Rabies is caused by several Lyssavirus species, a group of negative sense RNA viruses. Although rabies is preventable, it is often neglected particularly in developing countries in the face of many competing public and veterinary health priorities. Epidemiological information based on laboratory-based surveillance data is critical to adequately strategise control and prevention plans. In this regard the fluorescent antibody test for rabies virus antigen in brain tissues is still considered the basic requirement for laboratory confirmation of animal cases. Occasionally brain tissues from suspected rabid animals are still submitted in formalin, although this has been discouraged for a number of years. Immunohistochemical testing or a modified fluorescent antibody technique can be performed on such samples. However, this method is cumbersome and cannot distinguish between different Lyssavirus species. Owing to RNA degradation in formalin-fixed tissues, conventional RT-PCR methodologies have also been proven to be unreliable. This report is concerned with a rabies case in a domestic dog from an area in South Africa where rabies is not common. Typing of the virus involved was therefore important, but the only available sample was submitted as a formalin-fixed specimen. A real-time RT-PCR method was therefore applied and it was possible to confirm rabies and obtain phylogenetic information that indicated a close relationship between this virus and the canid rabies virus variants from another province (KwaZulu-Natal) in South Africa.

Nucleotide sequence analysis of a 200-bp region of the nucleoprotein (N) gene of rabies virus differentiated unique genetic groups of rabies virus from samples collected in areas where dog rabies is enzootic in Asia, Africa, Europe, and the Americas. Patterns of nucleotide sequence identified for an outbreak area were conserved in samples collected over three decades. Epidemiologic relationships among isolates were determined by patterns of conserved nucleotide sequence, and the degree of sequence divergence between samples from separate outbreak areas were measured. This approach suggested that a historical reconstruction of events leading to the introduction of rabies into an area would be possible. In this broader view of rabies epidemiology, the cultural legacy of European exploration and colonization may have also included zoonotic disease.

A heminested reverse transcriptase PCR (hnRT-PCR) protocol which is rapid and sensitive for the detection of rabies virus and rabies-related viruses is described. Sixty isolates from six of the seven genotypes of rabies and rabies-related viruses were screened successfully by hnRT-PCR and Southern blot hybridization. Of the 60 isolates, 93%(56 of 60) were positive by external PCR, while all isolates were detected by heminested PCR and Southern blot hybridization. We also report on a comparison of the sensitivity of the standard fluorescent-antibody test (FAT) for rabies antigen and that of hnRT-PCR for rabies viral RNA with degraded tissue infected with a genotype 1 virus. Results indicated that FAT failed to detect viral antigen in brain tissue that was incubated at 37 degrees C for greater than 72 h, while hnRT-PCR detected viral RNA in brain tissue that was incubated at 37 degrees C for 360 h.

The predominant role of Eptesicus serotinus in the epizootic of bat rabies in Europe was further outlined by the first isolation of the rabies virus from this species in France. The distribution of the virus was studied in naturally infected E. serotinus bats at the time of death and suggested that the papillae of the tongue and the respiratory mucosa may play a role in virus production and excretion. The analysis of 501 French rabies virus isolates from various animal species by antinucleocapsid monoclonal antibodies indicated that transmission of the disease from bats to terrestrial animals is unlikely. The antigenic profile of two isolates from French bats corresponded to that of European bat lyssavirus type 1 (EBL1). Comparisons of 12 different isolates from bats with antinucleocapsid and antiglycoprotein monoclonal antibodies and by direct sequencing of the polymerase chain reaction amplification product of the N gene indicated that EBL1, EBL2, Duvenhage virus (serotype 4 of lyssavirus), and the European fox rabies virus (serotype 1) are phylogenetically distant. They formed four tight genetic clusters named genotypes. EBL1 was shown to be antigenically and genetically more closely related to Duvenhage virus than to EBL2. We propose that EBL1 and EBL2 constitute two distinct genotypes which further serologic characterization will probably classify as new serotypes. We also report a simple method for the rapid characterization of EBL based on the digestion of the polymerase chain reaction product of the N gene by three restriction endonucleases.

Lagos bat virus and an isolate from shrews (IbAn 27377), both from Nigeria, were found to be bullet-shaped and to mature intracytoplasmically in association with a distinct matrix. They were related to, but readily distinguishable from, rabies virus and each other by complement fixation and neutralization tests. The three viruses, including rabies, form a subgrouping within the rhabdoviruses.

The evolution of rabies viruses of predominantly European origin was studied by comparing nucleotide sequences of the nucleoprotein and glycoprotein genes, and by typing isolates using RFLP. Phylogenetic analysis of the gene sequence data revealed a number of distinct groups, each associated with a particular geographical area. Such a pattern suggests that rabies virus has spread westwards and southwards across Europe during this century, but that physical barriers such as the Vistula river in Poland have enabled localized evolution. During this dispersal process, two species jumps took place - one into red foxes and another into raccoon dogs, although it is unclear whether virus strains are preferentially adapted to particular animal species or whether ecological forces explain the occurrence of the phylogenetic groups.

