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1.  Enhanced Passive Bat Rabies Surveillance in Indigenous Bat Species from Germany - A Retrospective Study 
In Germany, rabies in bats is a notifiable zoonotic disease, which is caused by European bat lyssaviruses type 1 and 2 (EBLV-1 and 2), and the recently discovered new lyssavirus species Bokeloh bat lyssavirus (BBLV). As the understanding of bat rabies in insectivorous bat species is limited, in addition to routine bat rabies diagnosis, an enhanced passive surveillance study, i.e. the retrospective investigation of dead bats that had not been tested for rabies, was initiated in 1998 to study the distribution, abundance and epidemiology of lyssavirus infections in bats from Germany. A total number of 5478 individuals representing 21 bat species within two families were included in this study. The Noctule bat (Nyctalus noctula) and the Common pipistrelle (Pipistrellus pipistrellus) represented the most specimens submitted. Of all investigated bats, 1.17% tested positive for lyssaviruses using the fluorescent antibody test (FAT). The vast majority of positive cases was identified as EBLV-1, predominately associated with the Serotine bat (Eptesicus serotinus). However, rabies cases in other species, i.e. Nathusius' pipistrelle bat (Pipistrellus nathusii), P. pipistrellus and Brown long-eared bat (Plecotus auritus) were also characterized as EBLV-1. In contrast, EBLV-2 was isolated from three Daubenton's bats (Myotis daubentonii). These three cases contribute significantly to the understanding of EBLV-2 infections in Germany as only one case had been reported prior to this study. This enhanced passive surveillance indicated that besides known reservoir species, further bat species are affected by lyssavirus infections. Given the increasing diversity of lyssaviruses and bats as reservoir host species worldwide, lyssavirus positive specimens, i.e. both bat and virus need to be confirmed by molecular techniques.
Author Summary
According to the World Health Organization rabies is considered both a neglected zoonotic and a tropical disease. The causative agents are lyssaviruses which have their primary reservoir in bats. Although bat rabies is notifiable in Germany, the number of submitted bats during routine surveillance is rarely representative of the natural bat population. Therefore, the aim of this study was to include dead bats from various sources for enhanced bat rabies surveillance. The results show that a considerable number of additional bat rabies cases can be detected, thus improving the knowledge on the frequency, geographical distribution and reservoir-association of bat lyssavirus infections in Germany. The overall proportion of positives was lower than during routine surveillance in Germany. While the majority of cases were found in the Serotine bat and characterized as European bat lyssavirus type 1 (EBLV-1), three of the four EBLV-2 infections detected in Germany were found in Myotis daubentonii during this study.
doi:10.1371/journal.pntd.0002835
PMCID: PMC4006713  PMID: 24784117
2.  Evidence of Two Lyssavirus Phylogroups with Distinct Pathogenicity and Immunogenicity 
Journal of Virology  2001;75(7):3268-3276.
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.
doi:10.1128/JVI.75.7.3268-3276.2001
PMCID: PMC114120  PMID: 11238853
3.  Monoclonal antibodies to Mokola virus for identification of rabies and rabies-related viruses. 
Journal of Clinical Microbiology  1988;26(12):2489-2494.
Rabies and rabies-related virus strains were studied by using a panel of monoclonal antibodies directed against either nucleocapsid proteins or cell surface antigens of Mokola virus (Mok-3). Each strain was used in parallel to infect cultured cells and mice. Then, the patterns of reactivity of the different monoclonal antibodies were determined by the immunofluorescent-antibody staining procedure. On cells, the monoclonal antibodies differentiated fixed rabies virus strains (serotype 1) from rabies-related virus strains. The seven fixed strains (CVS, PV4, PM, Flury LEP and HEP, ERA, and SAD) reacted identically. The previous serotype groupings (serotype 2, Lagos-bat virus; serotype 3, Mokola virus; serotype 4, Duvenhage virus) established with anti-rabies monoclonal antibodies were confirmed, except for that of Lagos-bat Kindia, which appeared to be related to the African subtype of the Duvenhage serotype (Duv-2). Within the Mokola (Mok-1, -2, -3, and -5 and Umhlanga) and the Lagos-bat (Lag-1 and -2, Zimbabwe, Pinetown, and Dakar) serotypes, each strain appeared to be distinct. The African subtype of the Duvenhage serotype reacted differently from the European subtype. Within the Duvenhage serotype, subtypes Duv-4, -5, and -6 and Denmark reacted identically, while subtypes Duv-1, -2, and -3 and German Democratic Republic appeared to be distinct. The monoclonal antibodies specific for the cell surface antigens were also used in neutralization tests with all the strains. Two of them neutralized the infectivity of Mokola virus.
