Development and validation of an all-in-one rabies virus Bat-Clade genomic sequencing and host identification protocol.

Rabies virus (RABV), remains a significant public health concern, with bat-maintained lineages accounting for all currently documented cases in Brazil. Despite the availability of pharmacological prophylaxis for humans and animals, the high genetic diversity of RABV in diverse natural bat hosts and continued circulation in multiple animals pose challenges for effective surveillance. Here, we developed and validated a novel, rapidly deployable amplicon-based sequencing approach for RABV genomic surveillance. This "all-in-one" protocol integrates whole RABV genome sequencing with host species identification through COI gene amplification and sequencing, addressing the challenges posed by RABV's high genetic diversity and complex transmission dynamics. We assessed the protocol's effectiveness by sequencing 25 near-complete RABV genomes from host species across four distinct families (Bovidae, Equidae, Felidae, and Microchiroptera) obtained from the Rabies Control and Surveillance Program from the Rio Grande do Sul State, Southern Brazil. The method achieved an average genome coverage of 91.4 % at a minimum 5x read depth, with a mean depth coverage of 816x across sequenced genomes. The results demonstrated significant Bat-Clade sublineage diversity, which was classified using the MADDOG RABV lineage system. The protocol successfully identified three bat species (Tadarida brasiliensis, Desmodus rotundus, and Myotis nigricans) among the samples, highlighting its capability for precise host identification. This study presents a powerful tool for high-resolution evaluation of RABV genomic features and host identification, enabling more targeted animal and human health interventions. This new approach has the potential to enhance RABV surveillance capabilities, contributing to more effective rabies control strategies within a One Health framework.