Detection of rabies virus antibodies in fruit bats (Eidolon helvum) from Nigeria

Fruit bats (Eidolon helvum) were collected from three different localities in Western Nigeria: Oti, Ibadan and Idanre. Fifty serum samples were analyzed using a modified rapid fluorescent focus inhibition technique against rabies, Mokola, Lagos bat and Duvenhage viruses. Twenty-five brain samples were screened for rabies and related lyssavirus antigens by direct fluorescent antibody microscopy and by an indirect fluorescent antibody technique with rabies anti-nucleocapsid monoclonal antibodies. All brain samples were negative. Two serum samples had relatively high anti-rabies activity; no neutralizing activity was detected against Mokola, Lagos bat or Duvenhage viruses.     

A modified rapid enzyme immunoassay for the detection of rabies and rabies-related viruses: RREID-lyssa.

This paper presents a modification of the previously described Rapid Rabies Enzyme Immuno-Diagnosis test (RREID) by using biotinylated antibodies, streptavidin conjugate and a mixture of monospecific polyclonal antibodies against several lyssaviruses. In the modified technique (RREID-lyssa), microplates were sensitized with a mixture of purified antibodies against ribonucleoprotein (RNP) from Pasteur virus (Lyssavirus serotype 1), European Bat Lyssavirus (EBL, unclassified) and Mokola virus (Lyssavirus serotype 3). Bound RNP was detected by the same antibodies labelled with biotin and peroxidase-strepavidin conjugate. These techniques were used for the detection of RNP of different Lyssavirus serotypes (rabies and rabies-related viruses). For lyssavirus specimens of serotype 1, the threshold of detection of RREID and RREID-lyssa were similar. However, a smaller amount of labelled antibodies was needed when biotinylated antibodies were used. For specimens infected by rabies-related strains (serotypes 2, 3, 4 and EBL), the threshold of detection of the RREID-lyssa was between two and 512 times lower than with the RREID. The sensitivity and the specificity of the RREID-lyssa for rabies virus (serotype 1) when tested on a small field trial (53 specimens) were found to be identical to the RREID. Consequently, RREID-lyssa can be a useful tool for diagnostic laboratories that receive specimens infected by rabies-related viruses.     

Failure to open the blood-brain barrier and deliver immune effectors to central nervous system tissues leads to the lethal outcome of silver-haired bat rabies virus infection

Rabies is a lethal disease caused by neurotropic viruses that are endemic in nature. When exposure to a potentially rabid animal is recognized, prompt administration of virus-neutralizing antibodies, together with active immunization, can prevent development of the disease. However, once the nonspecific clinical symptoms of rabies appear conventional postexposure treatment is unsuccessful. Over the last decade, rabies viruses associated with the silver-haired bat (SHBRV) have emerged as the leading cause of human deaths from rabies in the United States and Canada as a consequence of the fact that exposure to these viruses is often unnoticed. The need to treat SHBRV infection following the development of clinical rabies has lead us to investigate why the immune response to SHBRV fails to protect at a certain stage of infection. We have established that measurements of innate and adaptive immunity are indistinguishable between mice infected with the highly lethal SHBRV and mice infected with an attenuated laboratory rabies virus strain. While a fully functional immune response to SHBRV develops in the periphery of infected animals, the invasion of central nervous system (CNS) tissues by immune cells is reduced and, consequently, the virus is not cleared. Our data indicate that the specific deficit in the SHBRV-infected animal is an inability to enhance blood-brain barrier permeability in the cerebellum and deliver immune effectors to the CNS tissues. Conceivably, at the stage of infection where immune access to the infected CNS tissues is limited, either the provision or the development of antiviral immunity will be ineffective.     

Immune sera and antiglycoprotein monoclonal antibodies inhibit in vitro cell-to-cell spread of pathogenic rabies viruses.

