Genetic Diversity and Antigenic Variation of Rabies Viruses from Different Host Species in Eastern and Western Parts of Kenya

Abstract

Rabies, a viral illness that causes deadly encephalitis, claims over 59,000 lives globally each year, with approximately 2,000 deaths happening in Kenya despite effective countermeasures for both humans and dogs. This study sought to improve the understanding of the genetic differences of the rabies virus (RABV) collected from the brain stem tissues of animals with suspected rabies in two geographic rabies hotspots: Makueni county, in the eastern region, and Siaya county, in western Kenya. Furthermore, the study investigated discrepancies among the antigenic sites of the RABV vaccines currently approved for use in Kenya and the study samples. Between July 2021 and September 2022, 164 brain stem tissues were collected from animals suspected of rabies after testing positive for RABV antigens using rapid diagnostic kits. Subsequently, in the laboratory, the samples were re-screened for rabies ribonucleic acid (RNA) by quantitative Real-Time Reverse-transcription Polymerase Chain Reaction (qRT-PCR) that targets the RABV Large-structural protein gene (L gene). Negative samples by the L gene qRT-PCR were analyzed by conventional PCR that targets the RABV Nucleoprotein gene (N gene). Phylogenetic analysis was carried out using whole genome sequences (WGS), as well as single genes (nucleoprotein (N) and glycoprotein (G). The amino acid changes in the antigenic regions of individual proteins (N and G) in the research sequences were compared to three RABV vaccine sequences: Pitman-Moore L503 (PM), Challenge Virus Standard (CVS), and Pasteur vaccine (PV). The L gene qRT-PCR revealed positive results for 156 of the 164 brain stem tissues. Eight samples failed to amplify the L-gene but amplified by N gene. On sequencing, 141 samples produced sequences suitable for analysis. Phylogenetic analysis using WGS and single genes confirmed that the RABV strains belonged to the Cosmopolitan clade that branched into two distinct phylogeographic subclades: sequences from eastern Kenya predominantly grouped within the Africa 1b subclade with only three sequences in the Africa 1a subclade. Conversely, the sequences from western Kenya formed clusters within the Africa 1a subclade while three were in the 1b subclade. In keeping with this phylogeographic clustering, the western Kenya Africa 1a subclade's closest ancestor was found in Sudan, while the eastern Kenya Africa 1b subclade had its origin in Tanzania. Within the western Kenya 1a subclade, 8 lineages were observed and 5 within the eastern Kenya 1b subclade. The amino acid homologies of the N gene between the study sequences and the vaccines for RABV were found to be approximately 97.6% for the PV vaccine strains, 98.5% for PM, and 97.8% for CVS. Similarly, the homologies with the G gene were at least 92.95% for PV, 92.19% for PM, and 93.3% for CVS. Our findings corroborate the geographic segregation of RABV between the eastern and western regions of Kenya. The data indicates that there is restricted viral movement, likely facilitated by the transportation of domestic dogs by humans. Good vaccine efficacy is predicted by the low amino acid variations in the antigenic sites of RABV vaccines and the study samples, suggesting that a lack of programmatic vaccination coverage is a major contributor to the endemicity of RABV in Kenya.