Passage of the mouse-adapted rabies virus strain CVS-24 (where CVS is challenge virus standard) in BHK cells results in the rapid selection of a dominant variant designated CVS-B2c that differs genotypically and phenotypically from the dominant variant CVS-N2c present in mouse-brain- or neuroblastoma-cell-passaged CVS-24. The glycoprotein of CVS-B2c has 10 amino acid substitutions compared with that of CVS-N2c. Because CVS-B2c can be reproducibly selected in BHK cells, it is likely to be a conserved minor subpopulation of CVS-24. CVS-N2c is more neurotropic in vitro and in vivo than CVS-B2c, which replicates more readily in nonneuronal cells in vitro and in vivo. These characteristics appear to be relevant to the pathogenicity of the two variants. CVS-N2c is more pathogenic for adult mice than CVS-B2c. In contrast, CVS-B2c is more pathogenic for neonatal mice. These differences in pathogenicity are reflected in the selection pattern when mixtures of CVS-N2c and CVS-B2c were used to infect neonatal and adult mice. Although CVS-N2c was highly selected in adult mice, no selection for either variant was seen in neonates, suggesting that certain aspects of development, such as maturation of the nervous and immune systems, may contribute to the selection process. We speculate that the existence of different variants within a rabies virus strain may facilitate the virus in overcoming barriers to its spread, both within the host and between species.

Antigenic characterisation of over 350 chiropteran rabies viruses of the Americas, especially from species reported rabid in Canada, distinguished 13 viral types. In close accord with this classification, nucleotide sequencing of representative isolates, at both the N and G loci, identified four principal phylogenetic groups (I-IV), sub-groups of which circulated in particular bat species. Amongst the North American bat viruses, there was a notable division between group I specimens associated with colonial, non-migratory bats (Myotis sp. and Eptesicus fuscus) and those of group II harbored by solitary, migratory species (Lasiurus sp. and Lasionycteris noctivagans). Certain species of Myotis were clearly identified as rabies reservoirs, an observation often obscured previously by their frequent infection by viral variants of other chiroptera. An additional group (III) apparently circulates in E. fuscus, whilst viruses harbored by both insectivorous and haematophagus bats of Latin America clustered to a separate clade (group IV). Comparison of the predicted N and G proteins of these viruses with those of strains of terrestrial mammals indicated a similarity in structural organisation regardless of host species lifestyle. Finally, these sequences permitted examination of the evolutionary relationship of American bat rabies viruses within the Lyssavirus genus.

Prior to 1988, rabies was reported only sporadically in coyotes. However, in the final 4 months of 1988, Starr County, Tex, which is situated on the US-Mexico border, experienced an epizootic of canine rabies, consisting of 6 laboratory-confirmed cases of rabies in coyotes and of 2 cases in domestic dogs. The first 3 cases were detected in coyotes, and the first case in a domestic dog was observed 84 days after the index case. Adjacent Hidalgo County reported 9 cases of rabies in dogs during the same time that rabid dogs were being reported in Starr County. In 1989, the epizootic primarily involved dogs: 15 dogs in Starr County and 19 dogs in Hidalgo County. Five rabid coyotes were reported in Starr County in 1989, and 1 rabid coyote was reported from Hidalgo County. In 1990, rabies was reported in 3 coyotes and in 31 dogs in Starr County; cases were not detected in Hidalgo County. During 1991, the epizootic expanded approximately 160 km northward, resulting in laboratory-confirmed cases in 42 coyotes and 25 dogs in 10 counties. In 1992, Webb and Willacy Counties became involved; 70 rabid coyotes and 41 rabid dogs were reported in 1992 from the 12-county area. During the first 6 months of 1993, there were 31 rabid coyotes and 38 rabid dogs reported from the same 12 south Texas counties. In May 1993, a raccoon infected with the canine rabies ecotype was reported from Cameron County. Antigenic and genetic analysis revealed the virus ecotype affecting dogs and coyotes to be that associated with urban canine rabies along the US-Mexico border.

The nucleocapsid antigen of 204 strains of street rabies virus, which originated in Europe, Africa and Asia, was analyzed by fluorescent antibody staining technique with a panel of 20 monoclonal antibodies specific for nucleocapsid of rabies and rabies-related viruses. A definite pattern of reactivity was observed with strains of the same geographic origin with the exception of strains originating from Madagascar, Thailand and Iran which were more diversified. Mice immunized with a vaccine prepared from a Pasteur PV-11 strain of virus were well protected against challenge with representative strains from Europe and Africa, and a partial protection was observed following challenge with strains from Madagascar and Thailand.

A panel of 23 monoclonal antibodies to the nucleocapsid protein of rabies virus was used to study the antigenic character of isolates of rabies virus from raccoons in the mid-Atlantic region of the United States. Comparison of the reaction pattern of these isolates with that of isolates of rabies virus collected from areas of major rabies outbreaks (skunk rabies in the midwestern United States, fox rabies in the northeastern United States, and raccoon rabies in the southeastern United States) suggests that this new epizootic originated with the transportation of rabid raccoons from the southeastern United States.