Abstract
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.
Highlights
The sylvatic cycle of rabies is a significant sanitary burden in Central America
Optimization of sequencing protocol: Complete viral genome sequencing from clinical samples can be challenging due to the high percentage of host RNA compared to viral RNA and the structural elements intrinsic to viral genomes that hinder sequencing
To improve the coverage of viral genomes and get complete genomes from the study samples, we used a mixture of virusspecific designed primers, random hexamers, and previously published primers (Marston et al, 2013) during the single-stranded DNA (ssDNA) generation step; allowing us to obtain complete rabies virus genomes in all cases
Summary
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 extracted RNA from tissues of confirmed cases and generated ssDNA using several primers. 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. Conclusion: Our method produces valid viral assemblies from clinical specimens without target enrichment or viral isolation
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