Although the cell-to-cell spread of many viruses in vitro is inhibited by antibody, the effect of antibody on such spread of rabies viruses is uncertain. Thus, we examined the effects of anti-rabies virus immune sera and monoclonal antibodies (MAbs) on the in vitro spread of pathogenic rabies viruses in neuronal and nonneuronal cells. Both anti-rabies virus immune sera and neutralizing antiglycoprotein MAbs inhibited the cell-to-cell spread of street rabies virus, challenge virus standard, and ERA rabies viruses in cultures of neuroblastoma cells and of nonneuronal BHK-21 and chicken embryo-related cells. Furthermore, the cell-to-cell spread of virus was inhibited by greater than or equal to 75% with less than 1 IU/ml of human antirabies immunoglobulin. Nonneutralizing antinucleocapsid MAbs did not inhibit viral spread. After the immune serum was removed from the monolayers, virus spread rapidly to uninfected cells. Thus, antibody controlled the cell-to-cell spread of the virus but did not eliminate it from the cultures. Because antibody was more effective in inhibiting viral spread in fibroblast and epithelioid cells than in neuroblastoma cells infected at a high multiplicity of infection, we suggest that the inhibition of viral cell-to-cell spread by antibody in vivo would more likely occur at an initial site of exposure and before nerves are infected.     

Molecular epidemiology of rabies epizootics in Colombia, 1994-2002: evidence of human and canine rabies associated with chiroptera

Three urban rabies outbreaks have been reported in Colombia during the last two decades, one of which is ongoing in the Caribbean region (northern Colombia). The earlier outbreaks occurred almost simultaneously in Arauca (eastern Colombia) and in the Central region, ending in 1997. Phylogenetic relationships among rabies viruses isolated from the three areas were based on a comparison of cDNA fragments coding for the endodomain of protein G and a fragment of L protein obtained by RT-PCR. The sequenced amplicons which included the G-L intergenic region contained 902 base pairs. Phylogenetic analysis showed three distinct groups of viruses. Colombian genetic variant I viruses were isolated only from Arauca and the Central region, but are now apparently extinct. Colombian genetic variant II viruses were isolated in the Caribbean region and are still being transmitted in that area. The third group of bat rabies variants were isolated from two insectivorous bats, three domestic dogs and a human. This associates bat rabies virus with rabies in Colombian dogs and humans, and indicates bats to be a rabies reservoir of public health significance.     

Biological characterization of human monoclonal antibodies to rabies virus.

Rabies virus antigen-specific human monoclonal antibodies (MAbs) that recognized either viral glycoprotein, ribonucleoprotein, or matrix proteins were generated. Only glycoprotein-specific MAb neutralized a variety of rabies viruses and protected laboratory rodents against lethal rabies virus infection. The determinant recognized by this MAb does not appear to reside in previously defined antigenic sites of the viral glycoprotein.     

A simple and rapid immunochromatographic test kit for rabies diagnosis

In rabies endemic countries, funds and infrastructure are often insufficient to employ the approved gold standard for the definitive diagnosis of rabies: the direct fluorescent test. In the present study, two types (type 1 and 2) of an ICT kit were evaluated for detection of rabies. These were developed using monoclonal antibodies which recognize epitope II and III of the nucleoprotein of rabies virus. Both kits specifically detected all rabies virus strains and there was no cross reactivity with Lyssaviruses (Lagos, Mokola and Duvenhage), Rhabdovirus (VSV and Oita 296/1972) and other common canine-pathogenic viruses. In type 1, a single type of monoclonal antibody was used. It was capable of detecting recombinant nucleoprotein and showed sensitivity of 95.5% (42/44) and specificity of 88.9% (32/36) using brain samples from rabid dogs. In contrast, type 2 which was made of two different monoclonal antibodies had a lower sensitivity of 93.2% (41/44) and higher specificity of 100% (36/36). These ICT kits provide a simple and rapid method for rabies detection. They need neither cold chain for transportation nor complicated training for personnel. This diagnostic test is suitable for rabies screening, particularly in areas with a high prevalence of rabies and where the fluorescent antibody test is not available.     

Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations.     