Images
PMCID: PMC266931  PMID: 3068246
4.  Isolation of Irkut Virus from a Murina leucogaster Bat in China 
Background and objectives
Bats are recognized as a major reservoir of lyssaviruses; however, no bat lyssavirus has been isolated in Asia except for Aravan and Khujand virus in Central Asia. All Chinese lyssavirus isolates in previous reports have been of species rabies virus, mainly from dogs. Following at least two recent bat-associated human rabies-like cases in northeast China, we have initiated a study of the prevalence of lyssaviruses in bats in Jilin province and their public health implications. A bat lyssavirus has been isolated and its pathogenicity in mice and genomic alignment have been determined.
Results
We report the first isolation of a bat lyssavirus in China, from the brain of a northeastern bat, Murina leucogaster. Its nucleoprotein gene shared 92.4%/98.9% (nucleotide) and 92.2%/98.8% (amino acid) identity with the two known Irkut virus isolates from Russia, and was designated IRKV-THChina12. Following intracranial and intramuscular injection, IRKV-THChina12 produced rabies-like symptoms in adult mice with a short inoculation period and high mortality. Nucleotide sequence analysis showed that IRKV-THChina12 has the same genomic organization as other lyssaviruses and its isolation provides an independent origin for the species IRKV.
Conclusions
We have identified the existence of a bat lyssavirus in a common Chinese bat species. Its high pathogenicity in adult mice suggests that public warnings and medical education regarding bat bites in China should be increased, and that surveillance be extended to provide a better understanding of Irkut virus ecology and its significance for public health.
Author Summary
The Lyssavirus genus presently comprises 12 species and two unapproved species with different antigenic characteristics. Rabies virus is detectable worldwide; Lagos bat virus, Mokola virus, Duvenhage virus, Shimoni bat virus, and Ikoma lyssavirus circulate in Africa; European bat lyssavirus types 1 and 2, Irkut virus, West Caucasian bat virus, and Bokeloh bat lyssavirus are found in Europe; and Australian bat lyssavirus has been isolated in Australia. Only Aravan and Khujand viruses have been identified in central Asia. Bats are recognized as the most important reservoirs of lyssaviruses. In China, all lyssavirus isolates in previous reports have been rabies virus, mainly from dogs; none has been from bats. Recently, however, at least two bat-associated human rabies or rabies-like cases have been reported in northeast China. Therefore, we conducted a search for bat lyssaviruses in Jilin province, close to where the first bat-associated human rabies case was recorded. We isolated a bat lyssavirus, identified as an Irkut virus isolate with high pathogenicity in experimental mice. Our findings suggest that public warnings and medical education regarding bat bites in China should be increased, and that surveillance should be extended to provide a better understanding of Irkut virus ecology and its significance for public health.
doi:10.1371/journal.pntd.0002097
PMCID: PMC3591329  PMID: 23505588
5.  Bat Rabies in France: A 24-Year Retrospective Epidemiological Study 
PLoS ONE  2014;9(6):e98622.
Since bat rabies surveillance was first implemented in France in 1989, 48 autochthonous rabies cases without human contamination have been reported using routine diagnosis methods. In this retrospective study, data on bats submitted for rabies testing were analysed in order to better understand the epidemiology of EBLV-1 in bats in France and to investigate some epidemiological trends. Of the 3176 bats submitted for rabies diagnosis from 1989 to 2013, 1.96% (48/2447 analysed) were diagnosed positive. Among the twelve recognised virus species within the Lyssavirus genus, two species were isolated in France. 47 positive bats were morphologically identified as Eptesicus serotinus and were shown to be infected by both the EBLV-1a and the EBLV-1b lineages. Isolation of BBLV in Myotis nattereri was reported once in the north-east of France in 2012. The phylogenetic characterisation of all 47 French EBLV-1 isolates sampled between 1989 and 2013 and the French BBLV sample against 21 referenced partial nucleoprotein sequences confirmed the low genetic diversity of EBLV-1 despite its extensive geographical range. Statistical analysis performed on the serotine bat data collected from 1989 to 2013 showed seasonal variation of rabies occurrence with a significantly higher proportion of positive samples detected during the autumn compared to the spring and the summer period (34% of positive bats detected in autumn, 15% in summer, 13% in spring and 12% in winter). In this study, we have provided the details of the geographical distribution of EBLV-1a in the south-west of France and the north-south division of EBLV-1b with its subdivisions into three phylogenetic groups: group B1 in the north-west, group B2 in the centre and group B3 in the north-east of France.
doi:10.1371/journal.pone.0098622
PMCID: PMC4044004  PMID: 24892287
6.  First Human Rabies Case in French Guiana, 2008: Epidemiological Investigation and Control 
Background
Until 2008, human rabies had never been reported in French Guiana. On 28 May 2008, the French National Reference Center for Rabies (Institut Pasteur, Paris) confirmed the rabies diagnosis, based on hemi-nested polymerase chain reaction on skin biopsy and saliva specimens from a Guianan, who had never travelled overseas and died in Cayenne after presenting clinically typical meningoencephalitis.
