Molecular genetic analysis of some enzootic rabies viruses of southern Africa

Abstract

Rabies viruses are known to be able to infect a broad range of warm-blooded animals. In South Africa the disease is maintained in different animal species including dogs, jackals, bateared foxes and a variety of members of the viverridae family. These include the different mongoose species (principally the yellow mongoose), genets, suricates and a variety of small carmvores. The antigenic variation within the nucleoprotein gene has previously been investigated in efforts to characterise various isolates of rabies viruses in southern Africa. It was noted that two antigenically distinct groups are cocirculating in the country. In this study, the investigation into the epidemiology of rabies in South Africa was extended by molecular genetic analysis of a large number of isolates from wildlife and domestic animal hosts. Geographically and temporally distinct rabies viruses were previously studied by comparative nucleotide sequence analysis of DNA fragments encompassing the cytoplasmic domain of the glycoprotein and the G-L intergenic region. Based on this analysis two main rabies virus groups were identified. Members of the first group infect canid host species and are closely related to the European rabies strains, while the viruses belonging to the second group circulate within different viverrid species. Although isolates of mainly mongoose origin were initially analysed, considerable heterogeneity within this group was evident. The current study was consequently undertaken to include rabies virus isolates from other viverrid host species. Following the same approach as the previous investigators (Nel et al., 1993 & von Teichman et al., 1995) four genetically distinct clusters were indicated within the viverrid lineage. These clusters corresponded closely to the geographic origin of the virus isolates independent of specific viverrid host. The results suggest genetic divergence and independent evolution of the viverrid viruses within geographically isolated regions. Spillover or cross-infection, where a viverrid virus is recovered from a canid host and vice versa, could not be attributed to a new rabies virus and were most likely initiated by interspecies transmission events. These results suggest little modification of the virus following infection of an atypical host. A phylogeny of the rabies virus variants in southern Africa was established based on sequence variation within the abovementioned genome regions. Although such an approach is most informative when compared to other methods, it is time-consuming and laborious. The use of strain-specific oligonucleotides was therefore investigated for rapid strain differentiation. Two oligonucleotides were designed based on the nucleotide sequences of the cytoplasmic domain of the glycoprotein and the G-L intergenic region. These oligonucleotides together with a common downstream primer were used to amplify DNA fragments of characteristic size, allowing for discrimination between the two rabies biotypes.

Afrikaans: Dit is bekend dat die hondsdolheidsvirus verskeie warmbloedige diere kan infekteer. In suider Afrika word die siekte onderhou in gasheer spesies soos honde, jakkalse, bakoorvosse en lede van die Viverridae familie. Dit sluit verskeie meerkat spesies (hoofsaaklik die witkwasmuishond), kleinkolmuskejaatkat, stokstertmeerkat en 'n verskeidenheid klein karnivoorspesies in. Die antigeniese variasie binne die nukleoprotei"en geen is voorheen aangewend in 'n poging om hondsdolheidsvirus-isolate in suidelike Afrika te karakteriseer. Uit hierdie ondersoek het dit geblyk dat daar twee antigenies-onderskeibare virusgroepe in die land sirkuleer. In hierdie studie word die ondersoek na die epidemiologie van hondsdolheid in Suid-Afrika uitgebrei deur middel van genetiese analise van 'n groot aantal isolate afkomstig vanaf wilde- sowel as huishoudelike diere. Die genetiese variasie van geografies- en temporaal-verwyderde isolate is vantevore deur middel van vergelykende nukleotiedvolgorde-analise ondersoek. Hierdie vergelykings het die nuletensuurvolgordes van die sitoplasmiese gebied van die glikoprote1en en die G-L intergeniese gebied ingesluit. Hierdie analise het twee hoofgroepe hondsdolheidsvirusse aangedui. Lede van die eerste groep infekteer canid gasheer spesies en is nou verwant aan die Europese hondsdolheidsvirusse, terwyl die tweede groep binne verskillende viverrid spesies sirkuleer. Alhoewel daar aanvanklik virus isolate vanuit hoofsaaklik die witkwasmuishond ondersoek is, was daar heelwat heterogenisiteit binne die viverrid virusse gevind. Gevolglik is hierdie studie onderneem om hondsdolheidsvirusse vanaf antler viverrid gashere in te sluit. Deur gebruik te maak van dieselfde benadering as vorige navorsers (Nel et al., 1993; von Teichman et al., 1995) is vier geneties onderskeibare groepe binne die viverrid stamboom aangetoon. Hierdie groepe het nou ooreengestem met die geografiese oorsprong van die virus isolate, onafhanklik van 'n spesifieke gasheer. Hierdie resultate dui daarop dat onafhanklike ewolusie van die viverrid virusse binne ge1soleerde lokaliteite plaasvind. Kruis-infeksie, waar viverrid virusse canid gashere en omgekeerd infekteer, kon nie toegeskryf word aan 'n nuwe hondsdolheidsvirus me. Hierdie verskynsel word vermoedelik deur interspesie oordraag gebeure ge'inisieer. 'n Filogenetiese verwantskap van hondsdolheidsvirusse in suider Afrika is bepaal op grond van van die nuleotiedvolgorde variasie in bogenoemde genomiese gebiede. In vergeleke met antler metodes, is hierdie benadering baie informatief, maar dit kan tydrowend en moeisaam wees. Die gebruik van ras-spesifieke voorvoerders is daarom ondersoek as 'n alternatiewe metode vir differensiasie. Twee voorvoerders is ontwerp gebaseer op die volgordes van die sitoplasmiese gebied van die glikoprote1en en die G-L intergeniese gebied. Deur hierdie voorvoerders tesame met 'n gemeenskaplike stroom-af voorvoerder te gebruik word DNA fragmente van karakteristieke groottes geamplifiseer. Hierdie benadering voorsien dus 'n makliker manier vir onderskeiding tussen die twee virus groepe.