Phylogenetic relationships of a rabies virus isolate in Costa Rica

Phylogenetic relationships of a rabies virus isolate in Costa Rica




phylogenetic analysis, Next-generation Sequencing (NGS), lineage, Desmodus rotundus, Costa Rica


Introduction: The sylvatic cycle of rabies is a significant sanitary burden in Central America. The Costa Rican government monitors cases since 1985 and infections from bats are still reported for wild animals, livestock, and humans, generating a need of further pathogen characterization in the region. Objective: To compare rabies phylogenetic analyses from complete genomes with nucleoprotein gene studies. Methods: For the phylogenetic analyses we used four rabies tissue samples collected in 2018, and generated complete genomes by Next-Generation sequencing (NGS). We also extracted RNA from tissues of confirmed cases and generated ssDNA using several primers. Double-stranded DNA was generated and used to generate genomic libraries. Results: We describe, for the first-time, the complete genome of four sequences of the rabies virus isolated in Costa Rica in 2018. Complete genome trees resembled the topology of nucleoprotein gene trees. All isolates were related to Desmodus rotundus. One sample group into Lineage (L)2, and the remaining samples group in L1, matched previous reports from regional rabies viruses. Conclusion: Our method produces valid viral assemblies from clinical specimens without target enrichment or viral isolation.


NCBI Resource Coordinators. (2017). Database resources of the National Center for Biotechnology Information. Nucleic Acids Research, 46(D1), D8-D13.

Almeida, M. F., Martorelli, L. F. A., Aires, C. C., Sallum, P. C., Durigon, E. L., & Massad, E. (2005). Experimental rabies infection in haematophagous bats Desmodus rotundus. Epidemiology and Infection, 133(3), 523–527.

Amarilla, A. C. F., Pompei, J. C. A., Araujo, D. B., Vázquez, F. A., Galeano, R. R., Delgado, L. M., Bogado, G., Colman, M., Sanabria, L., Iamamoto, K., Garcia, R., Assis, D., Recalde, R., Martorelli, L. F., Quiñones, E., Cabello, A., Martini, M., Cosivi, O., Durigon, E. L., & Favoretto, S. R. (2018). Re-emergence of rabies virus maintained by canid populations in Paraguay. Zoonoses and Public Health, 65(1), 222–226.

Badilla, X., Pérez-Herra, V., Quirós, L., Morice, A., Jiménez, E., Sáenz, E., Salazar, F., Fernández, R., Orciari, L., Yager, P., Whitfield, S., & Rupprecht, C. E. (2003). Human rabies: A reemerging disease in Costa Rica? Emerging Infectious Diseases, 9(6), 721–723.

Bankevich, A., Nurk, S., Antipov, D., Gurevich, A. A., Dvorkin, M., Kulikov, A. S., Lesin, V. M., Nikolenko, S. I., Pham, S., Prjibelski, A. D., Pyshkin, A. V., Sirotkin, A. V., Vyahhi, N., Tesler, G., Alekseyev, M. A., & Pevzner, P. A. (2012). SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. Journal of Computational Biology, 19(5), 455–477.

Bano, I., Sajjad, H., Shah, A. M., Leghari, A., Mirbahar, K. H., Shams, S., & Soomro, M. (2016). A Review of Rabies Disease, its Transmission and Treatment. Journal of Animal Health and Production, 4(4), 140–144.

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

Brito-Hoyos, D. M., Brito, E., & Villalobos, R. (2013). Distribución geográfica del riesgo de rabia de origen silvestre y evaluación de los factores asociados con su incidencia en Colombia, 1982-2010. Revista Panamericana de Salud Pública, 33(1), 8–14.

Brown, J., Pirrung, M., & McCue, L. A. (2017). FQC Dashboard: Integrates FastQC results into a web-based, interactive, and extensible FASTQ quality control tool. Bioinformatics, 33(19), 3137–3139.

Brunker, K., Nadin-Davis, S., & Biek, R. (2018). Genomic sequencing, evolution and molecular epidemiology of rabies virus. Revue Scientifique et Technique (International Office of Epizootics), 37(2), 401–408.

Buchfink, B., Xie, C., & Huson, D. H. (2014). Fast and sensitive protein alignment using DIAMOND. Nature Methods, 12(1), 59–60.

Calderón, A., Guzmán, C., Mattar, S., Rodríguez, V., Acosta, A., & Martínez, C. (2019). Frugivorous bats in the Colombian Caribbean region are reservoirs of the rabies virus. Annals of Clinical Microbiology and Antimicrobials, 18(1), 1–8.

Capobianchi, M. R., Giombini, E., & Rozera, G. (2013). Next-generation sequencing technology in clinical virology. Clinical Microbiology and Infection, 19(1), 15–22.