Methodology/Principal Findings
Molecular typing of the virus identified a Lyssavirus (Rabies virus species), closely related to those circulating in hematophagous bats (mainly Desmodus rotundus) in Latin America. A multidisciplinary Crisis Unit was activated. Its objectives were to implement an epidemiological investigation and a veterinary survey, to provide control measures and establish a communications program. The origin of the contamination was not formally established, but was probably linked to a bat bite based on the virus type isolated. After confirming exposure of 90 persons, they were vaccinated against rabies: 42 from the case's entourage and 48 healthcare workers. To handle that emergence and the local population's increased demand to be vaccinated, a specific communications program was established using several media: television, newspaper, radio.
Conclusion/Significance
This episode, occurring in the context of a Department far from continental France, strongly affected the local population, healthcare workers and authorities, and the management team faced intense pressure. This observation confirms that the risk of contracting rabies in French Guiana is real, with consequences for population educational program, control measures, medical diagnosis and post-exposure prophylaxis.
Author Summary
Until 2008, rabies had never been described within the French Guianan human population. Emergence of the first case in May 2008 in this French Overseas Department represented a public health event that markedly affected the local population, healthcare workers and public health authorities. The antirabies clinic of French Guiana, located at Institut Pasteur de la Guyane, had to reorganize its functioning to handle the dramatically increased demand for vaccination. A rigorous epidemiological investigation and a veterinary study were conducted to identify the contamination source, probably linked to a bat bite, and the exposed population. Communication was a key factor to controlling this episode and changing the local perception of this formerly neglected disease. Because similar clinical cases had previously been described, without having been diagnosed, medical practices must be adapted and the rabies virus should be sought more systematically in similarly presenting cases. Sharing this experience could be useful for other countries that might someday have to manage such an emergence.
doi:10.1371/journal.pntd.0001537
PMCID: PMC3283561  PMID: 22363830
7.  European Bat Lyssavirus Infection in Spanish Bat Populations 
Emerging Infectious Diseases  2002;8(4):413-420.
From 1992 to 2000, 976 sera, 27 blood pellets, and 91 brains were obtained from 14 bat species in 37 localities in Spain. Specific anti-European bat lyssavirus 1 (EBL1)-neutralizing antibodies have been detected in Myotis myotis, Miniopterus schreibersii, Tadarida teniotis, and Rhinolophus ferrumequinum in the region of Aragon and the Balearic Islands. Positive results were also obtained by nested reverse transcription-polymerase chain reaction on brain, blood pellet, lung, heart, tongue, and esophagus-larynx-pharynx of M. myotis, Myotis nattereri, R. ferrumequinum, and M. schreibersii. Determination of nucleotide sequence confirmed the presence of EBL1 RNA in the different tissues. In one colony, the prevalence of seropositive bats over time corresponded to an asymmetrical curve, with a sudden initial increase peaking at 60% of the bats, followed by a gradual decline. Banded seropositive bats were recovered during several years, indicating that EBL1 infection in these bats was nonlethal. At least one of this species (M. schreibersii) is migratory and thus could be partially responsible for the dissemination of EBL1 on both shores of the Mediterranean Sea.
doi:10.3201/eid0804.010263
PMCID: PMC2730232  PMID: 11971777
Lyssavirus; polymerase chain reaction; serology; Spain
8.  Diagnosis and analysis of a recent case of human rabies in Canada 
BACKGROUND:
On September 30, 2000, staff at the Canadian Food Inspection Agency's Centre of Expertise for Rabies, located at the Animal Diseases Research Institute in Ottawa, Ontario, diagnosed rabies in a child from Quebec. This was the first case of rabies in a human in Canada in 15 years and in 36 years in the province of Quebec. After spending a week in intensive care in a Montreal hospital, the nine-year-old boy succumbed to this nearly always fatal disease. The boy had been exposed to a bat in late August 2000, while vacationing with his family in the Quebec countryside.
