Taiwan Bat Lyssavirus: In Vitro and In Vivo Assessment of the Ability of Rabies Vaccine-Derived Antibodies to Neutralise a Novel Lyssavirus.

Rabies is a neglected tropical disease. The prototype virus, the rabies virus, still causes tens of thousands of human fatalities annually. Rabies is one member of the genus Lyssavirus. The burden of other lyssaviruses is unclear. The continued emergence of novel lyssaviruses means that assessment of vaccine efficacy against these viruses is critical, as standard rabies vaccines are not efficacious against all lyssaviruses. Taiwan bat lyssavirus (TWBLV) was first reported in 2018 following isolation from Japanese house bats. Since the initial detection and genetic characterisation, no attempts have been made to antigenically define this virus. Due to the inaccessibility of the wildtype isolate, the successful generation of a live recombinant virus, cSN-TWBLV, is described, where the full-length genome clone of the RABV vaccine strain, SAD-B19, was constructed with the glycoprotein of TWBLV. In vitro and in vivo characterization of cSN-TWBLV was undertaken and demonstrated evidence for cross-neutralisation of cSN-TWBLV with phylogroup I -specific sera and rabies virus standard sera. For neutralisation equivalent to 0.5 IU/mL of WHO and World Organisation of Animal Health (WOAH) sera against CVS, 0.5 IU/mL of WOAH sera and 2.5 IU/mL of WHO sera were required to neutralise cSN-TWBLV. In addition, specific sera for ARAV and EBLV-1 exhibited the highest neutralising antibody titres against cSN-TWBLV, compared to other phylogroup I-specific sera.

Scaling-up the delivery of dog vaccination campaigns against rabies in Tanzania.

An increasing number of countries are committing to meet the global target to eliminate human deaths from dog-mediated rabies by 2030. Mass dog vaccination is central to this strategy. To interrupt rabies transmission from dogs to humans, the World Health Organization recommends that vaccination campaigns should be carried out every year in all dog-owning communities vaccinating 70% of their susceptible dogs. Monitoring and evaluation of dog vaccination campaigns are needed to measure progress towards elimination. In this study, we measured the delivery performance of large-scale vaccination campaigns implemented in 25 districts in south-east Tanzania from 2010 until 2017. We used regression modelling to infer the factors associated with, and potentially influencing the successful delivery of vaccination campaigns. During 2010-2017, five rounds of vaccination campaigns were carried out, vaccinating in total 349,513 dogs in 2,066 administrative vaccination units (rural villages or urban wards). Progressively more dogs were vaccinated over the successive campaigns. The campaigns did not reach all vaccination units each year, with only 16-28% of districts achieving 100% campaign completeness (where all units were vaccinated). During 2013-2017 when vaccination coverage was monitored, approximately 20% of vaccination units achieved the recommended 70% coverage, with average coverage around 50%. Campaigns were also not completed at annual intervals, with the longest interval between campaigns being 27 months. Our analysis revealed that districts with higher budgets generally achieved higher completeness, with a twofold difference in district budget increasing the odds of a vaccination unit being reached by a campaign by slightly more than twofold (OR: 2.29; 95% CI: 1.69-3.09). However, higher budgets did not necessarily result in higher coverage within vaccination units that were reached. We recommend national programs regularly monitor and evaluate the performance of their vaccination campaigns, so as to identify factors hindering their effective delivery and to guide remedial action.

A recombinase polymerase amplification assay for rapid detection of rabies virus.

