Typing Arctic and Africa-2 clades of rabies virus using clade-defining single nucleotide polymorphisms

Rabies is a deadly neurotropic, zoonotic disease with a mortality rate of 100% after symptoms appear. Rabies virus (RABV) is the primary cause of rabies disease in humans, and it mainly spreads via dog bites in developing countries. Over the course of RABV evolution, multiple RABV variants, called clades, have emerged. However, our understanding of these clades is limited, as the only method to identify a clade is sequencing, followed by phylogeny. In this study, we have developed a rapid, PCR-based method for typing two RABV clades. We utilised highly conserved amino acid changes specific to the Arctic and Africa-2 clades of the rabies virus (RABV). A single nucleotide substitution from adenine to thymine at position 178 within the nucleoprotein gene was found to be clade-specific in the Arctic clade. Similarly, adenine at position 638 is a distinctive marker for the Africa-2 clade. The assay demonstrated high specificity and offers the added benefit of PCR-based amplification, enabling virus detection even when viral titers are low. The assay can identify the Arctic clade and Africa-2 clade without sequencing and is highly specific and sensitive. Furthermore, this method can be adapted to detect other RABV clades and a wide range of viruses.