Complete Genome Sequences of Five Rabies Virus Strains Obtained from Domestic Carnivores in Liberia

Rabies virus (RABV) is the main etiological agent of rabies, an acute and always fatal form of encephalomyelitis which can potentially affect all mammalian species. This zoonotic virus belongs to the prototype species Rabies lyssavirus within the genus Lyssavirus, family Rhabdoviridae (order Mononegavirales) (1). Rabies viruses circulating in dogs are the main cause of human rabies, with an estimated 59,000 deaths worldwide each year, especially in Asia and Africa (2). As in other sub-Saharan countries, canine rabies remains endemic in Liberia (3). However, available data about the genetic diversity of RABV isolates circulating in this country remain limited.

Brain samples collected from four dogs and one cat suspected of rabies were collected from different regions of Liberia in 2017 and 2018, within the framework of a joint effort program to strengthen rabies surveillance in the country (Table 1) (3). All the samples were confirmed positive for rabies by fluorescence antibody test (FAT) (4) and by a modified version of a rapid immunochromatographic diagnostic test (RIDT) (5). For four samples, RNA was extracted locally from brain biopsy specimens (approximatively 0.5 cm³ each) using the Direct-zol RNA miniprep kit (Zymo Research) and then purified using Agencourt RNAClean XP beads (Beckman Coulter) at a 1:1.8 ratio. The last sample was extracted at Institut Pasteur using TRIzol reagent (Invitrogen) from an FTA card (Whatman FTA card technology; Sigma-Aldrich) impregnated with ground brain material as previously described (Table 1) (6). The five RNA samples were processed for next-generation sequencing (NGS) as previously described (7–9). Briefly, an rRNA depletion step was first carried out using Terminator 5′‐phosphate‐dependent exonuclease (Epicentre Biotechnologies). After purification, depleted RNA was reverse-transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen), and double-stranded DNA (dsDNA) was synthesized as already described (7–9). Finally, dsDNA libraries were constructed using the Nextera XT DNA library preparation kit (Illumina) and sequenced using a 2 × 150-nucleotide (nt) paired-end strategy on the NextSeq 500 platform (7–9). NGS data were analyzed using de novo assembly and mapping (both using CLC Assembly Cell; Qiagen), with a dedicated workflow built on the Institut Pasteur Galaxy platform (7–10). Contig sequences were assembled to produce the final consensus genome using Sequencher version 5.2.4 (Gene Codes Corporation). The quality and the accuracy of the final genome sequences were checked after a final mapping step of the original cleaned reads and visualized using Tablet (11). The leader and trailer sequences were verified after alignment with genetically close and available complete genomes (Fig. 1; Table 1). Identification of the open reading frames was performed using SnapGene software version 5.3.2. The nucleotide identity was determined using Ident and Sim software implemented in the Sequence Manipulation Suite (https://www.bioinformatics.org/sms2/ident_sim.html) (12). Maximum likelihood (ML) phylogenetic analysis was performed on the nearly complete genome sequences (11,800 to 11,804 nt) of the five RABV strains and different representative African strains using PhyML (13), after multiple alignment performed using ClustalW version 2.1 (14) implemented in the Institut Pasteur Galaxy platform (10). The ML phylogenetic tree was visualized using FigTree (http://tree.bio.ed.ac.uk/) (Fig. 1). All protocols were performed according to the manufacturer’s instructions, and all tools were run with default parameters, unless otherwise specified.

The genome sequences presented the five canonical genes encoding the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase (L) (Table 1). The leader and trailer sequences were 58 and 70 nucleotides long, respectively. The transcription initiation (TI) signal AACA and the transcription termination (TTP) TGA₇ was observed for all the genes, except for the G gene, which presented the AGA₇ motif for TTP. Three sequences presented a premature stop codon in the P gene. The nucleotide identity between four of the genome sequences was high (>99.1%), whereas strain 18018LIB was slightly more divergent (>97.5%). Genetic analysis confirmed that they clustered together in group H within the Africa 2 clade (3, 15, 16) (Fig. 1).

We thank the dog owners, the county livestock officers, the health surveillance officers, the members of the OH platform, and the laboratory staff for their great commitment. We are grateful to the sequencing facilities of the Plate-forme de microbiologie mutalisée (P2M) of Institut Pasteur (Paris, France) for technical assistance with NGS sequencing. We also acknowledge Lisa Crump for language editing.

This work was funded through the Global Alliance for Vaccines and Immunisation (GAVI) (VISLACRV19122014, work stream 3), the Wolfermann Nägeli Foundation, the Swiss African Research Cooperation (SARECO), research funds from Stay on Track of the University of Basel, the Stiftung für wissenschaftliche Forschung (SWF), the Freiwillige Akademische Gesellschaft (FAG), and the Novartis Foundation for Biomedical Research. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.