A simple sandwich ELISA (WELYSSA) for the detection of lyssavirus nucleocapsid in rabies suspected specimens using mouse monoclonal antibodies

Monoclonal antibody (MAb)-based capture enzyme-linked immunosorbent assays (ELISA) were developed for the diagnosis of rabies-suspect specimens. A combination of four mouse monoclonal antibodies directed against the rabies virus nucleocapsid was selected and used for the detection. The test was optimized and standardized so that maximum concordance could be maintained with the standard procedures of rabies diagnosis recommended by the WHO expert committee. Using prototype viruses from the different genotypes of lyssavirus and from various geographic origins and phylogenetic lineages, this paper presents a reliable, rapid and transferable diagnostic method, named WELYSSA that readily permits the detection of lyssaviruses belonging to the 7 genotypes of lyssavirus circulating in Europe, Africa, Asia and Oceania. The threshold of detection of lyssavirus nucleocapsids is low (0.8 ng/ml). With a panel of 1030 specimens received for rabies diagnostic testing, this test was found to be highly specific (0.999) and sensitive (0.970) when compared to other recommended rabies diagnostic methods.     

用lac基因筛选表达狂犬病毒糖蛋白的重组痘苗病毒

为了研制基因工程狂犬病疫苗,我国于1991年首次报道了在痘苗病毒天坛株中表达狂犬病毒糖蛋白,但报道中重组病毒的选择是先经人骨髓瘤细胞(TK-143)在诱变剂5-溴脱氧尿苷(BrudR)作用下通过标记拯救技术筛选出携带有同源基因的重组病毒,然后再利用重组病毒中携带的Lac基因为选择标记,通过噬斑纯化获得重组病毒,用这种选择方式获得的重组病毒,经过了TK-143细胞和BrudR,因此不宜发展成疫苗,本研究探索不经过TK-143细胞和BrudR,仅利用Lac基因为选择标记,直接在鸡胚细胞上通过噬斑纯化获得重组病毒,现将研究结果报道如下。     

The Production of Antibody by Invading B Cells Is Required for the Clearance of Rabies Virus from the Central Nervous System

Background

The pathogenesis of rabies is associated with the inability to deliver immune effectors across the blood-brain barrier and to clear virulent rabies virus from CNS tissues. However, the mechanisms that facilitate immune effector entry into CNS tissues are induced by infection with attenuated rabies virus.

Methodology/Principal Findings

Infection of normal mice with attenuated rabies virus but not immunization with killed virus can promote the clearance of pathogenic rabies virus from the CNS. T cell activity in B cell–deficient mice can control the replication of attenuated virus in the CNS, but viral mRNA persists. Low levels of passively administered rabies virus–neutralizing antibody reach infected cells in the cerebellum of B cell–deficient mice but are not sufficient to mediate virus clearance. Production of rabies virus-specific antibody by B cells invading CNS tissues is required for this process, and a substantial proportion of the B cells that accumulate in the CNS of mice infected with attenuated rabies virus produce virus-specific antibodies.

Conclusions/Significance

The mechanisms required for immune effectors to enter rabies virus-infected tissues are induced by infection with attenuated rabies virus but not by infection with pathogenic rabies viruses or immunization with killed virus. T cell activities can inhibit rabies virus replication, but the production of rabies virus–specific antibodies by infiltrating B cells, as opposed to the leakage of circulating antibody across the BBB, is critical to elimination of the virus. These findings suggest that a pathogenic rabies virus infection may be treatable after the virus has reached the CNS tissues, providing that the appropriate immune effectors can be targeted to the infected tissues.     

中国19个狂犬病病毒街毒分离株N基因的序列分析

测定了30年来从不同动物中分离的19个中国狂犬病病毒街毒株N基因的部分核酸序列,并对其核苷酸差异做了比较分析.可将中国狂犬病病毒街毒株分为4个组群,各组间的同源性为83.45%～88.62%.除广西地区分离的狂犬病病毒街毒株彼此差异较大外,其余街毒株的地理分布与其N基因核酸序列差异的距离是密切相关的,基本上可按其地理分布分为东、西二大组.     