METHODS:
Antemortem specimens taken from the patient were sent to the Canadian Food Inspection Agency laboratory for rabies diagnosis. Samples included saliva, eye secretions, corneal impressions, cerebral spinal fluid and skin. Specimens were examined by direct immunofluorescence microscopy, and results were confirmed using molecular biological techniques. Virus strain identification was performed by both genetic methods and phenotypic analysis with monoclonal antibodies.
RESULTS:
Initial results from direct immunofluorescence staining indicated that rabies virus was present in the skin biopsy specimen but not in the corneal impressions. This diagnosis of rabies was confirmed by polymerase chain reaction product analysis from several of the submitted specimens. Virus isolation from postmortem samples was not possible because fresh brain tissue was not available. Rabies virus was isolated from saliva and was determined to be similar to a variant that circulates in populations of silver-haired bats.
INTERPRETATION:
Intravitam diagnosis of rabies in humans is very dependent on the samples submitted for diagnosis and the method used for testing. Upon first examination, only skin specimens were positive for rabies virus antigen; using polymerase chain reaction analysis, only saliva yielded positive results for rabies virus genome. Extensive testing and retesting of specimens submitted for rabies diagnosis in humans must be done to avoid false negative results.
PMCID: PMC2094860  PMID: 18159382
Bat rabies; Rabies
9.  Rabies-related viruses. 
Five viruses related to rabies occur in Africa. Two of these, Obodhiang from Sudan and kotonkan from Nigeria, were found in insects and are only distantly related to rabies virus. The other three are antigenically more closely related to rabies. Mokola virus was isolated from shrews in Nigeria, Lagos bat virus from fruit bats in Nigeria, and Duvenhage virus from brain of a man bitten by a bat in South Africa. The public health significance of the rabies-related viruses was emphasized in Zimbabwe where in 1981 a rabies-related virus became epizootic in the dog and cat population. It is postulated that the ancestral origin of rabies virus was Africa where the greatest antigenic diversity occurs and that the ancestor may have been an insect virus. Questions are raised why rabies has not evolved more rapidly in the New World, given the frequency and ease with which antigenic changes can be induced in the laboratory, and how the virus became so extensively established in New World bats.
PMCID: PMC2596466  PMID: 6758373
10.  Rabies: ocular pathology. 
Ocular pathology in the first European case of human bat-borne rabies is described. The patient was a 30-year-old bat scientist who seven weeks after bat bite developed neurological symptoms and died 23 days later. Rabies virus antigens were detected in brain smears. After extensive virological studies the virus turned out to be a rabies-related virus, closely resembling the Duvenhage virus isolated from bats in South Africa in 1980. By light microscopy focal chronic inflammatory infiltration of the ciliary body and of the choroid was found. PAS-positive exudate was seen in the subretinal and in the outer plexiform layers of the retina, and retinal veins showed endothelial damage and perivascular inflammation. Many of the retinal ganglion cells were destroyed. The presence of rabies-related viral antigen in the retinal ganglion cells was shown by positive cytoplasmic immunofluorescence, though electron microscopy failed to identify definite viral structures in the retina. By immunohistochemistry glial fibrillary acidic protein was observed in the Müller's cells, which are normally negative for this antigen but express it as a reactive change when the retina is damaged. Synaptophysin, a constituent of presynaptic vesicles of normal retinal neurons, was not detected in the retina.
Images
PMCID: PMC1041645  PMID: 2920157
11.  New Lyssavirus Genotype from the Lesser Mouse-eared Bat (Myotis blythi), Kyrghyzstan 
Emerging Infectious Diseases  2003;9(3):333-337.
The Aravan virus was isolated from a Lesser Mouse-eared Bat (Myotis blythi) in the Osh region of Kyrghyzstan, central Asia, in 1991. We determined the complete sequence of the nucleoprotein (N) gene and compared it with those of 26 representative lyssaviruses obtained from databases. The Aravan virus was distinguished from seven distinct genotypes on the basis of nucleotide and amino acid identity. Phylogenetic analysis based on both nucleotide and amino acid sequences showed that the Aravan virus was more closely related to genotypes 4, 5, and—to a lesser extent—6, which circulates among insectivorus bats in Europe and Africa. The Aravan virus does not belong to any of the seven known genotypes of lyssaviruses, namely, rabies, Lagos bat, Mokola, and Duvenhage viruses and European bat lyssavirus 1, European bat lyssavirus 2, and Australian bat lyssavirus. Based on these data, we propose a new genotype for the Lyssavirus genus.
doi:10.3201/eid0903.020252
PMCID: PMC2958534  PMID: 12643828
Lyssavirus; genotype; N gene; phylogenetic analysis; bat; central Asia; research
12.  Intravenous Inoculation of a Bat-Associated Rabies Virus Causes Lethal Encephalopathy in Mice through Invasion of the Brain via Neurosecretory Hypothalamic Fibers 
PLoS Pathogens  2009;5(6):e1000485.