Rabies is a generally fatal encephalitis caused by a negative-sense single-stranded RNA lyssavirus transmitted to humans mainly from dog bite. Despite the recommendation by WHO and OIE to use the direct immunofluorescence test as standard method, molecular diagnostic assays like reverse transcription quantitative polymerase chain reaction (RT-qPCR) are increasing as a confirmatory method. However, both technologies are inaccessible in resource-limited settings. Moreover, the available point-of-need molecular assay is of poor detection limit for African strains. Herein, we developed a reverse transcription recombinase polymerase amplification (RT-RPA) assay as potential point-of-need diagnostic tool for rapid detection of various strains of rabies virus including locally isolated African strains. The sensitivity and specificity of the method was evaluated using a molecular RNA standard and different Rabies-related viruses belonging to the Rhabdoviridea family, respectively. The RABV-RPA performances were evaluated on isolates representative of the existing diversity and viral dilutions spiked in non-neural clinical specimen. The results were compared with RT-qPCR as a gold standard. The RABV-RPA detected down to 4 RNA molecules per reaction in 95% of the cases in less than 10 min. The RABV-RPA assay is highly specific as various RABV isolates were identified, but no amplification was observed for other member of the Rhabdoviridea family. The sample background did not affect the performance of the RABV-RPA as down to 11 RNA molecules were identified, which is similar to the RT-qPCR results. Our developed assay is suitable for use in low-resource settings as a promising alternative tool for ante-mortem rabies diagnosis in humans for facilitating timely control decisions.

Rabies control in Liberia: Joint efforts towards zero by 30.

Despite declaration as a national priority disease, dog rabies remains endemic in Liberia, with surveillance systems and disease control activities still developing. The objective of these initial efforts was to establish animal rabies diagnostics, foster collaboration between all rabies control stakeholders, and develop a short-term action plan with estimated costs for rabies control and elimination in Liberia. Four rabies diagnostic tests, the direct fluorescent antibody (DFA) test, the direct immunohistochemical test (dRIT), the reverse transcriptase polymerase chain reaction (RT-PCR) assay and the rapid immunochromatographic diagnostic test (RIDT), were implemented at the Central Veterinary Laboratory (CVL) in Monrovia between July 2017 and February 2018. Seven samples (n=7) out of eight suspected animals were confirmed positive for rabies lyssavirus, and molecular analyses revealed that all isolates belonged to the Africa 2 lineage, subgroup H. During a comprehensive in-country One Health rabies stakeholder meeting in 2018, a practical workplan, a short-term action plan and an accurately costed mass dog vaccination strategy were developed. Liberia is currently at stage 1.5/5 of the Stepwise Approach towards Rabies Elimination (SARE) tool, which corresponds with countries that are scaling up local-level interventions (e.g. dog vaccination campaigns) to the national level. Overall an estimated 5.3 - 8 million USD invested over 13 years is needed to eliminate rabies in Liberia by 2030. Liberia still has a long road to become free from dog-rabies. However, the dialogue between all relevant stakeholders took place, and disease surveillance considerably improved through implementing rabies diagnosis at the CVL. The joint efforts of diverse national and international stakeholders laid important foundations to achieve the goal of zero dog-mediated human rabies deaths by 2030.

A bioeconomic model for the optimization of local canine rabies control.

We present a new modeling tool that can be used to maximize the impact of canine rabies management resources that are available at the local level. The model is accessible through a web-based interface that allows for flexibility in the management strategies that can be investigated. Rabies vaccination, sterilization, chemo-contraception, and euthanasia can be specified and limited to specific demographic groups. Additionally, we allowed for considerable complexity in the specification of management costs. In many areas, the costs of contacting additional dogs increases as management effort increases, and this can have important strategic implications. We illustrated the application of the model by examining several alternative management strategies in an area of Mpumalanga Province, South Africa. Our results based on this dog population suggested that puppies should be vaccinated and sterilization would not be optimal if the spatial extent of management is not large (and perhaps not even then). Furthermore, given a sufficient budget, it was evident that vaccination campaigns should be repeated annually.

Rescue of a wild-type rabies virus from cloned cDNA and assessment of the proliferative capacity of recombinant viruses.