浙江地区鼬獾和犬源狂犬病病毒分离株全基因组测序与分析

测定浙江地区狂犬病病毒分离株(鼬獾和犬)全基因组序列,从分子水平对病毒进行遗传变异特征分析,了解狂犬病病毒在浙江的流行和变异情况以及目前浙江流行株的遗传学背景,以丰富中国狂犬病病毒街毒流行株的全基因组信息。脑内接种1~2日乳鼠分离狂犬病病毒,RT-PCR反应测定浙江地区狂犬病病毒分离株全基因组核苷酸序列,并进行编码蛋白和序列相似性比较及种系发生分析。测序获得狂犬病病毒浙江淳安鼬獾分离株F02、F04和松阳犬分离株D01、D02全基因组核苷酸序列信息:基因组全长11 923和11 925 nts,前导序列Leader长58nts,5个ORF为:NP(1 353 nts);PP(894 nts);MP(609 nts);GP(1 575 nts);LP(6 386 nts),N-P-M-G间隔序列长2、5、5 nts;G-L基因间的伪基因Ψ长423 nts;Trailer尾长70 nts。核酸BLAST及多重序列比对分析显示浙江地区4个狂犬病病毒分离株的全基因组序列的组成和结构符合弹状病毒科狂犬病病毒属的特征;中国毒株之间特别是浙江同种动物狂犬病病毒之间各个基因区域核苷酸与氨基酸序列相似性最高,浙江病毒全基因组序列编码蛋白氨基酸序列相似性高于核苷酸序列相似性,说明蛋白质编码基因的核苷酸变异大多属于同义突变;浙江病毒负链RNA基因组5个基因编码氨基酸的长度没有变异,5个编码蛋白仅表现较少的序列变化;浙江病毒与本研究选择的代表性引用街毒株或者来自街毒的减毒株的变异位点和变异类型相似,多重序列相似性的比较和种系发生分析显示所分离的狂犬病病毒浙江街毒株均属于基因1型,具有较独特的中国地域性特点,故本研究中的浙江地区分离株极有可能是自然界中固有的街毒株。     

A reduced panel of anti-nucleocapsid monoclonal antibodies for bat rabies virus identification in Europe

A reduced panel of 4 anti-nucleocapsid monoclonal antibodies (mAb) was set up to distinguish viruses of terrestrial mammal origin from viruses of bat origin in Europe. Four additional mAb were necessary to identify each one of the four serotypes of lyssavirus. These 8 mAb were selected out of 25 mAb secreted by hybridomas obtained form mice immunized with either serotype 1 lyssavirus (rabies virus PV4) or serotype 3 lyssavirus (Mokola). They were screened with 32 viruses representative of the four lyssavirus serotypes and the two types of European bat lyssavirus. The panel was tested by immunofluorescence assay with 25 cell-culture-adapted European wildlife isolates and in routine rabies identification with 65 rabid animal brain smears, Two isolates from Eptesicus serotinus in France were identified as European bat lyssavirus 1 with the reduced panel.     

Detection and genetic characterization of rabies virus from human patients

Saliva and blood were collected from two patients who had not received post exposure prophylaxis in the cities of Wenzhou and Xinning respectively. Both patients were confirmed as positive for rabies by detection of rabies virus specific nucleoprotein antibodies in the sera by Western Blot. However, rabies virus specific RNA was only identified in the saliva collected from the patient in Wenzhou. Furthermore, the isolate Zhejiang Wz0 (H) was obtained by inoculating one-day-old suckling mice. Both nucleoprotein (N) and glycoprotein (G) genes from the isolate were amplified by RT-PCR and sequenced. Phylogenetic analysis indicated that the isolate belonged to classic rabies virus, and shared a higher homology with the street viruses from dogs in the main endemic areas in China and the street virus from dogs in Indonesia than with other known strains. Further comparison of the deduced amino acid sequences between the isolate and the vaccine strains used in China showed that the virus had a higher level of homology with the vaccine strain CTN than with the other vaccine strains (3aG, PV, PM and ERA). In particular, amino acid residues substitutions located in antigenic site III in the G protein, which could react with the neutralizing antibodies, were observed. These results suggested that the virus belonged to the classic rabies virus, and both N and G genes diverged from the current vaccine strains used in China at either the nucleotide or the amino acid level. Foundation items: The project was supported by the Grants (2003BA712A08-02 and 2004BA718B03) from the Chinese Ministry of Science and Technology.     