The majority of rabies virus (RV) infections are caused by bites or scratches from rabid carnivores or bats. Usually, RV utilizes the retrograde transport within the neuronal network to spread from the infection site to the central nervous system (CNS) where it replicates in neuronal somata and infects other neurons via trans-synaptic spread. We speculate that in addition to the neuronal transport of the virus, hematogenous spread from the site of infection directly to the brain after accidental spill over into the vascular system might represent an alternative way for RV to invade the CNS. So far, it is unknown whether hematogenous spread has any relevance in RV pathogenesis. To determine whether certain RV variants might have the capacity to invade the CNS from the periphery via hematogenous spread, we infected mice either intramuscularly (i.m.) or intravenously (i.v.) with the dog-associated RV DOG4 or the silver-haired bat-associated RV SB. In addition to monitoring the progression of clinical signs of rabies we used immunohistochemistry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to follow the spread of the virus from the infection site to the brain. In contrast to i.m. infection where both variants caused a lethal encephalopathy, only i.v. infection with SB resulted in the development of a lethal infection. While qRT-PCR did not reveal major differences in virus loads in spinal cord or brain at different times after i.m. or i.v. infection of SB, immunohistochemical analysis showed that only i.v. administered SB directly infected the forebrain. The earliest affected regions were those hypothalamic nuclei, which are connected by neurosecretory fibers to the circumventricular organs neurohypophysis and median eminence. Our data suggest that hematogenous spread of SB can lead to a fatal encephalopathy through direct retrograde invasion of the CNS at the neurovascular interface of the hypothalamus-hypophysis system. This alternative mode of virus spread has implications for the post exposure prophylaxis of rabies, particularly with silver-haired bat-associated RV.
Author Summary
Rabies virus (RV) infects mammalian neurons and cycles in regionally distinct animal populations such as the red fox in Europe, domestic canines in Asia, or raccoons, skunks and bats in Northern America. Although human rabies can be prevented by pre- and post-exposure prophylaxis, more than 50,000 people die annually from the severe encephalopathy caused by RV. Recently, two cases of RV transmission by organ transplantation were reported. In our study, using intravenous inoculation of mice, we evaluated the pathogenetic relevance of virions that reach the bloodstream. Mice inoculated intravenously with a canine-derived RV survived the infection in contrast to intramuscularly injected mice, while mice infected with a silver-haired bat-related RV succumbed to the disease regardless of the route of inoculation. We found that the silver-haired bat-related RV was able to transit from the blood to the brain by invading neurosecretory fibers of the hypothalamus, which form neurohemal synapses lacking a blood-brain-barrier. This newly described route of brain invasion might reflect how RV reached the central nervous system from transplanted organs, since it takes longer to establish the neural connections between host and grafted tissue necessary for classical RV migration than the time until the infection became symptomatic in the two reported cases.
doi:10.1371/journal.ppat.1000485
PMCID: PMC2691950  PMID: 19543379
13.  Insights into Persistence Mechanisms of a Zoonotic Virus in Bat Colonies Using a Multispecies Metapopulation Model 
PLoS ONE  2014;9(4):e95610.
Rabies is a worldwide zoonosis resulting from Lyssavirus infection. In Europe, Eptesicus serotinus is the most frequently reported bat species infected with Lyssavirus, and thus considered to be the reservoir of European bat Lyssavirus type 1 (EBLV-1). To date, the role of other bat species in EBLV-1 epidemiology and persistence remains unknown. Here, we built an EBLV-1−transmission model based on local observations of a three-cave and four-bat species (Myotis capaccinii, Myotis myotis, Miniopterus schreibersii, Rhinolophus ferrumequinum) system in the Balearic Islands, for which a 1995–2011 serological dataset indicated the continuous presence of EBLV-1. Eptesicus serotinus was never observed in the system during the 16-year follow-up and therefore was not included in the model. We used the model to explore virus persistence mechanisms and to assess the importance of each bat species in the transmission dynamics. We found that EBLV-1 could not be sustained if transmission between M. schreibersii and other bat species was eliminated, suggesting that this species serves as a regional reservoir. Global sensitivity analysis using Sobol's method revealed that following the rate of autumn−winter infectious contacts, M. schreibersii's incubation- and immune-period durations, but not the infectious period length, were the most relevant factors driving virus persistence.
