Rapid identification of the raccoon rabies virus variant using a real-time reverse-transcriptase polymerase chain reaction.

The raccoon-associated variant of rabies virus (RRV) is enzootic throughout the eastern seaboard of the United States with frequent incursions into Canada. Many wildlife management agencies are actively engaged in control programmes targeting elimination of this disease and rapid identification of raccoon rabies cases is crucial to the success of these operations. This report documents the development of a reverse transcriptase real-time PCR (RT-qPCR) that specifically identifies this rabies virus variant (RRV RT-qPCR) and which can be readily multiplexed with a generic rabies virus RT-qPCR for use as a typing tool. Using a large collection of rabies virus samples representative of the variants circulating around the world, but with a focus on those occurring in the Americas, the RRV RT-qPCR was 100% sensitive and 99.31% specific. To further apply these assays for diagnostic purposes, addition of an RT-qPCR targeting the host ß-actin mRNA, which serves as an internal amplification control, in a triplex format was shown to yield highly comparable results using a subset of our viral collection. Use of these assays for early and accurate identification of this viral variant will help to optimize the utilization of resources required for control of this disease.

Geography but not alternative host species explain the spread of raccoon rabies virus in Vermont.

In North America, the raccoon-associated variant of rabies virus (RRV) is of special concern, given its relatively rapid spread throughout the eastern USA and its potential public health impact due to high raccoon host densities in urban areas. Northward expansion of this epizootic included an outbreak in the Canadian province of Quebec in 2006-2009 due to trans-border spread from the State of Vermont. To inform a more proactive approach to future control efforts, this study uses phylogenetic analyses to explore the role of geography and alternative carnivore hosts in the dynamics of RRV spread within Vermont. Specifically, we sought to examine whether striped skunks, a species frequently infected by RRV, could be part of the maintenance host community. Whole genome sequencing of 160 RRV samples from Vermont and neighbouring US states were used for fine-scale phylogeographic analyses. Results, together with the complete surveillance record of raccoon rabies since its entry into Vermont in 1994, document incursions by two distinct viral lineages and identify topographical features of the landscape which have significantly influenced viral spread, resulting in a complex distribution pattern of viral variants throughout the state. Results of phylogenetic cluster analysis and discrete state reconstruction contained some evidence of skunk-to-skunk and skunk-to-raccoon transmission but overall failed to support a role for skunks as alternative maintenance hosts.

A molecular epidemiological study of rabies in Cuba.

To investigate the emergence and current situation of terrestrial rabies in Cuba, a collection of rabies virus specimens was employed for genetic characterization. These data supported the monophyletic nature of all terrestrial rabies viruses presently circulating in Cuba but additionally delineated several distinct variants exhibiting limited spatial distribution which may reflect the history of rabies spread on the island. The strain of rabies currently circulating in Cuba, which emerged on the island in the early 20th century, has very close evolutionary ties to the Mexican dog type and is a member of the cosmopolitan lineage widely distributed during the colonial period. The Cuban rabies viruses, which circulate predominantly within the mongoose population, are phylogenetically distant from viruses circulating in mongooses in other parts of the world. These studies illustrate, at a global level, the adaptation of multiple strains of rabies to mongoose species which should be regarded as important wildlife hosts for rabies re-emergence. Given the recent emergence of human cases due to bat contact in Cuba, this study also included a single insectivorous bat specimen which was found to most closely resemble the rabies viruses known to circulate in Mexican vampire bats.

A molecular epidemiological analysis of the incursion of the raccoon strain of rabies virus into Canada.

Three physically separate incursions of the raccoon strain of rabies have entered Canada, two into eastern Ontario in 1999 and one into New Brunswick in 2000. The course of these epizootics is described. Phylogenetic analysis of the index cases from these two provinces with raccoon rabies viruses representative of this strain in the United States supported the independence of these incursions into Canada via cross-border transmission from the United States. Genetic characterization of 190 isolates from these two Canadian provinces over a 550-bp region of the variable central portion of the viral P gene distinguished 14 variants in Ontario and five in New Brunswick although in both regions the variant represented by the initial case was most commonly encountered. The quasi-species nature of the Ontario virus was analysed using isolates taken at different times during the main outbreak to examine whether viral variation was increasing with time as well as changing in nature. These data provide a framework for study of future incursions of this rabies strain into Canada.