Immobilization of specific antibody on SAM functionalized gold electrode for rabies virus detection by electrochemical impedance spectroscopy

The rabies constitutes one of the most dangerous viruses causing many death cases every year. Each year approximately 55,000 people die of rabies, with high percentage of children. High percentages (99%) of the registered cases were in Asia and Africa. In order to fight this dangerous disease, many techniques are usually used for diagnostic but are usually complex, heavy, expensive, difficult to implement, requiring high-qualified personnel, and this is a necessity of developing new detection process. In this work, we describe the development of an immunological sensor based on functionalized gold electrode allowing rabies antigen detection. The biosensor is based on the immobilization of specific rabies antibodies onto functionalized gold microelectrode. The affinity interaction of the immobilized antibody with the specific antigen can be measured with low limit detection and with a good reproducibility with impedance spectroscopy. The non-specific interaction has been tested using the Newcastle antigen.     

Antigenic and Functional Analyses of Glycoprotein of Rabies Virus Using Monoclonal Antibodies

Thirty-five monoclonal antibodies (MAbs) against glycoprotein (G protein) of the RC-HL strain of the rabies virus have been established. Using these MAbs, two antigenic sites (I and II) were delineated on the G protein of the RC-HL strain in a competitive binding assay. Of these, 34 MAbs recognized the epitopes on site IL Site II was further categorized into 10 subsites according to their patterns in a competitive binding assay. Each site II-specific MAb showed 5 to 23 nonreciprocal competitions. The reactivities of 35 MAbs to rabies and rabies-related viruses in an indirect immunofluorescent antibody test showed that six MAbs in group A binded to rabies and rabies-related viruses and eight MAbs in group E reacted only with rabies viruses, considering that the former represent the genus-specific of Lyssavirus and the latter are rabies virus-specific. From biological assays, 28 of the 35 MAbs showed neutralization activity, 31 showed hemagglutination inhibition (HI) activity, and 18 showed immunolysis (IL) activity. The MAbs recognizing neutralization epitopes fell into at least three groups: those exhibiting both HI and IL activity, those showing only HI activity, and those showing neither HI nor IL activity. All IL epitopes overlap with HA epitopes. Five of the nine MAbs which reacted with the antigen treated by sodium dodecyl sulfate in ELISA were not reduced, or reduced only slightly, in the titer. None of the MAbs reacted with 2-mercaptoethanol-treated antigen. Only one MAb that recognized site I reacted with the denatured G protein in a Western blotting assay, indicating that its epitope is linear. These results suggest that almost all of the epitopes on the G protein of the rabies virus are conformation-dependent and the G protein forms a complicated antigenic structure.     

Molecular Epidemiology of Rabies Viruses Circulating in Two Rabies Endemic Provinces of Laos, 2011–2012: Regional Diversity in Southeast Asia

BackgroundAlthough rabies is endemic in Laos, genetic characterization of the viruses in this country is limited. There are growing concerns that development in the region may have increased transport of dog through Laos for regional dog meat consumption, and that this may cause spillover of the viruses from dogs brought here from other countries. This study was therefore undertaken to evaluate the current rabies situation and the genetic characteristics of rabies viruses currently circulating in Laos.MethodsWe determined the rate of rabies-positive samples by analyzing data from animal samples submitted to the Lao Ministry of Agriculture and Forestry’s National Animal Health Centre rabies laboratory from 2004 through 2011. Twenty-three rabies-positive samples were used for viral genetic characterization. Full genome sequencing was performed on two rabies viruses.ResultsRabies-positive samples increased substantially from 40.5% in 2004 to 60.2% in 2009 and continued at this level during the study period. More than 99% of the samples were from dogs, followed by cats and monkeys. Phylogenetic analyses showed that three rabies virus lineages belonging to the Southeast Asian cluster are currently circulating in Laos; these are closely related to viruses from Thailand, Cambodia and Vietnam. Lineages of the circulating Laos rabies viruses diverged from common ancestors as recently as 44.2 years and as much as 55.3 years ago, indicating periodic virus invasions.ConclusionThere is an increasing trend of rabies in Laotian animals. Similar to other rabies-endemic countries, dogs are the main viral reservoir. Three viral lineages closely related to viruses from neighboring countries are currently circulating in Laos. Data provide evidence of periodic historic exchanges of the viruses with neighboring countries, but no recent invasion.     

