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 [...].

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.

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.

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.

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.

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.

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.

Characterization of epitopes on the rabies virus glycoprotein by selection and analysis of escape mutants.

The glycoprotein (G) is the only surface protein of the lyssavirus particle and the only viral product known to be capable of eliciting the production of neutralizing antibodies. In this study, the isolation of escape mutants resistant to monoclonal antibody (Mab) neutralization was attempted by a selection strategy employing four distinct rabies virus strains: the extensively passaged Evelyn Rokitnicki Abelseth (ERA) strain and three field isolates representing two bat-associated variants and the Western Canada skunk variant (WSKV). No escape mutants were generated from either of the bat-associated viral variants but two neutralization mutants were derived from the WSKV isolate. Seven independent ERA mutants were recovered using Mabs directed against antigenic sites I (four mutants) and IIIa (three mutants) of the glycoprotein. The cross-neutralization patterns of these viral mutants were used to determine the precise location and nature of the G protein epitopes recognized by these Mabs. Nucleotide sequencing of the G gene indicated that those mutants derived using Mabs directed to antigenic site (AS) III all contained amino acid substitutions in this site. However, of the four mutants selected with AS I Mabs, two bore mutations within AS I as expected while the remaining two carried mutations in AS II. WSKV mutants exhibited mutations at the sites appropriate for the Mabs used in their selection. All ERA mutant preparations were more cytopathogenic than the parental virus when propagated in cell culture; when in vivo pathogenicity in mice was examined, three of these mutants exhibited reduced pathogenicity while the remaining four mutants exhibited comparable pathogenic properties to those of the parent virus.

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.

Genetic characterization and phylogenetic analysis of skunk-associated rabies viruses in North America with special emphasis on the central plains.

Across North America the skunk acts as a reservoir for several rabies virus variants. Some of these variants are geographically restricted in range as is the case for the California skunk variant and two distinct variants present in Mexico. In contrast the North Central and South Central skunk rabies viruses are dispersed in overlapping ranges over large areas of the Midwestern region of the United States with the former extending into southern parts of the Canadian prairies. Despite this extensive range, there has been only very limited molecular characterization of these two viral variants. This study has examined the genetic diversity of the rabies viruses associated with North American skunks, with particular emphasis on the South Central skunk variant which was found to comprise three distinct geographically restricted groups of viruses that could in some cases be further sub-divided. The phylogenetic relationships of these groups and sub-groups allowed us to infer the likely direction of spread of these variants in some instances. Patterns of amino acid replacement of North American skunk-associated rabies viruses for both the nucleoprotein and glycoprotein products are also examined. These patterns reflect the virus phylogeny but no amino acid residues associated specifically with the skunk host were identified.

Recent emergence of the Arctic rabies virus lineage.

The rabies viruses that circulate in Arctic countries and in much of northern and central Asia are phylogenetically closely related and collectively referred to as the Arctic/Arctic-like (AL) lineage. The emergence and spread of this lineage is of significant interest given that rabies remains a serious zoonotic disease in many parts of Asia, especially in India where the prevalence of dog rabies leads to frequent human exposures and deaths. Previous molecular epidemiological studies of rabies viruses in India identified the AL lineage as the type circulating across much of the country. To further explore the relationship of Indian and Arctic rabies viruses, a collection of samples recovered from Rajasthan state in northern India was characterised at the N gene locus. Combination of these data with a larger collection of samples from India, central/northern Asia and the Arctic has permitted detailed phylogenetic analysis of this viral lineage and estimation of its time-frame of emergence. These analyses suggest that most current Indian viruses emerged from a common progenitor within the last 40 years and that the entire Arctic/AL lineage emerged within the last 200 years, a time-frame in accord with historical records of the invasion of Canada by the Arctic clade.

Generation and characterization of a panel of anti-phosphoprotein monoclonal antibodies directed against Mokola virus.

The generation of a new panel of 21 monoclonal antibodies (MAbs) reactive with the P protein of Mokola virus (MOKV) is described. Through competitive ELISA and immunoblotting analyses, these MAbs were classified into several groups. Consistent with prior studies on lyssavirus P protein antigenic structure, many of the sites recognized by these Mabs appear to correspond to sites identified previously. Studies on the reactivity of these anti-MOKV P MAbs against a collection of lyssaviruses identified MAbs that were broadly cross-reactive to all genus members and others that bound selectively to members of different species. In particular the utility of this MAb panel for differentiation of African lyssaviruses was explored. Such a panel will be useful for further examination of the extent of functional complementation between lyssavirus P proteins.