Reverse genetic systems (RGS) have been widely used for fixed rabies virus (RABV) strains. However, RGS, for wild-type (wt) strains, have been seldom reported despite the value of this approach in defining the biological characteristics of these strains. In this work, we developed a wt RGS using a swine-origin RABV strain (GD-SH-01) for the first time. In order to have a better understanding of the contribution and function of individual gene on viral proliferation for wt RABV isolates, we constructed a full-length cDNA clone of GD-SH-01 and exchanged the single genes encoding RABV protein of a highly attenuated RABV strain HEP-Flury with those of the virulent strain. Analysis of the viral growth kinetics, cell-to-cell spread, and genomic RNA (gRNA) synthesis of the both the rescued and parental virus strains revealed that replacement of the HEP-Flury N or L genes with those from GD-SH-01 resulted in higher proliferative capacity of both chimeric rHEP-shN and rHEP-shL while the former seemed to have a better viral gRNA synthesis ability, the latter spread faster. Replacement of HEP-Flury P gene with GD-SH-01 P gene resulted in reduction of the virus titer in cell culture supernatants with a poor replicative and spreading ability. However, replacement of HEP-Flury M or G genes with those from GD-SH-01 seemed to impact less on viral proliferation. Taken together, we show that we have successfully rescued a wt RABV strain, and assessed the impact of each gene on viral proliferative capacity using a series of single-gene-substituted viruses.

Assessment the Efficiency of the Constructed Minigenome of Rabies Virus using PV Strain as Helper Virus.

INTRODUCTION: Rabies is an acute viral disease that causes encephalomyelitis in mammals and human. The only way to prevent this disease is through vaccination before or after exposure. The aim of this study is to evaluate the efficiency of the Pasteur virus (PV) minigenome, using PV strain. MATERIALS AND METHODS: Enhanced Green Fluorescent Protein (EGFP) sequence was placed between the designed necessary elements (Hammerhead, HDV ribozyme, 3' Leader, and 5' Trailer sequences), which resemble the rabies virus PV strain (PV2061) genome and anti-genome. These constructs were placed between T7 polymerase promoter and T7 polymerase terminator sequences. The accuracy of the minigenome was confirmed by the expression of EGFP using the helper virus in T7-BHK cell line. RESULTS: The viral necessary elements of positive and negative sense strands were evaluated for the ability of EGFP expression in the presence of the helper virus. While the positive strand showed background results, no EGFP background was observed in the negative strand application. CONCLUSION: Establishment of minigenome system does not require advanced biosafety levels. Furthermore, using minigenome system eliminates many potential confounding factors that may be present in coding regions of the genome. Use of the minigenome system is easier and more feasible than the full genome rescue of the virus. This study successfully shows the efficiency of the constructed rabies virus minigenome in expression of inserted gene.

Development and evaluation of a recombinant-glycoprotein-based latex agglutination test for rabies virus antibody assessment.

The measurement of neutralizing antibodies induced by the glycoprotein of rabies virus is indispensable for assessing the level of neutralizing antibodies in animals or humans. A rapid fluorescent focus inhibition test (RFFIT) has been approved by WHO and is the most widely used method to measure the virus-neutralizing antibody content in serum, but a rapid test system would be of great value to screen large numbers of serum samples. To develop and evaluate a latex agglutination test (LAT) for measuring rabies virus antibodies, a recombinant glycoprotein was expressed in an insect cell system and purified, and the protein was coated onto latex beads at concentrations of 0.1, 0.25, 0.5, 0.75, and 1 mg/ml to find out the optimal concentration for coating latex beads. It was found that 0.5 mg/ml of recombinant protein was optimal for coating latex beads, and this concentration was used to sensitize the latex beads for screening of dog serum samples. Grading of LAT results was done with standard reference serum with known antibody titers. A total of 228 serum samples were tested, out of which 145 samples were positive by both RFFIT and LAT, and the specificity was found to be 100 %. In RFFIT, 151 samples were positive, the sensitivity was found to be 96.03 %, and the accuracy/concordance was found to be 97.39 %. A rapid field test-a latex agglutination test (LAT)-was developed and evaluated for rabies virus antibody assessment using recombinant glycoprotein of rabies virus expressed in an insect cell system.

