Processes Underlying Rabies Virus Incursions across US-Canada Border as Revealed by Whole-Genome Phylogeography.

Disease control programs aim to constrain and reduce the spread of infection. Human disease interventions such as wildlife vaccination play a major role in determining the limits of a pathogen's spatial distribution. Over the past few decades, a raccoon-specific variant of rabies virus (RRV) has invaded large areas of eastern North America. Although expansion into Canada has been largely prevented through vaccination along the US border, several outbreaks have occurred in Canada. Applying phylogeographic approaches to 289 RRV whole-genome sequences derived from isolates collected in Canada and adjacent US states, we examined the processes underlying these outbreaks. RRV incursions were attributable predominantly to systematic virus leakage of local strains across areas along the border where vaccination has been conducted but also to single stochastic events such as long-distance translocations. These results demonstrate the utility of phylogeographic analysis of pathogen genomes for understanding transboundary outbreaks.

Innate immune responses in raccoons after raccoon rabies virus infection.

Zoonotic wildlife diseases pose significant health risks not only to their primary vectors but also to humans and domestic animals. Rabies is a lethal encephalitis caused by rabies virus (RV). This RNA virus can infect a range of terrestrial mammals but each viral variant persists in a particular reservoir host. Active management of these host vectors is needed to minimize the negative impacts of this disease, and an understanding of the immune response to RV infection aids strategies for host vaccination. Current knowledge of immune responses to RV infection comes primarily from rodent models in which an innate immune response triggers activation of several genes and signalling pathways. It is unclear, however, how well rodent models represent the immune response of natural hosts. This study investigates the innate immune response of a primary host, the raccoon, to a peripheral challenge using the raccoon rabies virus (RRV). The extent and temporal course of this response during RRV infection was analysed using genes predicted to be upregulated during infection (IFNs; IFN regulatory factors; IL-6; Toll like receptor-3; TNF receptor). We found that RRV activated components of the innate immune system, with changes in levels of transcripts correlated with presence of viral RNA. Our results suggest that natural reservoirs of rabies may not mimic the immune response triggered in rodent models, highlighting the need for further studies of infection in primary hosts.

Special Issue "Advances in Rabies Research".

Rabies kills approximately 60,000 humans each year, with deaths mostly occurring in developing countries, where rabies lyssavirus (RABV) variants are maintained in dog populations [...].

Influence of genomic variations on glanders serodiagnostic antigens using integrative genomic and transcriptomic approaches.

Glanders is a highly contagious and life-threatening zoonotic disease caused by Burkholderia mallei (B. mallei). Without an effective vaccine or treatment, early diagnosis has been regarded as the most effective method to prevent glanders transmission. Currently, the diagnosis of glanders is heavily reliant on serological tests. However, given that markedly different host immune responses can be elicited by genetically different strains of the same bacterial species, infection by B. mallei, whose genome is unstable and plastic, may result in various immune responses. This variability can make the serodiagnosis of glanders challenging. Therefore, there is a need for a comprehensive understanding and assessment of how B. mallei genomic variations impact the appropriateness of specific target antigens for glanders serodiagnosis. In this study, we investigated how genomic variations in the B. mallei genome affect gene content (gene presence/absence) and expression, with a special focus on antigens used or potentially used in serodiagnosis. In all the genome sequences of B. mallei isolates available in NCBI's RefSeq database (accessed in July 2023) and in-house sequenced samples, extensive small and large variations were observed when compared to the type strain ATCC 23344. Further pan-genome analysis of those assemblies revealed variations of gene content among all available genomes of B. mallei. Specifically, differences in gene content ranging from 31 to 715 genes with an average of 334 gene presence-absence variations were found in strains with complete or chromosome-level genome assemblies, using the ATCC 23344 strain as a reference. The affected genes included some encoded proteins used as serodiagnostic antigens, which were lost due mainly to structural variations. Additionally, a transcriptomic analysis was performed using the type strain ATCC 23344 and strain Zagreb which has been widely utilized to produce glanders antigens. In total, 388 significant differentially expressed genes were identified between these two strains, including genes related to bacterial pathogenesis and virulence, some of which were associated with genomic variations, particularly structural variations. To our knowledge, this is the first comprehensive study to uncover the impacts of genetic variations of B. mallei on its gene content and expression. These differences would have significant impacts on host innate and adaptive immunity, including antibody production, during infection. This study provides novel insights into B. mallei genetic variants, knowledge which will help to improve glanders serodiagnosis.

