Mokola virus (MOKV), a rabies-related lyssavirus, represents one of 17 recognized species within the Lyssavirus genus, all of which are capable of causing the fatal encephalitic rabies disease. MOKV is exclusively endemic to Africa with only 30 sporadic cases reported since its discovery more than 50 years ago. The reservoir host for MOKV remains unknown, however, several hypotheses have been formulated. Small non-volant mammals (i.e. shrews, sengis and rodents) have been suggested as possible reservoir hosts with previous MOKV isolations from shrews (Crocidura spp.) and a single rodent (Lophuromys sikapusi) providing support of the first lyssavirus species that has an association with small non-volant mammals. To investigate further, nucleic acid- and serological surveillance were conducted in small non-volant mammals from Southern Africa (specifically South Africa and Mozambique). Nucleic acid surveillance with a pan-lyssavirus qRT-PCR assay of 355 brain samples did not identify any new MOKV infections. Serological surveillance using a micro-neutralization test of 287 serum samples identified 37 samples that were positive for the presence of MOKV virus neutralizing antibodies. These positive serum samples indicate previous MOKV exposure and were all collected from Bushveld gerbils (Gerbilliscus leucogaster) in both South Africa (n=36) and Mozambique (n=1). From all of the Bushveld gerbils that were tested, an overall MOKV seropositivity of 87.80% is observed for the gerbils that were caught at Meletse in Limpopo. Since MOKV have been shown to cross-react in serological assay with closely-related lyssaviruses, the seropositivity observed could have been due to exposure of another phylogroup II lyssavirus. Serological evidence of MOKV in this rodent species was previously observed in a study conducted in Zimbabwe in 1988, which raises their profile as a potential MOKV host candidate. Experimental pathogenicity studies support this notion due to significant amounts of MOKV found in their salivary glands that could be sufficient for transmission. To gain further insight of the phylogeny and genetic diversity of MOKV, complete genome sequences of three historic MOKV isolates from South Africa (MOKV 700/70, 229/97, and 12/458) were generated. Future studies are needed to expand surveillance, detection and characterization of lyssaviruses. / Dissertation (MSc (Medical Virology))--University of Pretoria, 2020. / The Centre for Disease Control and Prevention (CDC) Global Disease Detection (GDD) Programme (Corporate Agreement Number: 5 NU2GGH001874-02-00). / The South African Research Chair in Infectious Diseases of Animals (Zoonoses) from the National Research Foundation (NRF) of the Department of Science and Innovation (DSI), Prof. Wanda Markotter (UID98339), as well as additional grants awarded to Prof. Markotter by the NRF (UID92524, UID85756 & UID91496). / Medical Virology / MSc (Medical Virology) / Restricted