Rabid fox bites and human rabies in a village community in southern India: epidemiological and laboratory investigations, management and follow-up.

Human rabies transmitted from wild animals is rarely reported in endemic countries like India, where nearly 95% deaths occur due to bites from rabid dogs. In this paper, we report an incidence of rabid fox bites in a village in southern part of India involving 18 individuals, including 4 children. All people had category III exposures, including bites on the face and neck. The attacking fox was killed by the forest department and buried immediately. The victims of the fox bite did not receive appropriate and adequate postexposure treatment. Thirteen days after the bite, one of the bite victims developed typical symptoms of furious rabies and died 2 days later in a local hospital. His brain tissue, obtained at autopsy, was strongly positive for rabies by fluorescent antibody technique (FAT) and virus isolation. Panic prevailed in the community and the rest of the 17 cases were referred to our institute for advice and further management. Only 35% of them had protective levels of rabies virus neutralizing antibodies (RVNA). All of the patients were administered with an 8-site intradermal regimen with purified chick embryo cell (PCEC) vaccine and were followed up regularly. All of them developed adequate titers (>0.5 IU/mL) of RVNA 7 days later. They were under regular follow-up and after nearly 2 years none have developed rabies. The partial Nucleoprotein (N) gene sequencing of the virus isolate from the patient who died of rabies had close homology with species I (prototype rabies) sequences available in GenBank and our own past isolates from dogs and humans, thus confirming that virus spillover from wildlife to domestic dogs continues to occur. This episode should prompt health authorities to focus more attention on training rural medical practitioners in state-of-the-art modern prophylactic measures.

An assessment of ONRAB oral rabies vaccine persistence in free-ranging mammal populations in Ontario, Canada.

ONRAB is a rabies glycoprotein recombinant human adenovirus type 5 oral vaccine developed for application in baits to control rabies in wildlife populations. Prior to widespread use of ONRAB, both the safety and effectiveness of this vaccine required investigation. While previous research has focused on field performance and the persistence and pathogenicity of ONRAB in captive animals, we sought to examine persistence and shedding of ONRAB in populations of free-ranging target and non-target mammals. We collected oral and rectal swab samples from 84 red foxes, 169 striped skunks, and 116 raccoons during 2007 and 2008 in areas where ONRAB vaccine baits were distributed. We also analyzed 930 tissue samples, 135 oral swab and 138 rectal swab samples from 155 non-target small mammals from 10 species captured during 2008 at sites treated with high densities of ONRAB vaccine baits. Samples were screened for the presence and quantity of ONRAB DNA using quantitative real-time PCR. None of the samples that we analyzed from target and non-target species contained quantities of ONRAB greater than 10(3)EU/mL of ONRAB DNA which is a limit that has previously been applied to assess viral shedding. This study builds on similar research and suggests that replication of ONRAB in animals is short-lived and the likelihood of horizontal transmission to other organisms is low.

Expression and solubilization of insect cell-based rabies virus glycoprotein and assessment of its immunogenicity and protective efficacy in mice.

Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed in Spodoptera frugiperda (Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine.

[Temporal and spatial population dynamics of rabies virus isolates in China].

In order to study phylogeography, population dynamics and molecular evolution of rabies viruses (RABVs) isolates from China, especially spatio-temporal dynamics, the timescale of RABVs evolution and its pattern of migration, we performed an extensive comparative analysis of RABV N gene sequence data, representing 167 isolates sampled from 20 provinces in a 78-year period (from 1931 through 2009). The available Chinese isolates could be divided into two distinct clades:Phylogroup clades I comprised Chinese group 1-4; Phylogroup clades II contained Chinese group 5-8. We found no evidence for positive selection (dN/dS>1) acting at any codon and found strong selective constraints for N gene. Bayesian Markov Chain Monte Carlo (MCMC) analysis suggested that the Chinese rabies viruses originated within the last 2000 years and the mean rates of nucleotide substitution for the N gene were approximately 4 x 10(-4) substitutions per site per year. The analyses of the spatial and spatio-temporal evolution indicated that RABV isolates from China migrated among different Provinces.