doi:10.1371/journal.pone.0095610
PMCID: PMC3995755  PMID: 24755619
14.  Bat rabies surveillance in Finland 
Background
In 1985, a bat researcher in Finland died of rabies encephalitis caused by European bat lyssavirus type 2 (EBLV-2), but an epidemiological study in 1986 did not reveal EBLV-infected bats. In 2009, an EBLV-2-positive Daubenton’s bat was detected. The EBLV-2 isolate from the human case in 1985 and the isolate from the bat in 2009 were genetically closely related. In order to assess the prevalence of EBLVs in Finnish bat populations and to gain a better understanding of the public health risk that EBLV-infected bats pose, a targeted active surveillance project was initiated.
Results
Altogether, 1156 bats of seven species were examined for lyssaviruses in Finland during a 28–year period (1985–2012), 898 in active surveillance and 258 in passive surveillance, with only one positive finding of EBLV-2 in a Daubenton’s bat in 2009. In 2010–2011, saliva samples from 774 bats of seven species were analyzed for EBLV viral RNA, and sera from 423 bats were analyzed for the presence of bat lyssavirus antibodies. Antibodies were detected in Daubenton’s bats in samples collected from two locations in 2010 and from one location in 2011. All seropositive locations are in close proximity to the place where the EBLV-2 positive Daubenton’s bat was found in 2009. In active surveillance, no EBLV viral RNA was detected.
Conclusions
These data suggest that EBLV-2 may circulate in Finland, even though the seroprevalence is low. Our results indicate that passive surveillance of dead or sick bats is a relevant means examine the occurrence of lyssavirus infection, but the number of bats submitted for laboratory analysis should be higher in order to obtain reliable information on the lyssavirus situation in the country.
doi:10.1186/1746-6148-9-174
PMCID: PMC3846527  PMID: 24011337
EBLV; Lyssavirus; Rabies; Seroprevalence
15.  Screening of Active Lyssavirus Infection in Wild Bat Populations by Viral RNA Detection on Oropharyngeal Swabs 
Journal of Clinical Microbiology  2001;39(10):3678-3683.
Brain analysis cannot be used for the investigation of active lyssavirus infection in healthy bats because most bat species are protected by conservation directives. Consequently, serology remains the only tool for performing virological studies on natural bat populations; however, the presence of antibodies merely reflects past exposure to the virus and is not a valid marker of active infection. This work describes a new nested reverse transcription (RT)-PCR technique specifically designed for the detection of the European bat virus 1 on oropharyngeal swabs obtained from bats but also able to amplify RNA from the remaining rabies-related lyssaviruses in brain samples. The technique was successfully used for surveillance of a serotine bat (Eptesicus serotinus) colony involved in a case of human exposure, in which 15 out of 71 oropharyngeal swabs were positive. Lyssavirus infection was detected on 13 oropharyngeal swabs but in only 5 brains out of the 34 animals from which simultaneous brain and oropharyngeal samples had been taken. The lyssavirus involved could be rapidly identified by automatic sequencing of the RT-PCR products obtained from 14 brains and three bat oropharyngeal swabs. In conclusion, RT-PCR using oropharyngeal swabs will permit screening of wild bat populations for active lyssavirus infection, for research or epidemiological purposes, in line not only with conservation policies but also in a more efficient manner than classical detection techniques used on the brain.
doi:10.1128/JCM.39.10.3678-3683.2001
PMCID: PMC88406  PMID: 11574590
16.  Bat Rabies in Guatemala 
Rabies in bats is considered enzootic throughout the New World, but few comparative data are available for most countries in the region. As part of a larger pathogen detection program, enhanced bat rabies surveillance was conducted in Guatemala, between 2009 and 2011. A total of 672 bats of 31 species were sampled and tested for rabies. The prevalence of rabies virus (RABV) detection among all collected bats was low (0.3%). Viral antigens were detected and infectious virus was isolated from the brains of two common vampire bats (Desmodus rotundus). RABV was also isolated from oral swabs, lungs and kidneys of both bats, whereas viral RNA was detected in all of the tissues examined by hemi-nested RT-PCR except for the liver of one bat. Sequencing of the nucleoprotein gene showed that both viruses were 100% identical, whereas sequencing of the glycoprotein gene revealed one non-synonymous substitution (302T,S). The two vampire bat RABV isolates in this study were phylogenetically related to viruses associated with vampire bats in the eastern states of Mexico and El Salvador. Additionally, 7% of sera collected from 398 bats demonstrated RABV neutralizing antibody. The proportion of seropositive bats varied significantly across trophic guilds, suggestive of complex intraspecific compartmentalization of RABV perpetuation.
