Susan A. Nadin-Davis

Antigenic and genetic divergence of rabies viruses from bat species indigenous to Canada

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.

Polymerase chain reaction protocols for rabies virus discrimination.

The development of RT PCR methodology has facilitated greatly the genetic characterisation of many rabies viruses (RVs), distinct strains of which persist in certain host species reservoirs within geographically defined regions. The relative temporally conserved nature of certain regions of the RV genome, particularly the N gene, permits development of rapid molecular methods for RV typing. Two main strategies have been applied to viral discrimination: (1) restriction fragment length polymorphism (RFLP) of PCR products and (2) strain-specific PCR (SS-PCR), in which sequences of specific viral strains are amplified differentially using strain-specific primers. Both these approaches have yielded methods of value to rabies epidemiological studies and control programs in Ontario. These procedures have facilitated the identification of intra-strain variants of the arctic fox strain, the only terrestrial RV strain persisting in the area, and they allow rapid discrimination of this strain from those circulating in insectivorous bat reservoirs and from the foreign raccoon strain, which continues to spread throughout the northeastern US and threatens to enter Ontario. Such methods can be adapted readily for use in other regions harbouring multiple overlapping RV reservoirs.

A molecular epidemiological study of rabies virus in central Ontario and western Quebec

Rabies persists in Ontario wildlife in two predominant species: the red fox (Vulpes vulpes) and the striped skunk (Mephitis mephitis). A protocol applying reverse transcription/polymerase chain reaction (RT/PCR) and restriction endonuclease analysis (REA) to the rabies virus nucleoprotein gene was previously reported by Nadin-Davis et al. (Journal of General Virology 74, 829-837, 1993) to be useful for discrimination of rabies virus variants in Ontario. Four main types, which showed no host species specificity but which did exhibit different geographical distributions, were identified. Between 1989 and 1992 an area north and west of the city of North Bay experienced unusual and substantial rabies activity. In this report we describe the use of these molecular techniques to investigate the epidemiology of this recent rabies outbreak in central Ontario. It is shown that two of the four previously identified variants had invaded this region from the south and east, but in addition viruses very closely related to arctic isolates of rabies virus were found. The nucleoprotein and glycoprotein genes of this arctic type were sequenced and compared to those of its more southerly neighbours.

Identification of Regional Variants of the Rabies Virus within the Canadian Province of Ontario

Although rabies outbreaks in most parts of the world tend to be host species-specific the rabies currently enzootic in the Canadian province of Ontario is hosted by two wildlife species, the red fox and the striped skunk. Previous studies employing monoclonal antibody panels failed to identify any host-specific differences in Ontario rabies virus street isolates, but certain observations suggested the existence of more than one viral strain in terrestrial mammals of this region. The extent of variation of the rabies virus circulating within this region has been re-examined using molecular biology techniques. The N gene of several independent isolates was amplified using PCR and the resulting products were compared by restriction enzyme analysis and, in some cases, by DNA sequencing. This analysis confirmed that there was indeed no host-specific variation in the portion of the viral genome under study but there were, however, very clear and consistent differences in the virus from distinct geographical regions.

Detection of polyoma and corona viruses in bats of Canada.

Several instances of emerging diseases in humans appear to be caused by the spillover of viruses endemic to bats, either directly or through other animal intermediaries. The objective of this study was to detect, identify and characterize viruses in bats in the province of Manitoba and other regions of Canada. Bats were sampled from three sources: live-trapped Myotis lucifugus from Manitoba, rabies-negative Eptesicus fuscus, M. lucifugus, M. yumanensis, M. septentrionalis, M. californicus, M. evotis, Lasionycteris (L.) noctivagans and Lasiurus (Las.) cinereus, provided by the Centre of Expertise for Rabies of the Canadian Food Inspection Agency (CFIA), and L. noctivagans, Las. cinereus and Las. borealis collected from a wind farm in Manitoba. We attempted to isolate viruses from fresh tissue samples taken from trapped bats in cultured cells of bat, primate, rodent, porcine, ovine and avian origin. We also screened bat tissues by PCR using primers designed to amplify nucleic acids from members of certain families of viruses. We detected RNA of a group 1 coronavirus from M. lucifugus (3 of 31 animals) and DNA from an as-yet undescribed polyomavirus from female M. lucifugus (4 of 31 animals) and M. californicus (pooled tissues from two females).

Phylogeographic patterns exhibited by Ontario rabies virus variants.

A previous study on N gene variation of rabies viruses circulating in Ontario red foxes identified four viral variants. This study confirms the geographical localization of these variants and extends the analysis to the less conserved G gene of these viruses. A greater number of regionally localized variants was revealed and their phylogenetic relationships have been examined. Ongoing surveillance on recent disease outbreaks revealed that variants do not always persist in specific areas. The distribution of these variants did however appear to be influenced by topographical features of the study area likely to affect host animal movements and contacts. The majority of G gene base changes were synonymous and limited glycoprotein sequence variation predominantly to the C-terminal transmembrane and endo-domains. These data are most readily explained by random appearance of genetic viral variants followed by their spread throughout sub-populations of the fox host according to the easiest routes of transmission.

