Sandflies are known vectors of leishmaniasis. In the Old World, sandflies are also vectors of viruses while little is known about the capacity of New World insects to transmit viruses to humans. Here, we relate the identification of RNA sequences with homology to rhabdovirus nucleocapsids (NcPs) genes, initially in the Lutzomyia longipalpis LL5 cell lineage, named NcP1.1 and NcP2. The Rhabdoviridae family never retrotranscribes its RNA genome to DNA. The sequences here described were identified in cDNA and DNA from LL-5 cells and in adult insects indicating that they are transcribed endogenous viral elements (EVEs). The presence of NcP1.1 and NcP2 in the L. longipalpis genome was confirmed in silico. In addition to showing the genomic location of NcP1.1 and NcP2, we identified another rhabdoviral insertion named NcP1.2. Analysis of small RNA molecules derived from these sequences showed that NcP1.1 and NcP1.2 present a profile consistent with elements targeted by primary piRNAs, while NcP2 was restricted to the degradation profile. The presence of NcP1.1 and NcP2 was investigated in sandfly populations from South America and the Old World. These EVEs are shared by different sandfly populations in South America while none of the Old World species studied presented the insertions.

Departamento de Bioquímica e Imunologia Instituto de Ciências Biológicas Universidade Federal de Minas Gerais Belo Horizonte 31270 901 MG Brazil

Departamento de Imunologia Instituto Aggeu Magalhães Fiocruz Recife 50740 465 PE Brazil

Department of Immunology and Infectious Diseases Harvard School of Public Health Cambridge MA 02115 USA

Department of Parasitology Charles University 12800 Prague Czech Republic

Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular CNPq Rio de Janeiro 21040 360 RJ Brazil

Instituto Nacional de Medicina Tropical Ministerio de Salud de la Nación ANLIS Puerto Iguazu 3370 Misiones Argentina

Laboratório de Biologia Molecular de Insetos Instituto Oswaldo Cruz Fiocruz Rio de Janeiro 21040 360 RJ Brazil

Laboratório de Biologia Molecular de Parasitas e Vetores Instituto Oswaldo Cruz Fiocruz Rio de Janeiro 21040 360 RJ Brazil

Laboratório de Doenças Parasitárias Instituto Oswaldo Cruz Fiocruz Rio de Janeiro 21040 360 RJ Brazil

Laboratório de Ecologia de Doenças Transmissíveis na Amazônia Instituto Leônidas e Maria Deane Fiocruz Amazônia Manaus 69027 070 AM Brazil

Laboratório de Epidemiologia e Sistemática Molecular Instituto Oswaldo Cruz Fiocruz Rio de Janeiro 21040 360 RJ Brazil

See more in PubMed

Wang H., Naghavi M., Allen C., Barber R.M., Carter A., Casey D.C., Charlson F.J., Chen A.Z., Coates M.M., Coggeshall M., et al. Global, Regional, and National Life Expectancy, All-Cause Mortality, and Cause-Specific Mortality for 249 Causes of Death, 1980–2015: A Systematic Analysis for the Global Burden of Disease Study 2015. Lancet. 2016;388:1459–1544. doi: 10.1016/S0140-6736(16)31012-1. PubMed DOI PMC

Lainson R., Braga R.R., De Souza A.A., Pôvoa M.M., Ishikawa E.A., Silveira F.T. Leishmania (Viannia) shawi sp. n., a parasite of monkeys, sloths and procyonids in Amazonian Brazil. Ann. Parasitol. Hum. Comp. 1989;64:200–207. doi: 10.1051/parasite/1989643200. PubMed DOI

Killick-Kendrick R. Guide to the Identification and Geographic Distribution of Lutzomyia Sand Flies in Mexico, the West Indies, Central and South America (Diptera: Psychodidae). D. G. Young & M. A. Duncan. Memoirs of the American Entomological Institute no. 54. Gainesville, Florida, USA: Associated Publishers, 1994. 881 pp. US$ 85. ISBN 1-5666-054-2. Trans. R. Soc. Trop. Med. Hyg. 1995;89:125. doi: 10.1016/0035-9203(95)90687-8. DOI

Bruschi F., Gradoni L., editors. The leishmaniases: Old Neglected Tropical Diseases. Springer; Cham, Switzerland: 2018.