Ampliseq for Illumina Technology Enables Detailed Molecular Epidemiology of Rabies Lyssaviruses from Infected Formalin-Fixed Paraffin-Embedded Tissues.

Whole genome sequencing of rabies lyssaviruses (RABVs) has enabled the generation of highly detailed phylogenies that reveal viral transmission patterns of disease in reservoir species. Such information is highly important for informing best practices with respect to wildlife rabies control. However, specimens available only as formalin fixed paraffin embedded (FFPE) samples have been recalcitrant to such analyses. Due to the damage inflicted by tissue processing, only relatively short amplicons can be generated by standard RT-PCR methods, making the generation of full-length genome sequences very tedious. While highly parallel shotgun sequencing of total RNA can potentially overcome these challenges, the low percentage of reads representative of the virus may be limiting. Ampliseq technology enables massively multiplex amplification of nucleic acids to produce large numbers of short PCR products. Such a strategy has been applied to the sequencing of entire viral genomes but its use for rabies virus analysis has not been reported previously. This study describes the generation of an Ampliseq for Illumina primer panel, which was designed based on the global sequence diversity of rabies viruses, and which enables efficient viral genome amplification and sequencing of rabies-positive FFPE samples. The subsequent use of such data for detailed phylogenetic analysis of the virus is demonstrated.

Evaluation of the use of CRISPR loci for discrimination of Salmonella enterica subsp. enterica serovar Enteritidis strains recovered in Canada and comparison with other subtyping methods.

Salmonella enterica subsp. enterica serovar Enteritidis remains one of the most important foodborne pathogens worldwide. To minimise its public health impact when outbreaks of the disease occur, timely investigation to identify and recall the contaminated food source is necessary. Central to this approach is the need for rapid and accurate identification of the bacterial subtype epidemiologically linked to the outbreak. While traditional methods of S. Enteritidis subtyping, such as pulsed field gel electrophoresis (PFGE) and phage typing (PT), have played an important role, the clonal nature of this organism has spurred efforts to improve subtyping resolution and timeliness through molecular based approaches. This study uses a cohort of 92 samples, recovered from a variety of sources, to compare these two traditional methods for S. Enteritidis subtyping with recently developed molecular techniques. These latter methods include the characterisation of two clustered regularly interspaced short palindromic repeats (CRISPR) loci, either in isolation or together with sequence analysis of virulence genes such as fimH. For comparison, another molecular technique developed in this laboratory involved the scoring of 60 informative single nucleotide polymorphisms (SNPs) distributed throughout the genome. Based on both the number of subtypes identified and Simpson's index of diversity, the CRISPR method was the least discriminatory and not significantly improved with the inclusion of fimH gene sequencing. While PT analysis identified the most subtypes, the SNP-PCR process generated the greatest index of diversity value. Combining methods consistently improved the number of subtypes identified, with the SNP/CRISPR typing scheme generating a level of diversity comparable with that of PT/PFGE. While these molecular methods, when combined, may have significant utility in real-world situations, this study suggests that CRISPR analysis alone lacks the discriminatory capability required to support investigations of foodborne disease outbreaks.

Relationships between fox populations and rabies virus spread in northern Canada.

