Application of high-throughput sequencing to whole rabies viral genome characterisation and its use for phylogenetic re-evaluation of a raccoon strain incursion into the province of Ontario.
ABSTRACT: Raccoon rabies remains a serious public health problem throughout much of the eastern seaboard of North America due to the urban nature of the reservoir host and the many challenges inherent in multi-jurisdictional efforts to administer co-ordinated and comprehensive wildlife rabies control programmes. Better understanding of the mechanisms of spread of rabies virus can play a significant role in guiding such control efforts. To facilitate a detailed molecular epidemiological study of raccoon rabies virus movements across eastern North America, we developed a methodology to efficiently determine whole genome sequences of hundreds of viral samples. The workflow combines the generation of a limited number of overlapping amplicons covering the complete viral genome and use of high throughput sequencing technology. The value of this approach is demonstrated through a retrospective phylogenetic analysis of an outbreak of raccoon rabies which occurred in the province of Ontario between 1999 and 2005. As demonstrated by the number of single nucleotide polymorphisms detected, whole genome sequence data were far more effective than single gene sequences in discriminating between samples and this facilitated the generation of more robust and informative phylogenies that yielded insights into the spatio-temporal pattern of viral spread. With minor modification this approach could be applied to other rabies virus variants thereby facilitating greatly improved phylogenetic inference and thus better understanding of the spread of this serious zoonotic disease. Such information will inform the most appropriate strategies for rabies control in wildlife reservoirs.
Project description:Since the 1990s, oral rabies vaccination (ORV) has been used successfully to halt the westward spread of the raccoon rabies virus (RV) variant from the eastern continental USA. Elimination of raccoon RV from the eastern USA has proven challenging across targeted raccoon (<i>Procyon lotor</i>) and striped skunk (<i>Mephitis mephitis</i>) populations impacted by raccoon RV. Field trial evaluations of the Ontario Rabies Vaccine Bait (ONRAB) were initiated to expand ORV products available to meet the rabies management goal of raccoon RV elimination. This study describes the continuation of a 2011 trial in West Virginia. Our objective was to evaluate raccoon and skunk response to ORV occurring in West Virginia for an additional two years (2012-2013) at 75 baits/km<sup>2</sup> followed by three years (2014-2016) of evaluation at 300 baits/km<sup>2</sup>. We measured the change in rabies virus-neutralizing antibody (RVNA) seroprevalence in targeted wildlife populations by comparing levels pre- and post-ORV during each year of study. The increase in bait density from 75/km<sup>2</sup> to 300/km<sup>2</sup> corresponded to an increase in average post-ORV seroprevalence for raccoon and skunk populations. Raccoon population RVNA levels increased from 53% (300/565, 95% CI: 50-57%) to 82.0% (596/727, 95% CI: 79-85%) during this study, and skunk population RVNA levels increased from 11% (8/72, 95% CI: 6-20%) to 39% (51/130, 95% CI: 31-48%). The RVNA seroprevalence pre-ORV demonstrated an increasing trend across study years for both bait densities and species, indicating that multiple years of ORV may be necessary to achieve and maintain RVNA seroprevalence in target wildlife populations for the control and elimination of raccoon RV in the eastern USA.
Project description: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.
Project description:Background:Raccoon rabies is caused by a variant of the rabies virus found in raccoons but transmissible to other mammalian species, including humans. The disease of rabies caused by raccoon variant rabies virus is indistinguishable from rabies caused by other rabies virus variants. Objective:This paper describes the raccoon rabies outbreak in Ontario (identified in December 2015) and the control measures undertaken to curb the spread of the epizootic using the One Health approach. Investigation and Results:Representatives from local, provincial and federal agencies collectively activated a raccoon rabies response that involved policy updates, enhanced surveillance, a public education campaign and mass vaccination of wildlife and domestic animals. Between December 2015 and June 2017, 338 animals tested positive for raccoon rabies in Ontario. While the majority of the cases were raccoons, there was significant spillover into striped skunks, as well as other species including two cats, a fox and a llama. Viral genome sequencing determined that this epizootic was likely caused by long-distance translocation from the United States. Conclusion:This outbreak of raccoon rabies is by far the largest to have occurred in Canada and the first raccoon rabies outbreak documented in a densely populated urban area. This is also the first time this rabies virus variant has been identified in a domestic animal in Canada. A collaborative approach involving numerous stakeholders in the public and private sectors has been instrumental in addressing this epizootic. Though case incidence appears to be declining, several years will likely be required to reach elimination. Continued collaboration between these agencies is necessary to achieve this goal.
Project description:RABORAL V-RG® is an oral rabies vaccine bait that contains an attenuated ("modified-live") recombinant vaccinia virus vector vaccine expressing the rabies virus glycoprotein gene (V-RG). Approximately 250 million doses have been distributed globally since 1987 without any reports of adverse reactions in wildlife or domestic animals since the first licensed recombinant oral rabies vaccine (ORV) was released into the environment to immunize wildlife populations against rabies. V-RG is genetically stable, is not detected in the oral cavity beyond 48 h after ingestion, is not shed by vaccinates into the environment, and has been tested for thermostability under a range of laboratory and field conditions. Safety of V-RG has been evaluated in over 50 vertebrate species, including non-human primates, with no adverse effects observed regardless of route or dose. Immunogenicity and efficacy have been demonstrated under laboratory and field conditions in multiple target species (including fox, raccoon, coyote, skunk, raccoon dog, and jackal). The liquid vaccine is packaged inside edible baits (i.e., RABORAL V-RG, the vaccine-bait product) which are distributed into wildlife habitats for consumption by target species. Field application of RABORAL V-RG has contributed to the elimination of wildlife rabies from three European countries (Belgium, France and Luxembourg) and of the dog/coyote rabies virus variant from the United States of America (USA). An oral rabies vaccination program in west-central Texas has essentially eliminated the gray fox rabies virus variant from Texas with the last case reported in a cow during 2009. A long-term ORV barrier program in the USA using RABORAL V-RG is preventing substantial geographic expansion of the raccoon rabies virus variant. RABORAL V-RG has also been used to control wildlife rabies in Israel for more than a decade. This paper: (1) reviews the development and historical use of RABORAL V-RG; (2) highlights wildlife rabies control programs using the vaccine in multiple species and countries; and (3) discusses current and future challenges faced by programs seeking to control or eliminate wildlife rabies.