Antigenic and genetic characterization of rabies viruses isolated from domestic and wild animals of Brazil identifies the hoary fox as a rabies reservoir.

Fifty Brazilian rabies viruses, collected from many different animal species and several regions of the country, were characterized by partial sequencing of the central, variable region of the P gene, a locus useful for sensitive molecular epidemiological studies. Phylogenetic analysis of the sequences, which included comparison with other rabies strains recovered from throughout the Americas, identified three main groups of Brazilian viruses, arbitrarily designated BRL-1 to BRL-3. BRL-1 was found in terrestrial carnivores and clusters with other American strains of the cosmopolitan lineage. BRL-2 comprised two distinct isolates, recovered from two species of non-haematophagous bats, that had evolutionary links to insectivorous-bat-derived strains of North America. BRL-3 consisted of isolates from vampire bats and from livestock species probably infected via contact with vampire bats. The terrestrial group was further subdivided into three subtypes: BRL-1a was associated exclusively with dogs and cats, while BRL-1b and BRL-1c were found exclusively in hoary foxes. These observations strongly support the role of the Brazilian hoary fox as a rabies reservoir. Screening of representative Brazilian rabies viruses against a collection of anti-rabies monoclonal antibodies (mAbs) identified a small panel of mAbs that could be used to discriminate between all Brazilian subgroups as defined by genetic classification in this study.

Detection of multiple strains of rabies virus RNA using primers designed to target Mexican vampire bat variants.

A reverse transcription-polymerase chain reaction (RT-PCR), that uses primers specifically designed to amplify a portion of the N gene of vampire bat strains of rabies that circulate in Mexico, but also recognizing most of the rabies variants circulating in endemic areas, was established. This standardized PCR assay was able to detect viral RNA in tenfold serial dilutions up to a 10(7) dilution using stock virus at an original titre of 10(7.5) LD50. The assay was highly specific for rabies virus. Forty different rabies isolates recovered from different species and geographical regions in the country were diagnosed as positive and negative by the fluorescent antibody test (FAT). These same samples were re-examined by both PCR and the mouse inoculation test (MIT). Compared with MIT the PCR exhibited an epidemiological sensitivity of 86% and a specificity of 91% while its positive predictive value was 96%.

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.

Lyssavirus P gene characterisation provides insights into the phylogeny of the genus and identifies structural similarities and diversity within the encoded phosphoprotein.

A comprehensive phylogenetic analysis of the Lyssavirus genus, employing P gene sequences from 128 isolates recovered globally, is presented. While confirming prior suggestions of the genetic distinctness of the Australian bat lyssaviruses, these data also revealed a clear division within the rabies virus clade (Genotype 1) between globally distributed viruses circulating predominantly in canid species (subgroup 1-1), and American strains harbored by both chiropteran and terrestrial hosts (subgroup 1-2). Nucleotide substitution patterns within the P locus suggested differential selection operating along the length of the open reading frame (ORF) between rabies viruses of these two subgroups. Comparison of the deduced primary sequences of the encoded phosphoproteins of all isolates provided insights into the product's structural organization. Two conserved (CD1,2) and two variable (VD1,2) domains were evident; high variation in the VD2 region was reflected by differences in hydropathic profiles. Only two of five serine residues reported to function as phosphate acceptors in the P protein of the rabies challenge virus standard (CVS) strain were absolutely conserved; similarly, out of four internal methionines reported to direct internal translation initiation of the CVS strain to produce N-terminally truncated P proteins, only Met(20) was universally retained. In contrast, two sequence motifs, one believed to confer binding to the cytoplasmic dynein light chain LC8, and a lysine-rich sequence probably contributing to N protein binding, were conserved throughout the genus. Most rabies viruses of the carnivora (1-1) contain a potential C ORF in alternate frame to that of P, a feature limited or absent in most other isolates of the genus, an observation interpreted with consideration to the predicted course of lyssavirus evolution.