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.

Phylogeography of rabies virus isolated from dogs in Brazil between 1985 and 2006.

To establish the phylogeographic relationships in rabies viruses in Brazil, we studied a dataset retrieved from GenBank consisting of 71 genetic sequences from the coding region of the N gene of rabies viruses isolated in dogs over a period of 22 years. The Bayesian Markov chain Monte Carlo method available in the BEAST package was used with the GTR+G+Г4 evolutionary model in conjunction with the relaxed uncorrelated lognormal molecular clock model and an exponential growth tree prior. A discrete phylogeographic diffusion model was also analyzed using a standard continuous-time Markov chain viewed with Google Earth to provide a spatial projection of the diffusion of genetic lineages based on their phylogeographic relationships. The topology of the time and substitution phylogenetic trees agreed with the spatial dispersal of the lineages. It was possible to infer that the lineages in the southeastern region of Brazil in the 1970s are the closest to the most common recent ancestor and that all the lineages in the midwestern, northern and northeastern regions are more distant. The importance of this study lies in the fact that it can help with the planning of rabies control measures, as dogs continue to be the main reservoir of rabies virus throughout the world.

Host phylogeny constrains cross-species emergence and establishment of rabies virus in bats.

For RNA viruses, rapid viral evolution and the biological similarity of closely related host species have been proposed as key determinants of the occurrence and long-term outcome of cross-species transmission. Using a data set of hundreds of rabies viruses sampled from 23 North American bat species, we present a general framework to quantify per capita rates of cross-species transmission and reconstruct historical patterns of viral establishment in new host species using molecular sequence data. These estimates demonstrate diminishing frequencies of both cross-species transmission and host shifts with increasing phylogenetic distance between bat species. Evolutionary constraints on viral host range indicate that host species barriers may trump the intrinsic mutability of RNA viruses in determining the fate of emerging host-virus interactions.

Bayesian phylogeography finds its roots.

As a key factor in endemic and epidemic dynamics, the geographical distribution of viruses has been frequently interpreted in the light of their genetic histories. Unfortunately, inference of historical dispersal or migration patterns of viruses has mainly been restricted to model-free heuristic approaches that provide little insight into the temporal setting of the spatial dynamics. The introduction of probabilistic models of evolution, however, offers unique opportunities to engage in this statistical endeavor. Here we introduce a Bayesian framework for inference, visualization and hypothesis testing of phylogeographic history. By implementing character mapping in a Bayesian software that samples time-scaled phylogenies, we enable the reconstruction of timed viral dispersal patterns while accommodating phylogenetic uncertainty. Standard Markov model inference is extended with a stochastic search variable selection procedure that identifies the parsimonious descriptions of the diffusion process. In addition, we propose priors that can incorporate geographical sampling distributions or characterize alternative hypotheses about the spatial dynamics. To visualize the spatial and temporal information, we summarize inferences using virtual globe software. We describe how Bayesian phylogeography compares with previous parsimony analysis in the investigation of the influenza A H5N1 origin and H5N1 epidemiological linkage among sampling localities. Analysis of rabies in West African dog populations reveals how virus diffusion may enable endemic maintenance through continuous epidemic cycles. From these analyses, we conclude that our phylogeographic framework will make an important asset in molecular epidemiology that can be easily generalized to infer biogeogeography from genetic data for many organisms.

Elimination of terrestrial rabies in Western European countries.