Labbé F., Abdeladhim M., Abrudan J., Araki A.S., Araujo R.N., Arensburger P., Benoit J.B., Brazil R.P., Bruno R.V., Bueno da Silva Rivas G., et al. Genomic analysis of two phlebotomine sandfly vectors of Leishmania from the New and Old World. PLoS Negl. Trop. Dis. 2023;17:e0010862. doi: 10.1371/journal.pntd.0010862. PubMed DOI PMC

Jancarova M., Polanska N., Volf P., Dvorak V. The role of sandflies as vectors of viruses other than phleboviruses. J. Gen. Virol. 2023;104:001837. doi: 10.1099/jgv.0.001837. PubMed DOI

Depaquit J., Grandadam M., Fouque F., Andry P.E., Peyrefitte C. Arthropod-borne viruses transmitted by Phlebotomine sandflies in Europe: A review. Euro Surveill. 2010;15:19507. doi: 10.2807/ese.15.10.19507-en. PubMed DOI

Letchworth G.J., Rodriguez L.L., Del Cabarrera J. Vesicular stomatitis. Vet. J. 1999;157:239–260. doi: 10.1053/tvjl.1998.0303. PubMed DOI

Travassos da Rosa A.P.A., Shope R.E., Pinheiro F.P. Arbovirus research in the Brazilian Amazon. In: Uren M.F., Blok J., Manderson L.H., editors. Proceedings of the Fifth Symposium on Arbovirus Research in Australia; Brisbane, Australia. 28 August–1 September 1989; Brisbane, Australia: University of Queensland Medical School; 1989. pp. 4–8.

Carvalho M.S., de Lara Pinto A.Z., Pinheiro A., Rodrigues J.S.V., Melo F.L., da Silva L.A., Ribeiro B.M., Dezengrini-Slhessarenko R. Viola phlebovirus is a novel Phlebotomus fever serogroup member identified in Lutzomyia (Lutzomyia) longipalpis from Brazilian Pantanal. Parasit. Vectors. 2018;11:405. doi: 10.1186/s13071-018-2985-3. PubMed DOI PMC

Tesh R.B., Boshell J., Young D.G., Morales A., Ferra de Carrasquilla C., Corredor A., Modi G.B., Travassos da Rosa A.P., McLean R.G., de Rodriguez C., et al. Characterization of five new phleboviruses recently isolated from sandflies in tropical America. Am. J. Trop. Med. Hyg. 1989;40:529–533. doi: 10.4269/ajtmh.40-5529. PubMed DOI

Fonseca P., Ferreira F., da Silva F., Oliveira L.S., Marques J.T., Goes-Neto A., Aguiar E., Gruber A. Characterization of a Novel Mitovirus of the Sandfly Lutzomyia longipalpis Using Genomic and Virus-Host Interaction Signatures. Viruses. 2020;13:9. doi: 10.3390/v13010009. PubMed DOI PMC

Jern P., Coffin J.M. Effects of retroviruses on host genome function. Annu. Rev. Genet. 2008;42:709–732. doi: 10.1146/annurev.genet.42.110807.091501. PubMed DOI

Horie M., Honda T., Suzuki Y., Kobayashi Y., Daito T., Oshida T., Ikuta K., Jern P., Gojobori T., Coffin J.M., et al. Endogenous non-retroviral RNA virus elements in mammalian genomes. Nature. 2010;463:84–87. doi: 10.1038/nature08695. PubMed DOI PMC

Belyi V.A., Levine A.J., Skalka A.M. Unexpected inheritance: Multiple integrations of ancient bornavirus and ebolavirus/marburgvirus sequences in vertebrate genomes. PLoS Pathog. 2010;6:e1001030. doi: 10.1371/journal.ppat.1001030. PubMed DOI PMC

Gilbert C., Feschotte C. Genomic fossils calibrate the long-term evolution of hepadnaviruses. PLoS Biol. 2010;8:e1000495. doi: 10.1371/journal.pbio.1000495. PubMed DOI PMC

Liu H., Fu Y., Xie J., Cheng J., Ghabrial S.A., Li G., Peng Y., Yi X., Jiang D. Widespread endogenization of densoviruses and parvoviruses in animal and human genomes. J. Virol. 2011;85:9863–9876. doi: 10.1128/JVI.00828-11. PubMed DOI PMC

Veglia A.J., Bistolas K.S.I., Voolstra C.R., Hume B.C.C., Ruscheweyh H.J., Planes S., Allemand D., Boissin E., Wincker P., Poulain J., et al. Endogenous viral elements reveal associations between a non-retroviral RNA virus and symbiotic dinoflagellate genomes. Commun. Biol. 2023;6:566. doi: 10.1038/s42003-023-04917-9. PubMed DOI PMC