Rabies spreads in both Arctic (Vulpes lagopus) and red foxes (Vulpes vulpes) throughout the Canadian Arctic but limited wildlife disease surveillance, due to the extensive landmass of the Canadian north and its small widely scattered human population, undermines our knowledge of disease transmission patterns. This study has explored genetic population structure in both the rabies virus and its fox hosts to better understand factors that impact rabies spread. Phylogenetic analysis of 278 samples of the Arctic lineage of rabies virus recovered over 40 years identified four sub-lineages, A1 to A4. The A1 lineage has been restricted to southern regions of the Canadian province of Ontario. The A2 lineage, which predominates in Siberia, has also spread to northern Alaska while the A4 lineage was recovered from southern Alaska only. The A3 sub-lineage, which was also found in northern Alaska, has been responsible for virtually all cases across northern Canada and Greenland, where it further differentiated into 18 groups which have systematically evolved from a common predecessor since 1975. In areas of Arctic and red fox sympatry, viral groups appear to circulate in both hosts, but both mitochondrial DNA control region sequences and 9-locus microsatellite genotypes revealed contrasting phylogeographic patterns for the two fox species. Among 157 Arctic foxes, 33 mitochondrial control region haplotypes were identified but little genetic structure differentiating localities was detected. Among 162 red foxes, 18 control region haplotypes delineated three groups which discriminated among the Churchill region of Manitoba, northern Quebec and Labrador populations, and the coastal Labrador locality of Cartwright. Microsatellite analyses demonstrated some genetic heterogeneity among sampling localities of Arctic foxes but no obvious pattern, while two or three clusters of red foxes suggested some admixture between the Churchill and Quebec-Labrador regions but uniqueness of the Cartwright group. The limited population structure of Arctic foxes is consistent with the rapid spread of rabies virus subtypes throughout the north, while red fox population substructure suggests that disease spread in this host moves most readily down certain independent corridors such as the northeastern coast of Canada and the central interior. Interestingly the evidence suggests that these red fox populations have limited capacity to maintain the virus over the long term, but they may contribute to viral persistence in areas of red and Arctic fox sympatry.

A comparison of fourteen fully characterized mammalian-associated Campylobacter fetus isolates suggests that loss of defense mechanisms contribute to high genomic plasticity and subspecies evolution.

Campylobacter fetus is currently classified into three main subspecies, but only two of these, C. fetus subspecies fetus and C. fetus subsp. venerealis originate principally from ruminants where they inhabit different niches and cause distinct pathogenicity. Their importance as pathogens in international trade and reporting is also different yet the criteria defining these properties have never been fully substantiated nor understood. The situation is further compromised because the ability to differentiate between these two closely related C. fetus subspecies has traditionally been performed by phenotypic characterisation of isolates, methods which are limited in scope, time-consuming, tedious, and often yield inconsistent results, thereby leading to isolate misidentification. The development of robust genetic markers that could enable rapid discrimination between C. fetus subsp. fetus and subsp. venerealis has also been challenging due to limited differences in the gene complement of their genomes, high levels of sequence repetition, the small number of closed genome sequences available and the lack of standardisation of the discriminatory biochemical tests employed for comparative purposes. To yield a better understanding of the genomic differences that define these C. fetus strains, seven isolates were exhaustively characterised phenotypically and genetically and compared with seven previously well characterised isolates. Analysis of these 14 C. fetus samples clearly illustrated that adaption by C. fetus subsp. venerealis to the bovine reproductive tract correlated with increasing genome length and plasticity due to the acquisition and propagation of several mobile elements including prophages, transposons and plasmids harbouring virulence factors. Significant differences in the repertoire of the CRISPR (clustered regularly interspersed short palindromic repeats)-cas system of all C. fetus strains was also found. We therefore suggest that a deficiency in this adaptive immune system may have permitted the emergence of extensive genome plasticity and led to changes in host tropism through gene disruption and/or changes in gene expression. Notable differences in the sub-species complement of DNA adenine methylase genes may also have an impact. These data will facilitate future studies to better understand the precise genetic differences that underlie the phenotypic and virulence differences between these animal pathogens and may identify additional markers useful for diagnosis and sub-typing.

Spatial and temporal dynamics of rabies virus variants in big brown bat populations across Canada: footprints of an emerging zoonosis.