Project description: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.
Project description:The south-central skunk rabies virus (SCSK) is the most broadly distributed terrestrial viral lineage in North America. Skunk rabies has not been efficiently targeted by oral vaccination campaigns and represents a natural system of pathogen invasion, yielding insights to rabies emergence. In the present study we reconstructed spatiotemporal spread of SCSK in the whole territory of its circulation using a combination of Bayesian methods. The analysis based on 241 glycoprotein gene sequences demonstrated that SCSK is much more divergent phylogenetically than was appreciated previously. According to our analyses the SCSK originated in the territory of Texas ~170 years ago, and spread geographically during the following decades. The wavefront velocity in the northward direction was significantly greater than in the eastward and westward directions. Rivers (except the Mississippi River and Rio Grande River) did not constitute significant barriers for epizootic spread, in contrast to deserts and mountains. The mean dispersal rate of skunk rabies was lower than that of the raccoon and fox rabies. Viral lineages circulate in their areas with limited evidence of geographic spread during decades. However, spatiotemporal reconstruction shows that after a long period of stability the dispersal rate and wavefront velocity of SCSK are increasing. Our results indicate that there is a need to develop control measures for SCSK, and suggest how such measure can be implemented most efficiently. Our approach can be extrapolated to other rabies reservoirs and used as a tool for investigation of epizootic patterns and planning interventions towards disease elimination.
Project description:The major histocompatibility complex (MHC) presents a unique system to explore links between genetic diversity and pathogens, as diversity within MHC is maintained in part by pathogen driven selection. While the majority of wildlife MHC studies have investigated species that are of conservation concern, here we characterize MHC variation in a common and broadly distributed species, the North American raccoon (Procyon lotor). Raccoons host an array of broadly distributed wildlife diseases (e.g., canine distemper, parvovirus and raccoon rabies virus) and present important human health risks as they persist in high densities and in close proximity to humans and livestock. To further explore how genetic variation influences the spread and maintenance of disease in raccoons we characterized a fragment of MHC class II DRA exon 3 (250 bp) and DRB exon 2 (228 bp). MHC DRA was found to be functionally monomorphic in the 32 individuals screened; whereas DRB exon 2 revealed 66 unique alleles among the 246 individuals screened. Between two and four alleles were observed in each individual suggesting we were amplifying a duplicated DRB locus. Nucleotide differences between DRB alleles ranged from 1 to 36 bp (0.4-15.8% divergence) and translated into 1 to 21 (1.3-27.6% divergence) amino acid differences. We detected a significant excess of nonsynonymous substitutions at the peptide binding region (P = 0.005), indicating that DRB exon 2 in raccoons has been influenced by positive selection. These data will form the basis of continued analyses into the spatial and temporal relationship of the raccoon rabies virus and the immunogenetic response in its primary host.
Project description:Progressive elimination of rabies in wildlife has been a general strategy in Canada and the United States; common campaign tactics are trap-vaccinate-release (TVR), point infection control (PIC), and oral rabies vaccination (ORV). TVR and PIC are labor intensive and the most expensive tactics per unit area (approximately $616/km(2) [in 2008 Can$, converted from the reported $450/km(2) in 1991 Can$] and approximately $612/km(2) [$500/km(2) in 1999 Can$], respectively), but these tactics have proven crucial to elimination of raccoon rabies in Canada and to maintenance of ORV zones for preventing the spread of raccoon rabies in the United States. Economic assessments have shown that during rabies epizootics, costs of human postexposure prophylaxis, pet vaccination, public health, and animal control spike. Modeling studies, involving diverse assumptions, have shown that ORV programs can be cost-efficient and yield benefit:cost ratios >1.0.
Project description:Enhanced rabies surveillance (ERS) is essential for sound oral rabies vaccination (ORV) decisions to prevent the spread of specific rabies virus variants in meso-carnivores and to achieve disease elimination. Use of a direct rapid immunohistochemistry test (dRIT) in North America for timely, accurate rabies diagnosis in the field has facilitated greater ERS emphasis since 2005. ERS used in tandem with exposure-based public health surveillance provides a comprehensive understanding of the geographic distribution of rabies as an aid to formulate effective management strategies for raccoons and other meso-carnivores. In 2015, best management practices were implemented for improving, reinvigorating, and standardizing ERS. A point system for weighing ERS sample categories was evaluated, to determine whether sampling emphasis should be focused upon ill or strange-acting animals, the highest quality category. During 2016, 70.7% of rabid animals detected through ERS in raccoon rabies management states were obtained from strange-acting animals, followed by animals found dead (14.1%), road kills (9.1%), and nuisance-collected specimens (6.1%). Sample category weights may be adjusted based on additional evaluation to ensure continued emphasis on the highest value samples. High quality ERS, in conjunction with serologic evidence of population-based immunity, form the backbone for ORV decisions in the elimination of raccoon rabies.
Project description: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.