Development of real-time reverse transcriptase polymerase chain reaction methods for human rabies diagnosis.

To improve timely ante‐mortem human rabies diagnosis, methods to detect viral RNA by TaqMan‐based quantitative reverse transcriptase polymerase chain reactions (qRT‐PCRs) have been developed. Three sets of two primers and one internal dual‐labeled probe for each primer set that target distinct conserved regions of the rabies virus N gene were designed and evaluated. Using a collection of 203 isolates representative of the world‐wide diversity of rabies virus, all three primers/probe sets were shown to detect a wide range of rabies virus strains with very few detection failures; the RABVD1 set in particular was the most broadly reactive. These qRT‐PCR assays were shown to be quantitative over a wide range of viral titer and were 100–1,000 times more sensitive than nested RT‐PCR; however, both the standard and real‐time PCR methods yielded concordant results when used to test a collection of archived human suspect samples. The qRT‐PCR assay was employed to monitor virus load in the saliva of a rabies virus‐infected patient undergoing the Milwaukee treatment protocol. However in this case it would appear that reduction of the viral load in the patients saliva over time did not appear to correlate well with clearance of viral components from the brain. J. Med. Virol. 81:1484–1497, 2009.

Molecular and antigenic characterization of rabies viruses from Iran identifies variants with distinct epidemiological origins.

A molecular epidemiological study of 48 recent rabies isolates recovered from cases reported throughout Iran identified three distinct viral variants, the evolutionary origins of which were identified by phylogenetic comparison with rabies viruses originating from Europe and Asia. Members of group 1 (15 isolates) were recovered from the northern half of the country only, while those of group 2 (31 isolates) were widely dispersed; both groups clustered within the widely disseminated cosmopolitan lineage. The two isolates of group 3 were detected in the northeastern tip of the country only and belonged to the Arctic strain. Rapid variant discrimination tools, employing restriction fragment length polymorphisms applied to amplified fragments of the viral genome, were devised whilst antigenic characterization of representative viruses identified a small panel of monoclonal antibodies that were also discriminatory. The future application of such methods should provide valuable epidemiological information on rabies incidence in Iran.

ERA VACCINE-DERIVED CASES OF RABIES IN WILDLIFE AND DOMESTIC ANIMALS IN ONTARIO, CANADA, 1989–2004

A vaccination program for the control of terrestrial rabies in the province of Ontario, Canada, began in 1989. During the period between 1989 and 2004, over 13 million baits containing the live, attenuated rabies virus ERA-BHK21 were distributed across the province, with the aim of immunizing foxes by the oral route. Animals recovered from bait distribution areas were assayed by fluorescent antibody test for rabies virus infection. Immunoreactivity with a panel of monoclonal antibodies that discriminate between ERA and rabies virus variants known to circulate in Ontario, and molecular genetic analyses were used to identify animals infected with ERA. Nine cases of ERA variant rabies were identified over the 16-yr period of study; these did not appear to be stratified by species, year of discovery, or location of capture. The ERA-positive animals were found across the province in eight counties, all of which had been baited in the year of case discovery. The nine ERA-positive cases included four red foxes (Vulpes vulpes), two raccoons (Procyon lotor), two striped skunks (Mephitis mephitis), and one bovine calf (Bos taurus). Molecular phylogenetic analyses of the partial N gene sequences generated from these isolates indicated that these nine cases were due to infection with the ERA variant. The glycoprotein sequences were predicted from G gene sequencing of all nine field isolates and two laboratory stock ERA viruses. This revealed some heterogeneity at residue 120 (either arginine or histidine) in both field and laboratory stocks as well as a few other mutations in field isolates. The significance of this heterogeneity remains unclear. Our data demonstrate that the ERA vaccine distributed in Ontario carried residual pathogenicity; however, there does not appear to be any evidence of ERA establishment in wildlife populations over the 16-yr period. These results are consistent with previous reports of the rare detection of ERA vaccine–induced rabies and with laboratory studies of ERA pathogenicity.

The design of strain-specific polymerase chain reactions for discrimination of the raccoon rabies virus strain from indigenous rabies viruses of Ontario

Since its recognition as a discrete epizootic in Florida in the early 1950s, the raccoon strain of rabies virus (RV) has spread over almost the entire eastern seaboard of the US and now threatens to enter the southernmost regions of Canada. To characterise this RV strain in more detail, nucleotide sequencing of the N and G genes, encoding the nucleoprotein and glycoprotein, respectively, of representative isolates has been undertaken. This sequence information generated a conserved restriction map of the N gene, thereby permitting unequivocal identification of this strain by molecular techniques. Comparisons of the predicted nucleoprotein and glycoprotein products with those of other RV strains identified a number of amino acid sequence variations conserved only in the raccoon strain. This information was used to design strain-specific primers targeted to the N gene sequences encoding these residues. The incorporation of these primers into a multiplex polymerase chain reaction (PCR) protocol permitted easy and rapid discrimination between the raccoon RV strain and indigenous Ontario RVs.