Phylogenetic analysis of a collection of rabies viruses that currently circulate in Canadian big brown bats (Eptesicus fuscus) identified five distinct lineages which have emerged from a common ancestor that existed over 400 years ago. Four of these lineages are regionally restricted in their range while the fifth lineage, comprising two-thirds of all specimens, has emerged in recent times and exhibits a recent demographic expansion with rapid spread across the Canadian range of its host. Four of these viral lineages are shown to circulate in the US. To explore the role of the big brown bat host in dissemination of these viral variants, the population structure of this species was explored using both mitochondrial DNA and nuclear microsatellite markers. These data suggest the existence of three subpopulations distributed in British Columbia, mid-western Canada (Alberta and Saskatchewan) and eastern Canada (Quebec and Ontario), respectively. We suggest that these three bat subpopulations may differ by their level of female phylopatry, which in turn affects the spread of rabies viruses. We discuss how this bat population structure has affected the historical spread of rabies virus variants across the country and the potential impact of these events on public health concerns regarding rabies.

A long-distance translocation initiated an outbreak of raccoon rabies in Hamilton, Ontario, Canada.

Despite proactive measures to prevent raccoon rabies entering Canada from the United States, several incursions of this disease have occurred. The largest outbreak, first reported in December 2015 in the city of Hamilton, Ontario, has resulted in the reporting of 449 animal cases as of December 31, 2018. Initial phylogenetic studies on the index case suggested that this outbreak was not due to local cross-border spread from the Niagara region of the United States where raccoon rabies has persisted for several years. Phylogenetic analysis of whole genome sequences of a viral collection from the Hamilton area and several US states indicates that a long-distance translocation of a diseased animal from southeastern New York State was responsible for this incursion. The role of the skunk as a potential secondary host supporting persistence and / or spread of the virus is also examined.

Population- and Variant-Based Genome Analyses of Viruses from Vaccine-Derived Rabies Cases Demonstrate Product Specific Clusters and Unique Patterns.

Rabies in wildlife has been successfully controlled in parts of Europe and North America using oral rabies vaccination, i.e., the distribution of baits containing live-attenuated virus strains. Occasionally, these vaccines caused vaccine virus-induced rabies cases. To elucidate the mechanisms of genetic selection and the effect of viral populations on these rabies cases, a next generation sequencing approach as well as comprehensive data analyses of the genetic diversity of Street Alabama Dufferin (SAD) and ERA vaccine virus strains and vaccine-induced rabies cases from Canada and several European countries were conducted. As a result, twelve newly generated sets of sequencing data from Canada and Poland were added to a pool of previously investigated samples. While the population-based analysis showed a segregation of viruses of ERA vaccine-induced rabies cases from those of SAD Bern original (SAD Bernorig)-derived rabies cases, the in-depth variant analysis revealed three distinct combinations of selected variants for the ERA vaccine-induced cases, suggesting the presence of multiple replication-competent haplotypes in the investigated ERA-BHK21 vaccine. Our findings demonstrate the potential of a deep sequencing approach in combination with comprehensive analyses on the consensus, population, and variant level.

Rabies virus infection in a 21-year-old male presenting with ascending paralysis after a bat scratch.

A 21-year-old, previously healthy male presented to hospital following 1 week of bilateral asymmetric ascending paralysis, odynophagia, and dysphagia. Initial magnetic resonance imaging (MRI) of the spine revealed an abnormal increased T2 signal with predominant dorsal column involvement and sparing of white matter throughout the cervical cord and extending to T5. The initial presumptive diagnosis was an acute infectious, versus inflammatory, myelitis. On reviewing the history, family members recalled a bat scratch on the left hand, sustained months prior, for which the patient did not seek or receive post-exposure prophylaxis (PEP). Rabies virus (RABV) RNA was detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in two saliva samples, while nuchal skin biopsy and cerebrospinal fluid (CSF) were negative. Serum was negative for RABV neutralizing antibody. Sequencing and phylogenetic analyses identified the infecting RABV as a variant associated with silver-haired bats. Following risk assessment of exposure, 67 health care workers and several family members were offered PEP.