Evidence of two Lyssavirus phylogroups with distinct pathogenicity and immunogenicity

The genetic diversity of representative members of the Lyssavirus genus (rabies and rabies-related viruses) was evaluated using the gene encoding the transmembrane glycoprotein involved in the virus-host interaction, immunogenicity, and pathogenicity. Phylogenetic analysis distinguished seven genotypes, which could be divided into two major phylogroups having the highest bootstrap values. Phylogroup I comprises the worldwide genotype 1 (classic Rabies virus), the European bat lyssavirus (EBL) genotypes 5 (EBL1) and 6 (EBL2), the African genotype 4 (Duvenhage virus), and the Australian bat lyssavirus genotype 7. Phylogroup II comprises the divergent African genotypes 2 (Lagos bat virus) and 3 (Mokola virus). We studied immunogenic and pathogenic properties to investigate the biological significance of this phylogenetic grouping. Viruses from phylogroup I (Rabies virus and EBL1) were found to be pathogenic for mice when injected by the intracerebral or the intramuscular route, whereas viruses from phylogroup II (Mokola and Lagos bat viruses) were only pathogenic by the intracerebral route. We showed that the glycoprotein R333 residue essential for virulence was naturally replaced by a D333 in the phylogroup II viruses, likely resulting in their attenuated pathogenicity. Moreover, cross-neutralization distinguished the same phylogroups. Within each phylogroup, the amino acid sequence of the glycoprotein ectodomain was at least 74% identical, and antiglycoprotein virus-neutralizing antibodies displayed cross-neutralization. Between phylogroups, the identity was less than 64.5% and the cross-neutralization was absent, explaining why the classical rabies vaccines (phylogroup I) cannot protect against lyssaviruses from phylogroup II. Our tree-axial analysis divided lyssaviruses into two phylogroups that more closely reflect their biological characteristics than previous serotypes and genotypes.     

The Phylogeography of Rabies in Grenada,West Indies,and Implications for Control

In Grenada, West Indies, rabies is endemic, and is thought to be maintained in a wildlife host, the small Indian mongoose (Herpestes auropunctatus) with occasional spillover into other hosts. Therefore, the present study was undertaken to improve understanding of rabies epidemiology in Grenada and to inform rabies control policy. Mongooses were trapped island-wide between April 2011 and March 2013 and examined for the presence of Rabies virus (RABV) antigen using the direct fluorescent antibody test (dFAT) and PCR, and for serum neutralizing antibodies (SNA) using the fluorescent antibody virus neutralization test (FAVN). An additional cohort of brain samples from clinical rabies suspects submitted between April 2011 and March 2014 were also investigated for the presence of virus. Two of the 171 (1.7%) live-trapped mongooses were RABV positive by FAT and PCR, and 20 (11.7%) had SNAs. Rabies was diagnosed in 31 of the submitted animals with suspicious clinical signs: 16 mongooses, 12 dogs, 2 cats and 1 goat. Our investigation has revealed that rabies infection spread from the northeast to the southwest of Grenada within the study period. Phylogenetic analysis revealed that the viruses from Grenada formed a monophyletic clade within the cosmopolitan lineage with a common ancestor predicted to have occurred recently (6–23 years ago), and are distinct from those found in Cuba and Puerto Rico, where mongoose rabies is also endemic. These data suggest that it is likely that this specific strain of RABV was imported from European regions rather than the Americas. These data contribute essential information for any potential rabies control program in Grenada and demonstrate the importance of a sound evidence base for planning interventions.