Over the last decade, considerable progress has been made in unraveling RNA virus diversity. This has contributed to our understanding of the evolution of these viruses, which include emerging zoonotic human pathogens. Current success has been greatly facilitated by the development of next-generation sequencing platforms instrumental for meta-transcriptomic studies. However, due to the rapid evolution of RNA viruses, there are numerous "blind spots" waiting to be explored; one of those is the RNA virome of unicellular eukaryotes. Here, we present the pipeline, which has been successfully used to characterize various types of RNA viruses, including Leishbuviridae (Bunyaviricetes, Hareavirales) in the parasitic flagellates of the family Trypanosomatidae. The pipeline relies on axenic in vitro cell culture and double-stranded RNA enrichment, followed by direct RNA-sequencing. A detailed procedure description starting from the initial total RNA preparation to the final assembly of the viral segments is provided.

Central European Institute of Technology Masaryk University Brno Czechia

Life Science Research Centre Faculty of Science University of Ostrava Ostrava Czechia

Zoological Institute Russian Academy of Sciences St Petersburg Russia

Erratum v

Methods Mol Biol. 2025;2893:C1. doi: 10.1007/978-1-0716-4338-9_22. PubMed

Zobrazit více v PubMed

Rampersad S, Tennant P (2018) Replication and expression strategies of viruses. In: Tennant P, Fermin G, Foster JE (eds) Viruses: molecular biology, host interactions, and applications to biotechnology. Academic Press/Elsevier, London, pp 55–82

Baltimore D (1971) Expression of animal virus genomes. Bacteriol Rev 35(3):235–241 PubMed PMC

Field AK, Lampson GP, Tytell AA, Hilleman MR (1972) Demonstration of double-stranded ribonucleic acid in concentrates of RNA viruses. Proc Soc Exp Biol Med 141(2):440–444 PubMed

Zinzula L, Tramontano E (2013) Strategies of highly pathogenic RNA viruses to block dsRNA detection by RIG-I-like receptors: hide, mask, hit. Antivir Res 100(3):615–635 PubMed

Baltimore D (1966) Purification and properties of poliovirus double-stranded ribonucleic acid. J Mol Biol 18(3):421–428 PubMed

Muthukrishnan S, Shatkin AJ (1975) Reovirus genome RNA segments: resistance to S PubMed

Diaz-Ruiz JR, Kaper JM (1978) Isolation of viral double-stranded RNAs using a LiCl fractionation procedure. Prep Biochem 8(1):1–17 PubMed

Ghabrial SA, Castón JR, Jiang D, Nibert ML, Suzuki N (2015) 50-plus years of fungal viruses. Virology 479–480:356–368 PubMed

García-Pedrajas MD, Cañizares MC, Sarmiento-Villamil JL, Jacquat AG, Dambolena JS (2019) Mycoviruses in biological control: from basic research to field implementation. Phytopathology 109(11):1828–1839 PubMed

Hong Y, Cole TE, Brasier CM, Buck KW (1998) Evolutionary relationships among putative RNA-dependent RNA polymerases encoded by a mitochondrial virus-like RNA in the Dutch elm disease fungus, Ophiostoma novo-ulmi, by other viruses and virus-like RNAs and by the Arabidopsis mitochondrial genome. Virology 246(1):158–169 PubMed

Crawford LJ, Osman TA, Booy FP, Coutts RH, Brasier CM, Buck KW (2006) Molecular characterization of a partitivirus from Ophiostoma himal-ulmi. Virus Genes 33(1):33–39 PubMed

Osaki H, Nakamura H, Sasaki A, Matsumoto N, Yoshida K (2006) An endornavirus from a hypovirulent strain of the violet root rot fungus, Helicobasidium mompa. Virus Res 118(1–2):143–149 PubMed

Kozlakidis Z, Hacker CV, Bradley D, Jamal A, Phoon X, Webber J, Brasier CM, Buck KW, Coutts RH (2009) Molecular characterisation of two novel double-stranded RNA elements from Phlebiopsis gigantea. Virus Genes 39(1):132–136 PubMed

Magae Y (2012) Molecular characterization of a novel mycovirus in the cultivated mushroom, Lentinula edodes. Virol J 9:60 PubMed PMC

Kanhayuwa L, Kotta-Loizou I, Ozkan S, Gunning AP, Coutts RH (2015) A novel mycovirus from Aspergillus fumigatus contains four unique dsRNAs as its genome and is infectious as dsRNA. Proc Natl Acad Sci USA 112(29):9100–9105 PubMed PMC