Un homme de 21 ans auparavant en santé a consulté à l'hôpital parce qu'il souffrait de paralysie ascendante, asymétrique et bilatérale, d'odynophagie et de dysphagie depuis une semaine. Une première imagerie par résonance magnétique (IRM) du rachis a révélé une augmentation anormale du signal T2 avec atteinte prédominante de la colonne dorsale et épargne de la matière blanche dans toute la colonne cervicale jusqu'à la vertèbre T5. Le diagnostic provisoire en était un de myélite infectieuse, et non inflammatoire. À la prise de l'histoire, les membres de la famille se sont souvenus d'une égratignure de chauve-souris sur la main gauche du patient plusieurs mois auparavant, qui n'a pas été suivie d'une prophylaxie postexposition (PPE). Les chercheurs ont décelé l'ARN du virus de la rage (RABV) par amplification en chaîne par polymérase quantitative de transcription inverse (RT-qPCR) dans deux échantillons de salive, mais constaté un résultat négatif de la biopsie de la peau nucale et du liquide céphalorachidien. Le sérum était négatif à l'anticorps neutralisant du RABV. Les analyses de séquençage et de phylogénétique ont confirmé une contamination par une variante du RABV associée aux chauves-souris argentées. Après une évaluation du risque d'exposition, 67 travailleurs de la santé et plusieurs membres de la famille se sont fait offrir une PPE.

An Unusual Salmonella Enteritidis Strain Carrying a Modified Virulence Plasmid Lacking the prot6e Gene Represents a Geographically Widely Distributed Lineage.

This study identifies a strain of Salmonella enterica subspecies enterica serovar Enteritidis that harbors a highly unusual virulence plasmid. During the characterisation of a group of S. Enteritidis isolates, 10 isolates recovered from Canadian duck production facilities, of which seven were phage type 9b and three were closely related atypical phage types, failed detection by a PCR targeting the prot6e gene, a marker located on the virulence plasmid often employed for identification of this serovar. Comparison to prot6e+ isolates by several standard genetic typing tools, further revealed their distinctive genomic makeup. Both short read and long read whole genome sequencing were completed on six of these isolates. In addition to loss of the prot6e gene, the virulence plasmid of each isolate was found to be exceptionally large (86.5 Kb) due to a 28 Kb insertion of S. Typhimurium plasmid sequence that encodes multiple genes of the incF operon. Interrogation of the chromosome sequence data of these isolates using a SNP-based typing tool and MLST both indicated their close genetic relatedness. One additional isolate carrying this plasmid was identified in an in-house collection of S. Enteritidis isolates. Finally, the identification of this unusual plasmid sequence in additional isolates submitted to public repositories of Salmonella sequence data was explored. All these analyses indicated that a very distinctive but rarely reported strain of S. Enteritidis was widely distributed across North America and the United Kingdom with one additional report involving a case from Brazil. With increased use of genetic methods for Salmonella identification, the loss of the prot6e sequence may confound correct identification of this serovar while also potentially altering the mode of transmission to humans given the gene's role in facilitating propagation of this bacterium in eggs. Accordingly, this strain may present certain challenges with respect to public health investigations. Our studies also suggest this strain is often associated with duck hosts thereby providing a possible mechanism by which this strain has spread over an extensive geographical area.

Mobility of ß-Lactam Resistance Under Bacterial Co-infection and Ampicillin Treatment in a Mouse Model.

