Eleven different lyssavirus species, four of which occur on the African continent, are presently recognized. These viruses cause rabies, the burden of which is highest in the developing world, where routine laboratory diagnosis is often not available. From an epidemiological and control perspective, it is necessary that diagnostic methods detect the diversity of lyssaviruses present in different regions of the world. A published and widely used heminested reverse transcription-PCR (hnRT-PCR) was evaluated for its ability to detect a panel of diverse African lyssaviruses. Due to the limitations experienced for this assay, an alternative hnRT-PCR was developed. The new assay was found to be accurate and sensitive in the detection of African lyssavirus RNA in a variety of clinical specimens. The assay was further adapted to a real-time PCR platform to allow rapid, one-step, quantitative, and single-probe detection, and an internal control for the verification of sample preparation was included. The limit of detection of the real-time PCR assay was 10 RNA copies per reaction, with inter-and intra-assay variability below 4%. Subsequently, in demonstrating utility, both assays were successfully applied to antemortem rabies diagnosis in humans. We believe that the quantitative real-time PCR assay could find application as a routine confirmatory test for rabies diagnosis in the future and that it will serve as a valuable research tool in the biology of African lyssaviruses. Alternatively, the hnRT-PCR assay can be used in laboratories that do not have access to expensive real-time PCR equipment for sensitive diagnosis of lyssaviruses.

In Africa, bat-borne zoonoses emerged in the past few decades resulting in large outbreaks or just sporadic spillovers. In addition, hundreds of more viruses are described without any information on zoonotic potential. We discuss important characteristics of bats including bat biology, evolution, distribution and ecology that not only make them unique among most mammals but also contribute to their potential as viral reservoirs. The detection of a virus in bats does not imply that spillover will occur and several biological, ecological and anthropogenic factors play a role in such an event. We summarize and critically analyse the current knowledge on African bats as reservoirs for corona-, filo-, paramyxo-and lyssaviruses. We highlight that important information on epidemiology, bat biology and ecology is often not available to make informed decisions on zoonotic spillover potential. Even if knowledge gaps exist, it is still important to recognize the role of bats in zoonotic disease outbreaks and implement mitigation strategies to prevent exposure to infectious agents including working safely with bats. Equally important is the crucial role of bats in various ecosystem services. This necessitates a multidisciplinary One Health approach to close knowledge gaps and ensure the development of responsible mitigation strategies to not only minimize risk of infection but also ensure conservation of the species.

Mokola virus (MOKV) appears to be exclusive to Africa. Although the first isolates were from Nigeria and other Congo basin countries, all reports over the past 20 years have been from southern Africa. Previous phylogenetic studies analyzed few isolates or used partial gene sequence for analysis since limited sequence information is available for MOKV and the isolates were distributed among various laboratories. The complete nucleoprotein, phosphoprotein, matrix and glycoprotein genes of 18 MOKV isolates in various laboratories were sequenced either using partial or full genome sequencing using pyrosequencing and a phylogenetic analysis was undertaken. The results indicated that MOKV isolates from the Republic of South Africa, Zimbabwe, Central African Republic and Nigeria clustered according to geographic origin irrespective of the genes used for phylogenetic analysis, similar to that observed with Lagos bat virus. A Bayesian Markov-Chain-Monte-Carlo- (MCMC) analysis revealed the age of the most recent common ancestor (MRCA) of MOKV to be between 279 and 2034 years depending on the genes used. Generally, all MOKV isolates showed a similar pattern at the amino acid sites considered influential for viral properties.

Bats (order Chiroptera) are the principal reservoir host for 14 of the 16 officially recognised lyssavirus species. Rabies virus is the only lyssavirus that is well established in terrestrial carnivores (worldwide), as well as bats (but only in the Americas). The other bat lyssaviruses occur only outside the Americas. They have a distinct geographical distribution and association with specific bat species, with limited cross-species transmission to other animals and humans, resulting in deadend infections. The nucleoprotein gene is well conserved between all lyssavirus species. Therefore, gold-standard diagnostic techniques detect all lyssaviruses but do not discriminate between viral species. Lyssaviruses are divided into at least three phylogroups, based on their immunogenic and phylogenic properties. Owing to the diversity of glycoproteins among phylogroups, rabies vaccines and immunoglobulins only provide protection against phylogroup I, excluding several of the bat lyssaviruses. Africa hosts a high diversity of lyssaviruses, leading to the hypothesis that this region was the site of emergence; however, this has been challenged by more recent phylogenetic analysis, suggesting a Palearctic origin. Serological evidence indicates a more widespread and even higher diversity of lyssaviruses in bats, suggesting that the incidence of known lyssaviruses is underestimated and several new lyssavirus species are yet to be discovered. Most bats are, however, not able to transmit the virus and therefore pose a low risk to human and animal populations.

3592-App1.pdf) of 41 species across South Africa (Figure 1; Appendix Tables 1, 2). Most bats were collected as part of a broader biosurveillance program in collaboration with bat taxonomists for species identification and classification. Among the bats collected, 562 appeared healthy and 28 were dead. Another 12 exhibited signs of disease or abnormal behavior, and 3 had been involved in human contact; we submitted these samples for rabies testing. We anesthetized the sampled bats with isoflurane inhalant (Safeline Pharmaceuticals, https://safeline. co.za), exsanguinated them by cardiac puncture, and then performed full necropsies. We identified bats on the basis of morphologic (4) and genetic characteristics (5). The sampling protocol was approved by the University of Pretoria Animal Ethics Committee (approval no. EC054-14). Permission to conduct research was obtained from the Department of Agriculture, Land Reform and Rural Development (formerly Department of Agriculture, Forestry, and Fisheries) of the Republic of South Africa under Section 20 of the Animal Diseases Act 1984 with additional provincial permits granted (Appendix Table 3). We extracted total RNA from the bats' brain material and subjected it to real-time reverse transcription PCR selective for 126 bp of the nucleoprotein gene