Genetic tracking of the raccoon variant of rabies virus in eastern North America.

To gain insight into the incursion of the raccoon variant of rabies into the raccoon population in three Canadian provinces, a collection of 192 isolates of the raccoon rabies virus (RRV) strain was acquired from across its North American range and was genetically characterized. A 516-nucleotide segment of the non-coding region between the G and L protein open reading frames, corresponding to the most variable region of the rabies virus genome, was sequenced. This analysis identified 119 different sequences, and phylogenetic analysis of the dataset supports the documented history of RRV spread. Three distinct geographically restricted RRV lineages were identified. Lineage 1 was found in Florida, Alabama and Georgia and appears to form the ancestral lineage of the raccoon variant of rabies. Lineage 2, represented by just two isolates, was found only in Florida, while the third lineage appears broadly distributed throughout the rest of the eastern United States and eastern Canada. In New York State, two distinct spatially segregated variants were identified; the one occupying the western and northern portions of the state was responsible for an incursion of raccoon rabies into the Canadian province of Ontario. Isolates from New Brunswick and Quebec form distinct, separate clusters, consistent with their independent origins from neighboring areas of the United States. The data are consistent with localized northward incursion into these three separate areas with no evidence of east-west viral movement between the three Canadian provinces.

Molecular phylogenetics of the lyssaviruses--insights from a coalescent approach.

Technical improvements over the past 2 decades have enormously facilitated the generation of nucleotide sequence data for lyssavirus collections. These databases are amenable to methods of phylogenetic analysis, which attempt to define the taxonomic structure of this genus and predict the evolutionary relationships of current circulating strains. Coupled with a range of mathematical tools to explore the appropriateness of nucleotide substitution models and test for positive selection, the evolutionary process is being explored in detail. Despite the potential for high viral mutation levels, the operation of purifying selection appears to effectively constrain lyssavirus evolution. The recent development of coalescent theory has provided additional approaches to data analysis whereby the time frame of emergence of viral lineages can be most reliably estimated. Such studies suggest that all currently circulating rabies viruses have emerged within the past 1500 years. Moreover, through the capability of analyzing viral population dynamics and determining patterns of population size variation, coalescent approaches can provide insight into the demographics of viral outbreaks. Whereas human-assisted movement of reservoir host species has clearly facilitated transfer of rabies between continents, topographical landscape features significantly influence the rate and extent of contiguous disease spread. Together with empirical studies on virus diversity, the application of coalescent approaches will help to better understand lyssavirus emergence, evolution, and spread. In particular, such methods are presently facilitating exploration of the factors operating to limit the ability of lyssaviruses to establish new persistent virus-host associations and ultimately control the emergence of new species of this genus.

Re-assessment of direct fluorescent antibody negative brain tissues with a real-time PCR assay to detect the presence of raccoon rabies virus RNA.

The first report of the raccoon variant of rabies virus was in Ontario, Canada in 1999. As part of the control of this outbreak a Point Infection Control (PIC) strategy of trapping and euthanizing vector species was implemented. To evaluate whether this strategy was indeed removing diseased animals, rabies diagnosis was performed on these specimens. During a PIC program conducted in 2003, 721 animals (raccoons, striped skunks and red foxes) were collected and euthanized and brain material from each specimen was divided into two halves; one half was submitted for rabies diagnosis by a direct fluorescent antibody (DFA) test while the other was tested using a sensitive real-time reverse-transcriptase polymerase chain reaction (RT-qPCR), to detect raccoon rabies virus (RRV) RNA. This latter assay can detect less than ten viral copies in 200ng of total cellular RNA. All 721 PIC brain samples were negative by the DFA test but ten of them (5 raccoons, 5 skunks) tested positive for raccoon rabies virus by the RT-qPCR assay albeit at low levels. Three of these samples were confirmed by sequencing of the PCR products. Little correlation was observed between clinical rabies DFA positive scoring categories and viral copy number as determined by RT-qPCR.

Characterization of a panel of anti-phosphoprotein monoclonal antibodies generated against the raccoon strain of rabies virus.