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.

A panel of monoclonal antibodies targeting the rabies virus phosphoprotein identifies a highly variable epitope of value for sensitive strain discrimination.

A recombinant rabies virus phosphoprotein fusion product (GST-P) was used to generate a series of monoclonal antibodies (MAbs) with anti-P reactivity. Competitive binding assays classified 27 of these MAbs into four groups (I to IV), and 24 of them were deemed to recognize linear epitopes, as judged by their reaction in immunoblots. The linear epitope recognized in each case was mapped by using two series of N- and C-terminally deleted recombinant phosphoproteins. Assessment of the reactivities of representative MAbs to a variety of lyssavirus isolates by an indirect fluorescent antibody test indicated that group I MAbs, which recognized a highly conserved N-terminal epitope, were broadly cross-reactive with all lyssaviruses assayed, while group III MAbs, which reacted with a site overlapping that of group I MAbs, exhibited variable reactivities and group IV MAbs reacted with most isolates of genotypes 1, 6, and 7 only. In contrast, group II MAbs, which recognized an epitope located within a highly divergent central portion of the protein, were exquisitely strain specific. These anti-P MAbs are potentially useful tools for lyssavirus identification and discrimination.

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.

Site-directed mutagenesis of large plasmids.

A protocol combining recombination PCR and long-distance PCR is demonstrated to be highly accurate and rapid for site-directed mutagenesis of large (> 10 kb) plasmids. Application of this protocol to the generation of mutant rabies virus glycoproteins expressed by the baculovirus/insect cell system illustrates the usefulness of this approach in facilitating structure-function relationships in this important eukaryotic expression system.

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.

Polymorphism of rabies viruses within the phosphoprotein and matrix protein genes.

Although the rabies virus P and M genes, encoding the viral phosphoprotein and matrix protein respectively, have been characterised for a few laboratory-adapted strains, there is essentially no information on the variability of these two genes in wild-type rabies viruses. In this study rabies viruses, responsible for epizootics in different wildlife species in three geographically distinct areas of North America, have been characterised at the P and M gene loci. These data reveal that the M gene and its encoded product are much more conserved than the P gene and its encoded phosphoprotein. The latter product harbors two variable domains which contribute to different hydropathy profiles for this protein for each of the rabies virus strains studied. Phylogenetic analysis of the nucleotide sequences generated in this study, together with data generated previously on the N and G genes of these rabies virus strains, indicated that similar evolutionary relationships are predicted regardless of the portion of the viral genome targeted.

The design of strain-specific polymerase chain reactions for discrimination of the racoon 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.

Bovine herpesvirus 1 isolates contain variable copy numbers of GC-rich tandem repeats in the gI non-coding regions of their genomes.

A polymerase chain reaction (PCR) targeted to the central portion of the bovine herpesvirus 1 (BHV1) genome, and overlapping the 3' untranslated end of the gI glycoprotein, was used to amplify BHV1 genomic sequences. PCR products generated from cell cultures infected with BHV1.1 were consistently smaller than the corresponding products from cells infected with BHV1.2. The nature of the sequence differences between these isolates within the target region was found to be a consequence of variable numbers of small GC rich repeats, particularly the sequence 5'-G(A/T)CC-3', present in the region downstream of the gI coding region. Based on these differences a modified PCR protocol which readily discriminated between several BHV1.1 and BHV1.2 strains was devised.

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.

Isolation and structure of an acetolactate synthase gene from Schizosaccharomyces pombe and complementation of the ilv2 mutation in Saccharomyces cerevisiae.

A gene encoding a functional acetolactate synthase (ALS) subunit has been isolated from the fission yeast Schizosaccharomyces pombe, and has been structurally and genetically characterized. The approximate 5-kbp cloned DNA segment was found to contain a 2007-bp open reading frame capable of encoding a 669 aminoacid polypeptide which exhibited 57.1% similarity to the corresponding ALS subunit from Saccharomyces cerevisiae. The putative ilv1 isolated from S. pombe was shown to encode a functional subunit of acetolactate synthase by complementation of an S. cerevisiae strain deleted for the ILV2 locus.