Fort P., Albertini A., Van-Hua A., Berthomieu A., Roche S., Delsuc F., Pasteur N., Capy P., Gaudin Y., Weill M. Fossil rhabdoviral sequences integrated into arthropod genomes: Ontogeny, evolution, and potential functionality. Mol. Biol. Evol. 2012;29:381–390. doi: 10.1093/molbev/msr226. PubMed DOI

Vasilakis N., Widen S., Mayer S.V., Seymour R., Wood T.G., Popov V., Guzman H., da Rosa A.P., Ghedin E., Holmes E.C. Niakha virus: A novel member of the family Rhabdoviridae isolated from phlebotomine sandflies in Senegal. Virology. 2013;444:80–89. doi: 10.1016/j.virol.2013.05.035. PubMed DOI PMC

Ter Horst A.M., Nigg J.C., Dekker F.M., Falk B.W. Endogenous Viral Elements Are Widespread in Arthropod Genomes and Commonly Give Rise to PIWI-Interacting RNAs. J. Virol. 2019;93:e02124-18. doi: 10.1128/JVI.02124-18. PubMed DOI PMC

Wallau G.L. RNA virus EVEs in insect genomes. [(accessed on 1 February 2022)];Curr. Opin. Insect Sci. 2022 49:42–47. doi: 10.1016/j.cois.2021.11.005. Available online: https://www.sciencedirect.com/science/article/pii/S2214574521001267. PubMed DOI

Fujino K., Horie M., Honda T., Merriman D.K., Tomonaga K. Inhibition of Borna disease virus replication by an endogenous bornavirus-like element in the ground squirrel genome. Proc. Natl. Acad. Sci. USA. 2014;111:13175–13180. doi: 10.1073/pnas.1407046111. PubMed DOI PMC

Goic B., Stapleford K.A., Frangeul L., Doucet A.J., Gausson V., Blanc H., Schemmel-Jofre N., Cristofari G., Lambrechts L., Vignuzzi M., et al. Virus-derived DNA drives mosquito vector tolerance to arboviral infection. Nat. Commun. 2016;7:12410. doi: 10.1038/ncomms12410. PubMed DOI PMC

Volf P., Volfova V. Establishment and maintenance of sand fly colonies. J. Vector Ecol. 2011;36:S1–S9. doi: 10.1111/j.1948-7134.2011.00106.x. PubMed DOI

Thompson J.D., Higgins D.G., Gibson T.J. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. doi: 10.1093/nar/22.22.4673. PubMed DOI PMC

Jones D.T., Taylor W.R., Thornton J.M. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 1992;8:275–282. doi: 10.1093/bioinformatics/8.3.275. PubMed DOI

Tamura K., Stecher G., Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 2021;38:3022–3027. doi: 10.1093/molbev/msab120. PubMed DOI PMC

Aguiar E.R., Olmo R.P., Marques J.T. Virus-derived small RNAs: Molecular footprints od host-pahogen interaction. Wiley Interdiscip. Rev. RNA. 2016;7:824–837. doi: 10.1002/wrna.1361. PubMed DOI PMC

Lourenço-de-Oliveira R., Marques J.T., Sreenu V.B., Atyame Nten C., Aguiar E.R.G.R., Varjak M., Kohl A., Failloux A.B. Culex quinquefasciatus mosquitoes do not support replication of Zika virus. J. Gen. Virol. 2018;99:258–264. doi: 10.1099/jgv.0.000949. PubMed DOI PMC

Marconcini M., Pischedda E., Houé V., Palatini U., Lozada-Chávez N., Sogliani D., Failloux A.B., Bonizzoni M. Profile of Small RNAs, vDNA Forms and Viral Integrations in Late Chikungunya Virus Infection of Aedes albopictus Mosquitoes. Viruses. 2021;13:553. doi: 10.3390/v13040553. PubMed DOI PMC

Olmo R.P., Todjro Y.M., Aguiar E.R., de Almeida J.P.P., Ferreira F.V., Armache J.N., de Faria I.J., Ferreira A.G., Amadou S.C., Silva A.T.S., et al. Mosquito vector competence for dengue is modulated by insect-specific viruses. Nat. Microbiol. 2023;8:135–149. doi: 10.1038/s41564-022-01289-4. PubMed DOI