The generation of a new panel of 32 monoclonal antibodies (MAbs) reactive with the P protein of the raccoon strain of rabies virus is described. Through a series of analyses employing competitive ELISA and immunoblotting, these MAbs were classified into eight groups, each defining an antigenic site, thereby increasing the number of sites now recognized along the length of the P protein. Studies on MAb reactivity with a collection of diverse lyssaviruses identified sites that were highly conserved, moderately conserved and highly variable. Several groups of MAbs were highly specific for the raccoon rabies virus (RRV) strain and may be useful for inclusion into panels used for antigenic typing of rabies viruses. The utility of these MAbs to detect truncated versions of the P product may facilitate more fundamental studies on the function of this rabies virus protein.

Transmission dynamics of rabies in China over the last 40 years: 1969-2009.

BACKGROUND: Rabies is a serious reemerging zoonosis in China. The molecular evolution and transmission patterns of rabies virus inferred from historical data can provide guidelines for better disease control and prevention in the future. OBJECTIVES: To investigate the epidemiology and evolutionary dynamics of the rabies virus in China. STUDY DESIGN: The molecular evolution of 132 viral glycoprotein gene sequences of Chinese rabies viruses collected in 17 provinces and 3 municipalities between 1969 and 2009 was analyzed. RESULTS: Phylogenetic analysis revealed that Chinese rabies viruses are subdivided into 6 lineages (A-F) within Lyssavirus genotype 1. Lineage A represents the widely dispersed cosmopolitan lineage while lineage B is closely related to Arctic-like rabies viruses. The remaining lineages (C-F) are typical of those circulating across much of Southeast Asia. The evolutionary rate for Chinese rabies virus was 1.532 x 10(-4) substitutions per site per year, and the corresponding common ancestor was in about 1115. CONCLUSIONS: The phylogeographic structure demonstrated Chinese rabies viruses have been transmitted intra-provincially and extra-provincially due to human-related activities.

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.

Development of a reverse genetics system for a human rabies virus vaccine strain employed in China.

The CTN rabies virus was isolated from a human in China in 1953 and subsequently attenuated by multiple passaging to a vaccine strain now approved by the WHO. In this study, we describe the development of a reverse genetics system for the CTN rabies virus strain. The recombinant full-length genomic cDNA was flanked by a hammerhead ribozyme (HamRz) and the hepatitis delta virus ribozyme (HdvRz) while the non-coding G-L region was replaced with a green fluorescent protein (GFP) gene. A set of helper plasmids encoding nucleoprotein (N), phosphoprotein (P), and large protein (L) were constructed and co-transfected with recombinant full-length genome plasmid into BHK-21 cells. Recombinant virus was successfully recovered from cloned cDNA under control of the CMV promoter driven by RNA polymerase II. The recombinant virus, CTN-GFP, stably expressed GFP as detected by fluorescence microscopy. A group of 1-day-old suckling mice was challenged with the CTN-GFP strain by intracerebral inoculation, resulting in 100% morbidity and GFP expression was detected in brain tissues. The recombinant virus CTN-GFP strain recovered from cloned cDNA will be useful as a viral vector to express other foreign genes.

Safety studies on an adenovirus recombinant vaccine for rabies (AdRG1.3-ONRAB) in target and non-target species.

A replication-competent human adenovirus vector in which the rabies virus glycoprotein gene was inserted (AdRG1.3-ONRAB) was given by direct instillation into the oral cavity to representatives of three wildlife vector species of concern in Ontario (red fox, raccoon and striped skunk) and to a variety of non-target wildlife species, domestic and laboratory species. Despite use of a relatively high dose of vaccine, no untoward clinical signs were observed. Subsequent to vaccine exposure, detection of vaccine virus in lung, spleen, intestine, liver, kidney and brain of each animal was attempted using an ONRAB-specific assay combining PCR with Southern blotting (PCR-SB). Of the 1280 tissue samples obtained from vaccinates or contact animals, 18 (1.4%) were found to be PCR-SB positive. Virus isolation attempts were performed utilizing cell culture for all PCR-SB positive tissues and a selection of PCR-SB negative tissues. Histological examination performed on all PCR-SB positive tissues failed to identify lesions attributed to the vaccine. A quantitative real-time PCR was used to determine the excretion of the vaccine in feces and in the oral cavity with 0.8% of oral swabs and 6.8% of fecal specimens found to be positive. The low rates of recovery of vaccine virus from tissues, feces and the oral cavity suggest that the likelihood of ONRAB causing a negative impact on wildlife species is unlikely.