Author Summary
In this study we provide results of the first active and extensive surveillance effort for rabies virus (RABV) circulation among bats in Guatemala. The survey included multiple geographic areas and multiple species of bats, to assess the broader public and veterinary health risks associated with rabies in bats in Guatemala. RABV was isolated from vampire bats (Desmodus rotundus) collected in two different locations in Guatemala. Sequencing of the isolates revealed a closer relationship to Mexican and Central American vampire bat isolates than to South American isolates. The detection of RABV neutralizing antibodies in 11 species, including insectivorous, frugivorous, and sanguivorous bats, demonstrates viral circulation in both hematophagous and non-hematophagous bat species in Guatemala. The presence of bat RABV in rural communities requires new strategies for public health education regarding contact with bats, improved laboratory-based surveillance of animals associated with human exposures, and novel techniques for modern rabies prevention and control. Additionally, healthcare practitioners should emphasize the collection of a detailed medical history, including questions regarding bat exposure, for patients presenting with clinical syndromes compatible with rabies or any clinically diagnosed progressive encephalitis.
doi:10.1371/journal.pntd.0003070
PMCID: PMC4117473  PMID: 25080103
17.  European Bat Lyssaviruses, the Netherlands 
Emerging Infectious Diseases  2005;11(12):1854-1859.
Genotype 5 lyssaviruses are endemic in the Netherlands, and can cause fatal infections in humans.
To study European bat lyssavirus (EBLV) in bat reservoirs in the Netherlands, native bats have been tested for rabies since 1984. For all collected bats, data including species, age, sex, and date and location found were recorded. A total of 1,219 serotine bats, Eptesicus serotinus, were tested, and 251 (21%) were positive for lyssavirus antigen. Five (4%) of 129 specimens from the pond bat, Myotis dasycneme, were positive. Recently detected EBLV RNA segments encoding the nucleoprotein were sequenced and analyzed phylogenetically (45 specimens). All recent serotine bat specimens clustered with genotype 5 (EBLV1) sequences, and homologies within subgenotypes EBLV1a and EBLV1b were 99.0%–100% and 99.2%–100%, respectively. Our findings indicate that EBLVs of genotype 5 are endemic in the serotine bat in the Netherlands. Since EBLVs can cause fatal infections in humans, all serotine and pond bats involved in contact incidents should be tested to determine whether the victim was exposed to EBLVs.
doi:10.3201/eid1112.041200
PMCID: PMC3367619  PMID: 16485470
EBLV; lyssavirus; the Netherlands; bat; Eptesicus serotinus; Myotis dasycneme; Europe; research
18.  Mokola virus glycoprotein and chimeric proteins can replace rabies virus glycoprotein in the rescue of infectious defective rabies virus particles. 
Journal of Virology  1995;69(3):1444-1451.
A reverse genetics approach which allows the generation of infectious defective rabies virus (RV) particles entirely from plasmid-encoded genomes and proteins (K.-K. Conzelmann and M. Schnell, J. Virol. 68:713-719, 1994) was used to investigate the ability of a heterologous lyssavirus glycoprotein (G) and chimeric G constructs to function in the formation of infectious RV-like particles. Virions containing a chloramphenicol acetyltransferase (CAT) reporter gene (SDI-CAT) were generated in cells simultaneously expressing the genomic RNA analog, the RV N, P, M, and L proteins, and engineered G constructs from transfected plasmids. The infectivity of particles was determined by a CAT assay after passage to helper virus-infected cells. The heterologous G protein from Eth-16 virus (Mokola virus, lyssavirus serotype 3) as well as a construct in which the ectodomain of RV G was fused to the cytoplasmic and transmembrane domains of the Eth-16 virus G rescued infectious SDI-CAT particles. In contrast, a chimeric protein composed of the amino-terminal half of the Eth-16 virus G and the carboxy-terminal half of RV G failed to produce infectious particles. Site-directed mutagenesis was used to convert the antigenic site III of RV G to the corresponding sequence of Eth-16 G. This chimeric protein rescued infectious SDI-CAT particles as efficiently as RV G. Virions containing the chimeric protein were specifically neutralized by an anti-Eth-16 virus serum and escaped neutralization by a monoclonal antibody directed against RV antigenic site III. The results show that entire structural domains as well as short surface epitopes of lyssavirus G proteins may be exchanged without affecting the structure required to mediate infection of cells.