Niu Y, Yuan Y, Mao J, Yang Z, Cao Q, Zhang T, Wang S, Liu D (2018) Characterization of two novel mycoviruses from Penicillium digitatum and the related fungicide resistance analysis. Sci Rep 8(1):5513 PubMed PMC

Weber F, Wagner V, Rasmussen SB, Hartmann R, Paludan SR (2006) Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J Virol 80(10):5059–5064 PubMed PMC

Pearson MN, Beever RE, Boine B, Arthur K (2009) Mycoviruses of filamentous fungi and their relevance to plant pathology. Mol Plant Pathol 10(1):115–128 PubMed

Khan HA, Nerva L, Bhatti MF (2023) The good, the bad and the cryptic: the multifaceted roles of mycoviruses and their potential applications for a sustainable agriculture. Virology 585:259–269 PubMed

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(17):6460–6469 PubMed 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(5):E811–E819 PubMed PMC

Cashdollar LW, Esparza J, Hudson GR, Chmelo R, Lee PW, Joklik WK (1982) Cloning the double-stranded RNA genes of reovirus: sequence of the cloned S2 gene. Proc Natl Acad Sci USA 79(24):7644–7648 PubMed PMC

Imai M, Richardson MA, Ikegami N, Shatkin AJ, Furuichi Y (1983) Molecular cloning of double-stranded RNA virus genomes. Proc Natl Acad Sci USA 80(2):373–377 PubMed PMC

Niu Y, Zhang T, Zhu Y, Yuan Y, Wang S, Liu J, Liu D (2016) Isolation and characterization of a novel mycovirus from Penicillium digitatum. Virology 494:15–22 PubMed

Sterky F, Lundeberg J (2000) Sequence analysis of genes and genomes. J Biotechnol 76(1):1–31 PubMed

Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M, Macmanes MD, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R, Leduc RD, Friedman N, Regev A (2013) De novo transcript sequence reconstruction from RNA-seq using the trinity platform for reference generation and analysis. Nat Protoc 8(8):1494–1512 PubMed

Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19(5):455–477 PubMed PMC

Li CX, Shi M, Tian JH, Lin XD, Kang YJ, Chen LJ, Qin XC, Xu J, Holmes EC, Zhang YZ (2015) Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses. elife 4

Shi M, Lin XD, Tian JH, Chen LJ, Chen X, Li CX, Qin XC, Li J, Cao JP, Eden JS, Buchmann J, Wang W, Xu J, Holmes EC, Zhang YZ (2016) Redefining the invertebrate RNA virosphere. Nature 540(7634):539–543 PubMed

Shi M, Lin XD, Chen X, Tian JH, Chen LJ, Li K, Wang W, Eden JS, Shen JJ, Liu L, Holmes EC, Zhang YZ (2018) The evolutionary history of vertebrate RNA viruses. Nature 556(7700):197–202 PubMed

Zayed AA, Wainaina JM, Dominguez-Huerta G, Pelletier E, Guo J, Mohssen M, Tian F, Pratama AA, Bolduc B, Zablocki O, Cronin D, Solden L, Delage E, Alberti A, Aury JM, Carradec Q, da Silva C, Labadie K, Poulain J, Ruscheweyh HJ, Salazar G, Shatoff E, Tara Oceans Coordinatorsdouble, d, Bundschuh R, Fredrick K, Kubatko LS, Chaffron S, Culley AI, Sunagawa S, Kuhn JH, Wincker P, Sullivan MB, Acinas SG, Babin M, Bork P, Boss E, Bowler C, Cochrane G, de Vargas C, Gorsky G, Guidi L, Grimsley N, Hingamp P, Iudicone D, Jaillon O, Kandels S, Karp-Boss L, Karsenti E, Not F, Ogata H, Poulton N, Pesant S, Sardet C, Speich S, Stemmann L, Sullivan MB, Sungawa S, Wincker P (2022) Cryptic and abundant marine viruses at the evolutionary origins of Earth’s RNA virome. Science 376(6589):156–162 PubMed PMC

Kukurba KR, Montgomery SB (2015) RNA sequencing and analysis. Cold Spring Harb Protoc 2015(11):951–969 PubMed PMC

Yang IS, Kim S (2015) Analysis of whole transcriptome sequencing data: workflow and software. Genomics Inform 13(4):119–125 PubMed PMC

Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120 PubMed PMC

Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4):357–359 PubMed PMC

Ramirez-Gonzalez RH, Bonnal R, Caccamo M, Maclean D (2012) Bio-SAMtools: ruby bindings for SAMtools, a library for accessing BAM files containing high-throughput sequence alignments. Source Code Biol Med 7(1):6 PubMed PMC

Bushnell B, Rood J, Singer E (2017) BBMerge – accurate paired shotgun read merging via overlap. PLoS One 12(10):e0185056 PubMed PMC

Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and cufflinks. Nat Protoc 7(3):562–578 PubMed PMC

Au KF, Jiang H, Lin L, Xing Y, Wong WH (2010) Detection of splice junctions from paired-end RNA-seq data by SpliceMap. Nucleic Acids Res 38(14):4570–4578 PubMed PMC

Cross ST, Michalski D, Miller MR, Wilusz J (2019) RNA regulatory processes in RNA virus biology. Wiley Interdiscip Rev RNA 10(5):e1536 PubMed PMC

Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL (2009) BLAST+: architecture and applications. BMC Bioinformatics 10:421 PubMed PMC

Buchfink B, Reuter K, Drost HG (2021) Sensitive protein alignments at tree-of-life scale using DIAMOND. Nat Methods 18(4):366–368 PubMed PMC

Suzek BE, Wang Y, Huang H, McGarvey PB, Wu CH, UniProt C (2015) UniRef clusters: a comprehensive and scalable alternative for improving sequence similarity searches. Bioinformatics 31(6):926–932 PubMed

Prakash A, Jeffryes M, Bateman A, Finn RD (2017) The HMMER web server for protein sequence similarity search. Curr Protoc Bioinformatics 60: 3.15.11–13.15.23

Steinegger M, Meier M, Mirdita M, Vöhringer H, Haunsberger SJ, Söding J (2019) HH-suite3 for fast remote homology detection and deep protein annotation. BMC Bioinformatics 20(1):473 PubMed PMC

Wolf YI, Silas S, Wang Y, Wu S, Bocek M, Kazlauskas D, Krupovic M, Fire A, Dolja VV, Koonin EV (2020) Doubling of the known set of RNA viruses by metagenomic analysis of an aquatic virome. Nat Microbiol 5(10):1262–1270 PubMed PMC

Neri U, Wolf YI, Roux S, Camargo AP, Lee B, Kazlauskas D, Chen IM, Ivanova N, Zeigler Allen L, Paez-Espino D, Bryant DA, Bhaya D, Consortium RNAVD, Krupovic M, Dolja VV, Kyrpides NC, Koonin EV, Gophna U (2022) Expansion of the global RNA virome reveals diverse clades of bacteriophages. Cell 185(21):4023–4037 PubMed

Grybchuk D, Akopyants NS, Kostygov AY, Konovalovas A, Lye LF, Dobson DE, Zangger H, Fasel N, Butenko A, Frolov AO, Votýpka J, d'Avila-Levy CM, Kulich P, Moravcová J, Plevka P, Rogozin IB, Serva S, Lukeš J, Beverley SM, Yurchenko V (2018) Viral discovery and diversity in trypanosomatid protozoa with a focus on relatives of the human parasite Leishmania. Proc Natl Acad Sci USA 115(3):E506–E515 PubMed

Grybchuk D, Kostygov AY, Macedo DH, Votýpka J, Lukeš J, Yurchenko V (2018) RNA viruses in Blechomonas (Trypanosomatidae) and evolution of Leishmaniavirus. MBio 9(5):e01932–e01918 PubMed PMC

Grybchuk D, Macedo DH, Kleschenko Y, Kraeva N, Lukashev AN, Bates PA, Kulich P, Leštinová T, Volf P, Kostygov AY, Yurchenko V (2020) The first non-LRV RNA virus in Leishmania. Viruses 12(2):168 PubMed PMC

Macedo DH, Grybchuk D, Režnarová J, Votýpka J, Klocek D, Yurchenko T, Ševčík J, Magri A, Urda Dolinská M, Záhonová K, Lukeš J, Servienė E, Jászayová A, Serva S, Malysheva MN, Frolov AO, Yurchenko V, Kostygov AY (2023) Diversity of RNA viruses in the cosmopolitan monoxenous trypanosomatid Leptomonas pyrrhocoris. BMC Biol 21(1):191 PubMed PMC

Klocek D, Grybchuk D, Macedo DH, Galan A, Votýpka J, Schmid-Hempel R, Schmid-Hempel P, Yurchenko V, Kostygov AY (2023) RNA viruses of Crithidia bombi, a parasite of bumblebees. J Invertebr Pathol 201:107991 PubMed