Since the late 1930s, the red fox (Vulpes vulpes) has been the main vector of rabies in Europe. Practically, decimation of fox population did not prevent the spread of the disease. The only efficient method to control wildlife rabies consisted in using oral vaccination by depositing vaccine baits containing a capsule or a plastic sachet filled with an attenuated anti-rabies liquid vaccine throughout fox habitats. Several live virus vaccines have been and are currently being used: the SAD B19 and SAD P5/88 are rabies strains attenuated in cell culture, the SAG1 and SAG2 strains are apathogenic mutants of an already attenuated rabies strain, and the VRG vaccine is an attenuated vaccinia virus recombinant coding for the rabies glycoprotein gene. These vaccines have different residual pathogenicity. SAG1 and SAG2 are pathogenic only for suckling mice inoculated by intracerebral and oral routes. VRG presents absolutely no rabies risk to humans and the environment and the residual pathogenicity of the vaccinia vector virus is very low even for humans. Other parameters such as the thermostability of the vaccine and the melting point of the bait casing are of utmost importance to guarantee the success of oral vaccination campaigns. Additionally, VRG is the only vaccine that did not interfere with maternal immunity in fox cubs, an important issue for spring campaigns. Successes and failure of national programmes confirm that whatever the ecological conditions, the same rules must be strictly followed to ensure the success of rabies elimination programmes: (i) considering the strategy, any rabies vaccination programme must be organised with the support of a national scientific team specially designated for the task that will have to apply the only methods that have already been proved successful elsewhere in Europe, including rabies surveillance, bait distribution calendar and pattern, and monitoring of this distribution; (ii) vaccination must be pursued for at least two years after the last reported case of rabies in the area; (iii) the choice of a low cost but poorly efficient and poorly stable vaccine does not prove to be cost-beneficial for successful elimination of rabies. Several European countries have become rabies-free: Belgium, Luxembourg, France, Italy, Switzerland, Finland and the Netherlands. Since the European Union is going comprise 25 countries from May 2004, all the scientific knowledge is available for establishing efficient and adapted oral programmes aimed at eliminating terrestrial rabies from this area.

Assessment of template quality by the incorporation of an internal control into a RT-PCR for the detection of rabies and rabies-related viruses.

A method is described to assess RNA template quality by the incorporation of a ribosomal RNA (rRNA) internal (in tube) control into a standard rabies and rabies-related virus specific RT-PCR. Specific virus and rRNA templates were co-amplified in a duplex reaction from RNA extracts derived from 60 isolates representing all six of the established lyssavirus genotypes. To ensure a wide species applicability of this technique we demonstrated that the rRNA assay was capable of functioning using the cells or tissues of 14 different mammals. Parallel studies between the duplex and the unlinked lyssavirus assay demonstrated only a minor reduction in the sensitivity of the former test. The ribosomal and viral targets (unlike beta-actin RNA) were shown to have similar degradation kinetics making rRNA amplification a good control for viral target integrity. As a consequence, the use of this system would reduce the likelihood of obtaining false negative RT-PCR results from lyssavirus infected material.

Current issues in rabies prevention in the United States health dilemmas. Public coffers, private interests.

OVER THE LAST 100 years, rabies in the United States has changed dramatically. More than 90% of all animal rabies cases reported annually to the CDC now occur in wildlife, whereas before 1960 the majority were in domestic animals. The principal rabies hosts today are wild carnivores and bats infected with several viral variants. Annual human deaths have fallen from more than a hundred at the turn of the century to one to two per year despite major outbreaks of animal rabies in several geographic areas. Modern day prophylaxis has proven nearly 100% successful; most human fatalities now occur in people who fail to seek medical treatment, usually because they do not recognize a risk in the animal contact leading to the infection. Although these human rabies deaths are rare, the estimated public health costs associated with disease detection, prevention, and control have risen, exceeding millions of dollars each year. Cost considerations must be weighed along with other factors in addressing issues such as the appropriate handling of nontraditional and exotic pets, future guidelines for rabies prophylaxis, and novel methods of disease prevention.