Cargnelutti, J. F., de Quadros, J. M., Martins, M., Batista, H. B. C. R., Weiblen, R., & Flores, E. F. (2017). Glycoprotein-G-gene-based molecular and phylogenetic analysis of rabies viruses associated with a large outbreak of bovine rabies in southern Brazil. Archives of Virology, 162(12), 3697–3704.

Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., & Sayers, E. W. (2016). GenBank. Nucleic Acids Research, 44(D1), D67–D72.

de Almeida Campos, A. C., Romano, C. M., Melo, F. L., Araújo, D. B., Cunha, E. M. S., Sacramento, D. R. V., Durigon, E. L., & Lazarini, S. R. F. (2020). Phylogenetic analysis of near full-length sequences of the Desmodus rotundus genetic lineage of rabies virus. Infection, Genetics and Evolution, 80, 104179.

Deforche, K. (2017). An alignment method for nucleic acid sequences against annotated genomes. BioRxiv, 200394.

Delmas, O., Holmes, E. C., Talbi, C., Larrous, F., Dacheux, L., Bouchier, C., & Bourhy, H. (2008). Genomic diversity and evolution of the lyssaviruses. PLoS ONE, 3(4), e2057.

Desai, A., Marwah, V. S., Yadav, A., Jha, V., Dhaygude, K., Bangar, U., Kulkarni, V., & Jere, A. (2013). Identification of Optimum Sequencing Depth Especially for De Novo Genome Assembly of Small Genomes Using Next Generation Sequencing Data. PLoS ONE, 8(4), e60204.

Desjardins, P., & Conklin, D. (2010). NanoDrop microvolume quantitation of nucleic acids. Journal of Visualized Experiments, 45, 1–4.

Drummond, A. J., Nicholls, G. K., Rodrigo, A. G., & Solomon, W. (2002). Estimating Mutation Parameters, Population History and Genealogy Simultaneously From Temporally Spaced Sequence Data. Genetics, 161(July), 1307–1320.

Drummond, A. J., Suchard, M. A., Xie, D., & Rambaut, A. (2012). Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution, 29(8), 1969–1973.

Ellison, J. A., Gilbert, A. T., Recuenco, S., Moran, D., Alvarez, D. A., Kuzmina, N., Garcia, D. L., Peruski, L. F., Mendonça, M. T., Lindblade, K. A., & Rupprecht, C. E. (2014). Bat Rabies in Guatemala. PLoS Neglected Tropical Diseases, 8(7), e3070.

Escobar, L. E., Peterson, T., Favi, M., Yung, V., & Medina-Vogel, G. (2015). Bat-borne rabies in Latin America. Revista Do Instituto de Medicina Tropical de Sao Paulo, 57(1), 63–72.

Garcés-Ayala, F., Aréchiga-Ceballos, N., Ortiz-Alcántara, J. M., González-Durán, E., Pérez-Agüeros, S. I., Méndez-Tenorio, A., Torres-Longoria, B., López-Martínez, I., Hernández-Rivas, L., Díaz-Quiñonez, J. A., & Ramírez-González, J. E. (2017). Molecular characterization of atypical antigenic variants of canine rabies virus reveals its reintroduction by wildlife vectors in southeastern Mexico. Archives of Virology, 162(12), 3629–3637.

Gorbalenya, A. E., & Lauber, C. (2017). Phylogeny of Viruses. Reference Module in Biomedical Sciences.

Hall, T., Biosciences, I., & Carlsbad, C. (2011). BioEdit: An important software for molecular biology. GERF Bulletin of Biosciences, 2(1), 60–61.

Houldcroft, C. J., Beale, M. A., & Breuer, J. (2017). Clinical and biological insights from viral genome sequencing. Nature Reviews Microbiology, 15(3), 183–192.

Hutter, S. E., Brugger, K., Sancho V. H., González, R., Aguilar, O., León, B., Tichy, A., Firth, C. L., & Rubel, F. (2016). Rabies in Costa Rica: Documentation of the Surveillance Program and the Endemic Situation from 1985 to 2014. Vector-Borne and Zoonotic Diseases, 16(5), 334–341.

Johnson, N., Aréchiga-Ceballos, N., & Aguilar-Setien, A. (2014). Vampire bat rabies: Ecology, epidemiology and control. Viruses, 6(5), 1911–1928.

Katoh, K., & Standley, D. M. (2013). MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution, 30(4), 772–780.

Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35(6), 1547–1549.

Kuzmin, I. V., Shi, M., Orciari, L. A., Yager, P. A., Velasco-Villa, A., Kuzmina, N. A., Streicker, D. G., Bergman, D. L., & Rupprecht, C. E. (2012). Molecular inferences suggest multiple host shifts of rabies viruses from bats to mesocarnivores in Arizona during 2001-2009. PLoS Pathogens, 8(6), e1002786.

Lavergne, A., Darcissac, E., Bourhy, H., Tirera, S., de Thoisy, B., & Lacoste, V. (2016). Complete genome sequence of a vampire bat rabies virus from French Guiana. Genome Announcements, 4(2), e00188-16.

