Genetic analysis of rabies and rabies-related viruses in southern Africa, with emphasis on virus isolates associated with atypical infection patterns

Abstract

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.