Ingestion of food- or waterborne antibiotic-resistant bacteria may lead to the dissemination of antibiotic-resistance genes in the gut microbiota and the development of antibiotic-resistant bacterial infection, a significant threat to animal and public health. Food or water may be contaminated with multiple resistant bacteria, but animal models on gene transfer were mainly based on single-strain infections. In this study, we investigated the mobility of ß-lactam resistance following infection with single- versus multi-strain of resistant bacteria under ampicillin treatment. We characterized three bacterial strains isolated from food-animal production systems, Escherichia coli O80:H26 and Salmonella enterica serovars Bredeney and Heidelberg. Each strain carries at least one conjugative plasmid that encodes a ß-lactamase. We orally infected mice with each or all three bacterial strain(s) in the presence or absence of ampicillin treatment. We assessed plasmid transfer from the three donor bacteria to an introduced E. coli CV601gfp recipient in the mouse gut, and evaluated the impacts of the bacterial infection on gut microbiota and gut health. In the absence of ampicillin treatment, none of the donor or recipient bacteria established in the normal gut microbiota and plasmid transfer was not detected. In contrast, the ampicillin treatment disrupted the gut microbiota and enabled S. Bredeney and Heidelberg to colonize and transfer their plasmids to the E. coli CV601gfp recipient. E. coli O80:H26 on its own failed to colonize the mouse gut. However, during co-infection with the two Salmonella strains, E. coli O80:H26 colonized and transferred its plasmid to the E. coli CV601gfp recipient and a residential E. coli O2:H6 strain. The co-infection significantly increased plasmid transfer frequency, enhanced Proteobacteria expansion and resulted in inflammation in the mouse gut. Our findings suggest that single-strain infection models for evaluating in vivo gene transfer may underrepresent the consequences of multi-strain infections following the consumption of heavily contaminated food or water.

Molecular characterization of the complete genome of a street rabies virus isolated in China.

In this study, the complete genomic sequence of a rabies virus isolate HN10, recovered from brain tissue of a rabid patient in China, was determined. This is the first Chinese street isolate that has been fully characterized. The overall organization of this virus is typical of that observed for all other rabies viruses. Alignments of amino acid sequences of the phosphoprotein, glycoprotein and large protein of HN10 with those of other rabies viruses were used to examine the extent of conservation of known functional regions. Phylogenetic analysis using either the complete or partial genomic sequence of HN10 determined that this isolate is most closely associated with viruses previously shown to circulate in Guangxi and Hunan provinces. In addition, of all vaccine strains used for comparison, the attenuated Chinese vaccine strain CTN181 is most closely related to HN10.

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 patient's saliva over time did not appear to correlate well with clearance of viral components from the brain.

Origins of the arctic fox variant rabies viruses responsible for recent cases of the disease in southern Ontario.

A subpopulation of the arctic fox lineage of rabies virus has circulated extensively in red fox populations of Ontario, Canada, between the 1960s and 1990s. An intensive wildlife rabies control program, in which field operations were initiated in 1989, resulted in elimination of the disease in eastern Ontario. However in southwestern Ontario, as numbers of rabid foxes declined the proportion of skunks confirmed to be infected with this rabies virus variant increased and concerted control efforts targeting this species were employed to eliminate the disease. Since 2012 no cases due to this viral variant were reported in southwestern Ontario until 2015 when a single case of rabies due to the arctic fox variant was reported in a bovine. Several additional cases have been documented subsequently. Since routine antigenic typing cannot discriminate between the variants which previously circulated in Ontario and those from northern Canada it was unknown whether these recent cases were the result of a new introduction of this variant or a continuation of the previous enzootic. To explore the origins of this new outbreak whole genome sequences of a collection of 128 rabies viruses recovered from Ontario between the 1990s to the present were compared with those representative of variants circulating in the Canadian north. Phylogenetic analysis shows that the variant responsible for current cases in southwestern Ontario has evolved from those variants known to circulate in Ontario previously and is not due to a new introduction from northern regions. Thus despite ongoing passive surveillance the persistence of wildlife rabies went undetected in the study area for almost three years. The apparent adaptation of this rabies virus variant to the skunk host provided the opportunity to explore coding changes in the viral genome which might be associated with this host shift. Several such changes were identified including a subset for which the operation of positive selection was supported. The location of a small number of these amino acid substitutions in or close to protein motifs of functional importance suggests that some of them may have played a role in this host shift.

ONRAB® oral rabies vaccine is shed from, but does not persist in, captive mammals.