León, B., Fallas González, S., Miranda Solís, L., Ramírez-Cardoce, M., Moreira-Soto, A., Cordero-Solórzano, J. M., Elisabeth Hutter, S., González-Barrientos, R., & Rupprecht, C. E. (2021). Rabies in Costa Rica – Next Steps Towards Controlling Bat-Borne Rabies After its Elimination in Dogs. Yale Journal of Biology and Medicine, 94(2), 311-329.

Marston, D. A., McElhinney, L. M., Ellis, R. J., Horton, D. L., Wise, E. L., Leech, S. L., David, D., de Lamballerie, X., & Fooks, A. R. (2013). Next generation sequencing of viral RNA genomes. BMC Genomics, 14(1), 1-12.

Martin, D. P., Murrell, B., Golden, M., Khoosal, A., & Muhire, B. (2015). RDP4: Detection and analysis of recombination patterns in virus genomes. Virus Evolution, 1(1), vev003.

Maurier, F., Beury, D., Fléchon, L., Varré, J.-S., Touzet, H., Goffard, A., Hot, D., & Caboche, S. (2019). A complete protocol for whole-genome sequencing of virus from clinical samples: Application to coronavirus OC43. Virology, 531, 141–148.

Miller, M. A., Pfeiffer, W., & Schwartz, T. (2010). 2010 Gateway Computing Environments Workshop, GCE 2010. 2010 Gateway Computing Environments Workshop, GCE 2010.

Mochizuki, N., Kobayashi, Y., Sato, G., Hirano, S., Itou, T., Ito, F. H., & Sakai, T. (2011). Determination and molecular analysis of the complete genome sequence of two wild-type rabies viruses isolated from a haematophagous bat and a frugivorous bat in Brazil. Journal of Veterinary Medical Science, 73(6), 759–766.

Moser, L. A., Ramirez-Carvajal, L., Puri, V., Pauszek, S. J., Matthews, K., Dilley, K. A., Mullan, C., McGraw, J., Khayat, M., Beeri, K., Yee, A., Dugan, V., Heise, M.T., Frieman, M.B., Rodriguez, L.L., Bernard, K.A., Wentworth, D.E., Stockwell, T.B., Shabman, R.S. (2016). A universal next-generation sequencing protocol to generate noninfectious containment RNA viruses. MSystems, 1(3), e00039-15.

Oyola, S. O., Gu, Y., Manske, M., Otto, T. D., O’Brien, J., Alcock, D., MacInnis, B., Berriman, M., Newbold, C. I., Kwiatkowski, D. P., Swerdlow, H. P., & Quail, M. A. (2013). Efficient depletion of host DNA contamination in malaria clinical sequencing. Journal of Clinical Microbiology, 51(3), 745–751.

Sadeuh-Mba, S. A., Momo, J. B., Besong, L., Loul, S., & Njouom, R. (2017). Molecular characterization and phylogenetic relatedness of dog-derived Rabies Viruses circulating in Cameroon between 2010 and 2016. PLoS Neglected Tropical Diseases, 11(10), 1–21.

Salomão, C., Nacima, A., Cuamba, L., Gujral, L., Amiel, O., Baltazar, C., Cliff, J., & Gudo, E. S. (2017). Epidemiology, clinical features and risk factors for human rabies and animal bites during an outbreak of rabies in Maputo and Matola cities, Mozambique, 2014: Implications for public health interventions for rabies control. PLoS Neglected Tropical Diseases, 11(7), 1–16.

Soltis, P. S., & Soltis, D. E. (2003). Applying the Bootstrap in Phylogeny Reconstruction. Statistical Science, 18(2), 256–267.

Streicker, D. G., Fallas González, S. L., Luconi, G., Barrientos, R. G., & Leon, B. (2019). Phylodynamics reveals extinction–recolonization dynamics underpin apparently endemic vampire bat rabies in Costa Rica. Proceedings of the Royal Society B: Biological Sciences, 286(1912).

Thompson, J. D., Higgins, D. G., & Gibson, T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22(22), 4673–4680.

Vilsker, M., Moosa, Y., Nooij, S., Fonseca, V., Ghysens, Y., Dumon, K., Pauwels, R., Alcantara, L. C., Vanden Eynden, E., Vandamme, A. M., Deforche, K., & de Oliveira, T. (2019). Genome Detective: An automated system for virus identification from high-throughput sequencing data. Bioinformatics, 35(5), 871–873.

Yaguana, J., & López, M. (2017). La Rabia canina: Su historia, epidemiología y sus medidas de control. Revista Electrónica de Veterinaria, 18(9), 1-13.



How to Cite

Castro Rodríguez, L., León, B., & Ramírez Carvajal, L. (2021). Phylogenetic relationships of a rabies virus isolate in Costa Rica. UNED Research Journal, 14(1), e3713.