The African continent sustains a variety of lyssaviruses and this study focused on two of these lyssaviruses that are unique to the continent namely rabies virus mongoose biotype and Mokola virus (MOKV). Rabies virus (RABV) belongs to genotype (gt) 1 of the Lyssavirus genus in the family Rhabdoviridae, order Mononegavirales, while Mokola virus belongs to gt3 of this genus. Both these viruses cause fatal rabies encephalitis in vertebrate animals. Genotype 1 (rabies virus) isolates from southern African countries display great genetic diversity and are grouped into two main biotypes i.e. canid and mongoose biotypes. Due to the difference in the epidemiology and pathogenesis of these biotypes, it has been hypothesized that the two biotypes were introduced into Africa at different times. The objective was to study the molecular phylogeny of representative rabies virus isolates of the mongoose biotype, isolated in South Africa and Zimbabwe over a period of 27 years, towards a better understanding of the origin of this group. In this study the complete nucleoprotein (1353 nucleotides) and glycoprotein (1575 nucleotides) genes were sequenced. The evolutionary dynamics of this virus variant was investigated using Bayesian methodology, allowing for rate variation among the different viral lineages. The phylogenetic analysis of this dataset confirms previous findings of extended evolutionary adaptation of isolates in specific geographic areas. Furthermore when these isolates are analyzed together with rabies virus isolates from across the world, they still form an independent cluster separate from any other African rabies virus isolates, thereby hinting towards a separate introduction to the continent before that of canid rabies. Molecular clock analysis estimates the age of the mongoose biotype to be approximately 200 years old, which is in concurrence with literature describing rabies in mongooses since the early 1800’s. In addition, a phylogenetic analysis of Mokola virus isolates (gt3) from South Africa, Zimbabwe, Cameroon and Central African Republic is described. All the South African isolates before 2008, as well as most of the Zimbabwean isolates (except isolate 21846) were included in this analysis. The complete nucleoprotein gene (1353nt) was amplified and sequenced. Phylogenetic analysis showed virus grouping to correspond to their geographic location. Further analysis showed Mokola virus isolates to display genetic diversity similar to that found in representative gt1 isolates. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

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

The lyssavirus genus of the Rhabdovirus family is divided into seven genotypes. Genotype 3, Mokola virus, has only been found on the African continent, and has been reported to infect rodents, cats, dogs and humans. The first Mokola virus identification in South Africa was made in 1970, on the east coast of the KwaZulu-Natal province. After 25 years, Mokola virus was again identified in three cats, 650 km south-west of the previous isolation. In 1997 two more Mokola infections were identified in Pinetown, only about 23 km south-west of the 1970 isolation. Phylogenetic analysis of the nucleic acid sequences of the nucleoprotein gene region of the Mokola genome, indicated that the Mokola viruses from the same geographical region were more closely related, irrespective of the time of isolation. The identification of these two distinct clusters of Mokola in South Africa leads i us to believe that this virus is more widespread than previously thought, but that the reservoir host species remains to be identified. Genotype 1 in the Rhabdovirus family, rabies virus, is found on all continents, except Australia, New Zealand, Papua New Guinea, Japan, Hawaii, Taiwan, United Kingdom, Ireland, etc. An ongoing rabies enzootic in southern Africa is associated with two genetically distinct groups of viruses, called the canid biotype (infecting carnivores of the family Canidae) and the viverrid biotype (infecting carnivores of the subfamily Viverrinae). We identified the first cases of spillover of canid biotype virus into viverrid hosts, using monoclonal antibody and nucleic acid sequence analysis. Genetic analysis of the G-L intergenic region of the rabies virus genome, showed that these spillover events do not bring about any significant change on this part of the virus genome. All of these spillover isolates maintained a typical canid virus phylogeny. Rabies viruses associated with the family Viverridae form a highly diverse group of viruses, which can be divided into four distinct phylogenetic groups, each associated with a specific geographical area in South Africa. The canid biotype of rabies virus is divided into three specific groups, based on geographic location and the associated reservoir species, namely KwaZulu-Natal province (with domestic dogs as its main vector), the western parts of South Africa (bat-eared foxes) and the northern parts of South Africa (black-backed jackals). In order to determine the degree of genetic change in the virus over a period of time, we identified two endemic canid rabies regions (KwaZulu-Natal and the northern parts of South Africa) and analysed the nucleic acid sequence variation 0f the viruses over 15 years. Phylogenetic analysis of the variable G-L intergenic region of t e virus genome indicated that the canid rabies biotype changed less than 1% over the period studied. This implies that the highly diverse viverrid biotype has been circulating in the southern African wildlife for a very long time. In order to obtain a faster, more economical, and reliable method for rabies virus biotype identification, a competitive, hemi-nested PCR assay was developed. In a single tube, two biotype specific oligonucleotides (developed by Jaftha, 1997), and a common downstream primer were -used in the biotype specific, second round amplification. The specific virus biotypes were identified on the basis of specific amplicon sizes for each biotype. A third biotype specific primer was designed to target a region of the Nucleoprotein gene, this primer was used in a second round hemi-nested reaction. Despite having been designed to specifically amplify canid biotype viruses, this primer amplified all rabies biotypes non¬specifically. We conclude that the nucleoprotein genes are too conserved to make this part of the genome a good target for a biotype-specific PCR diagnostic assay. / Dissertation (MSc (Agric) Microbiology)--University of Pretoria, 1997. / Microbiology and Plant Pathology / unrestricted