Chiba S., Kondo H., Tani A., Saisho D., Sakamoto W., Kanematsu S., Suzuki N. Widespread endogenization of genome sequences of non-retroviral RNA viruses into plant genomes. PLoS Pathog. 2011;7:e1002146. doi: 10.1371/journal.ppat.1002146. PubMed DOI PMC

Aguiar E.R.G.R., de Almeida J.P.P., Queiroz L.R., Oliveira L.S., Olmo R.P., de Faria I.J.D.S., Imler J.L., Gruber A., Matthews B.J., Marques J.T. A single unidirectional piRNA cluster similar to the flamenco locus is the major source of EVE-derived transcription and small RNAs in Aedes aegypti mosquitoes. RNA. 2020;26:581–594. doi: 10.1261/rna.073965.119. PubMed DOI PMC

Kryukov K., Ueda M.T., Imanishi T., Nakagawa S. Systematic survey of non-retroviral virus-like elements in eukaryotic genomes. Virus Res. 2019;262:30–36. doi: 10.1016/j.virusres.2018.02.002. PubMed DOI

Katzourakis A., Gifford R.J. Endogenous viral elements in animal genomes. PLoS Genet. 2010;6:e1001191. doi: 10.1371/journal.pgen.1001191. PubMed DOI PMC

Suzuki Y., Frangeul L., Dickson L.B., Blanc H., Verdier Y., Vinh J., Lambrechts L., Saleh M.C. Uncovering the Repertoire of Endogenous Flaviviral Elements in Aedes mosquito Genomes. J. Virol. 2017;91:e00571-17. doi: 10.1128/JVI.00571-17. PubMed DOI PMC

Cox D.N., Chao A., Baker J., Chang L., Qiao D., Lin H. A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev. 1998;12:3715–3727. doi: 10.1101/gad.12.23.3715. PubMed DOI PMC

Léger P., Lara E., Jagla B., Sismeiro O., Mansuroglu Z., Coppée J.Y., Bonnefoy E., Bouloy M. Dicer-2- and Piwi-mediated RNA interference in Rift Valley fever virus-infected mosquito cells. J. Virol. 2013;87:1631–1648. doi: 10.1128/JVI.02795-12. PubMed DOI PMC

Varjak M., Maringer K., Watson M., Sreenu V.B., Fredericks A.C., Pondeville E., Donald C.L., Sterk J., Kean J., Vazeille M., et al. Aedes aegypti Piwi4 Is a Noncanonical PIWI Protein Involved in Antiviral Responses. mSphere. 2017;2:e00144-17. doi: 10.1128/mSphere.00144-17. PubMed DOI PMC

Ferreira F.V., Aguiar E.R.G.R., Olmo R.P., de Oliveira K.P.V., Silva E.G., Sant’Anna M.R.V., Gontijo N.F., Kroon E.G., Imler J.L., Marques J.T. The small non-coding RNA response to virus infection in the Leishmania vector Lutzomyia longipalpis. PLoS Negl. Trop. Dis. 2018;12:e0006569. doi: 10.1371/journal.pntd.0006569. PubMed DOI PMC

Martins-da-Silva A., Telleria E.L., Batista M., Marchini F.K., Traub-Csekö Y.M., Tempone A.J. Identification of Secreted Proteins Involved in Nonspecific dsRNA-Mediated Lutzomyia longipalpis LL5 Cell Antiviral Response. Viruses. 2018;10:43. doi: 10.3390/v10010043. PubMed DOI PMC

Souza Freitas M.T., Ríos-Velasquez C.M., Costa C.R., Jr., Figueirêdo C.A., Jr., Aragão N.C., da Silva L.G., de Aragão Batista M.V., Balbino T.C., Pessoa F.A., de Queiroz Balbino V. Phenotypic and genotypic variations among three allopatric populations of Lutzomyia umbratilis, main vector of Leishmania guyanensis. Parasit. Vectors. 2015;8:448. doi: 10.1186/s13071-015-1051-7. PubMed DOI PMC

Sousa-Paula L.C., da Silva L.G., da Silva Junior W.J., Figueiredo Júnior C.A.S., Costa C.H.N., Pessoa F.A.C., Dantas-Torres F. Genetic structure of allopatric populations of Lutzomyia longipalpis sensu lato in Brazil. Acta Trop. 2021;222:106031. doi: 10.1016/j.actatropica.2021.106031. PubMed DOI