde G, Wang H. 2010. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res 38:D457–62. doi:10.1093/nar/gkp851. PubMed DOI PMC

Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842. doi:10.1093/bioinformatics/btq033. PubMed DOI PMC

Robinson MD, McCarthy DJ, Smyth GK. 2010. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140. doi:10.1093/bioinformatics/btp616. PubMed DOI PMC

Raudvere U, Kolberg L, Kuzmin I, Arak T, Adler P, Peterson H, Vilo J. 2019. g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res 47:W191–W198. doi:10.1093/nar/gkz369. PubMed DOI PMC

Szklarczyk D, Gable AL, Lyon D, Junge A, Wyder S, Huerta-Cepas J, Simonovic M, Doncheva NT, Morris JH, Bork P, Jensen LJ, Mering CV. 2019. STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res 47:D607–D613. doi:10.1093/nar/gky1131. PubMed DOI PMC

A novel strain of Leishmania braziliensis harbors not a toti- but a bunyavirus

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

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

Diversity of RNA viruses in the cosmopolitan monoxenous trypanosomatid Leptomonas pyrrhocoris

Revisiting epidemiology of leishmaniasis in central Asia: lessons learnt