ONRAB® is a human adenovirus rabies glycoprotein recombinant vaccine developed to control rabies in wildlife. To support licensing and widespread use of the vaccine, safety studies are needed to assess its potential residual impact on wildlife populations. We examined the persistence of the ONRAB® vaccine virus in captive rabies vector and non-target mammals. This research complements work on important rabies vector species (raccoon, striped skunk, and red fox) but also adds to previous findings with the addition of some non-target species (Virginia opossum, Norway rats, and cotton rats) and a prolonged period of post vaccination monitoring (41 days). Animals were directly inoculated orally with the vaccine and vaccine shedding was monitored using quantitative real-time PCR applied to oral and rectal swabs. ONRAB® DNA was detected in both oral and rectal swabs from 6 h to 3 days post-inoculation in most animals, followed by a resurgence of shedding between days 17 and 34 in some species. Overall, the duration over which ONRAB® DNA was detectable was shorter for non-target mammals, and by day 41, no animal had detectable DNA in either oral or rectal swabs. All target species, as well as cotton rats and laboratory-bred Norway rats, developed robust humoral immune responses as measured by competitive ELISA, with all individuals being seropositive at day 31. Similarly, opossums showed good response (89% seropositive; 8/9), whereas only one of nine wild caught Norway rats was seropositive at day 31. These results support findings of other safety studies suggesting that ONRAB® does not persist in vector and non-target mammals exposed to the vaccine. As such, we interpret these data to reflect a low risk of adverse effects to wild populations following distribution of ONRAB® to control sylvatic rabies.

In-depth genome analyses of viruses from vaccine-derived rabies cases and corresponding live-attenuated oral rabies vaccines.

Live-attenuated rabies virus strains such as those derived from the field isolate Street Alabama Dufferin (SAD) have been used extensively and very effectively as oral rabies vaccines for the control of fox rabies in both Europe and Canada. Although these vaccines are safe, some cases of vaccine-derived rabies have been detected during rabies surveillance accompanying these campaigns. In recent analysis it was shown that some commercial SAD vaccines consist of diverse viral populations, rather than clonal genotypes. For cases of vaccine-derived rabies, only consensus sequence data have been available to date and information concerning their population diversity was thus lacking. In our study, we used high-throughput sequencing to analyze 11 cases of vaccine-derived rabies, and compared their viral population diversity to the related oral rabies vaccines using pairwise Manhattan distances. This extensive deep sequencing analysis of vaccine-derived rabies cases observed during oral vaccination programs provided deeper insights into the effect of accidental in vivo replication of genetically diverse vaccine strains in the central nervous system of target and non-target species under field conditions. The viral population in vaccine-derived cases appeared to be clonal in contrast to their parental vaccines. The change from a state of high population diversity present in the vaccine batches to a clonal genotype in the affected animal may indicate the presence of a strong bottleneck during infection. In conclusion, it is very likely that these few cases are the consequence of host factors and not the result of the selection of a more virulent genotype. Furthermore, this type of vaccine-derived rabies leads to the selection of clonal genotypes and the selected variants were genetically very similar to potent SAD vaccines that have undergone a history of in vitro selection.

Complete genome sequence of a raccoon rabies virus isolate.

The entire genome of a mid-Atlantic raccoon strain rabies virus (RRV) isolated in Canada was sequenced; this is the second North American wildlife rabies virus isolate to be fully characterized. The overall organization and length of the genome was similar to that of other lyssaviruses. The nucleotide sequence identity of the raccoon strain ranged between 32.7% and 85.0% when compared to other lyssaviruses, while the deduced amino acid sequence identity ranged between 22.9% and 94.2% with the nucleoprotein and polymerase being the most conserved. Notable features of RRV include the phosphoprotein's four amino acid extension compared to most other rabies viruses, and a nucleotide substitution immediately prior to the normal start codon that results in an additional methionine at the beginning of the L protein. This is the first report of the RRV L gene sequence and its 2128 amino acid product. Rates of non-synonymous and synonymous nucleotide changes within the lyssavirus L gene identified the conserved blocks II, III and IV as being most constrained. Analysis of L gene codon substitution patterns favoured models that supported positive selection, but only one site, corresponding to Leu62 of the RRV L protein, was identified as being under weak positive selection.