PMCID: PMC188731  PMID: 7853476
19.  Molecular Inferences Suggest Multiple Host Shifts of Rabies Viruses from Bats to Mesocarnivores in Arizona during 2001–2009 
PLoS Pathogens  2012;8(6):e1002786.
In nature, rabies virus (RABV; genus Lyssavirus, family Rhabdoviridae) represents an assemblage of phylogenetic lineages, associated with specific mammalian host species. Although it is generally accepted that RABV evolved originally in bats and further shifted to carnivores, mechanisms of such host shifts are poorly understood, and examples are rarely present in surveillance data. Outbreaks in carnivores caused by a RABV variant, associated with big brown bats, occurred repeatedly during 2001–2009 in the Flagstaff area of Arizona. After each outbreak, extensive control campaigns were undertaken, with no reports of further rabies cases in carnivores for the next several years. However, questions remained whether all outbreaks were caused by a single introduction and further perpetuation of bat RABV in carnivore populations, or each outbreak was caused by an independent introduction of a bat virus. Another question of concern was related to adaptive changes in the RABV genome associated with host shifts. To address these questions, we sequenced and analyzed 66 complete and 20 nearly complete RABV genomes, including those from the Flagstaff area and other similar outbreaks in carnivores, caused by bat RABVs, and representatives of the major RABV lineages circulating in North America and worldwide. Phylogenetic analysis demonstrated that each Flagstaff outbreak was caused by an independent introduction of bat RABV into populations of carnivores. Positive selection analysis confirmed the absence of post-shift changes in RABV genes. In contrast, convergent evolution analysis demonstrated several amino acids in the N, P, G and L proteins, which might be significant for pre-adaptation of bat viruses to cause effective infection in carnivores. The substitution S/T242 in the viral glycoprotein is of particular merit, as a similar substitution was suggested for pathogenicity of Nishigahara RABV strain. Roles of the amino acid changes, detected in our study, require additional investigations, using reverse genetics and other approaches.
Author Summary
Host shifts of the rabies virus (RABV) from bats to carnivores are important for our understanding of viral evolution and emergence, and have significant public health implications, particularly for the areas where “terrestrial” rabies has been eliminated. In this study we addressed several rabies outbreaks in carnivores that occurred in the Flagstaff area of Arizona during 2001–2009, and caused by the RABV variant associated with big brown bats (Eptesicus fuscus). Based on phylogenetic analysis we demonstrated that each outbreak resulted from a separate introduction of bat RABV into populations of carnivores. No post-shift changes in viral genomes were detected under the positive selection analysis. Trying to answer the question why certain bat RABV variants are capable for host shifts to carnivores and other variants are not, we developed a convergent evolution analysis, and implemented it for multiple RABV lineages circulating worldwide. This analysis identified several amino acids in RABV proteins which may facilitate host shifts from bats to carnivores. Precise roles of these amino acids require additional investigations, using reverse genetics and animal experimentation. In general, our approach and the results obtained can be used for prediction of host shifts and emergence of other zoonotic pathogens.
doi:10.1371/journal.ppat.1002786
PMCID: PMC3380930  PMID: 22737076
20.  Laboratory Surveillance of Rabies in Humans, Domestic Animals, and Bats in Madagascar from 2005 to 2010 
Background. Rabies virus (RABV) has circulated in Madagascar at least since the 19th century. Objectives. To assess the circulation of lyssavirus in the island from 2005 to 2010. Materials and Methods. Animal (including bats) and human samples were tested for RABV and other lyssavirus using antigen, ribonucleic acid (RNA), and antibodies detection and virus isolation. Results. Half of the 437 domestic or tame wild terrestrial mammal brains tested were found RABV antigen positive, including 54% of the 341 dogs tested. This percentage ranged from 26% to 75% across the period. Nine of the 10 suspected human cases tested were laboratory confirmed. RABV circulation was confirmed in 34 of the 38 districts sampled. No lyssavirus RNA was detected in 1983 bats specimens. Nevertheless, antibodies against Lagos bat virus were detected in the sera of 12 among 50 Eidolon dupreanum specimens sampled. Conclusion. More than a century after the introduction of the vaccine, rabies still remains endemic in Madagascar.
doi:10.4061/2011/727821
PMCID: PMC3170745  PMID: 21991442
21.  High Diversity of Rabies Viruses Associated with Insectivorous Bats in Argentina: Presence of Several Independent Enzootics 
Background
Rabies is a fatal infection of the central nervous system primarily transmitted by rabid animal bites. Rabies virus (RABV) circulates through two different epidemiological cycles: terrestrial and aerial, where dogs, foxes or skunks and bats, respectively, act as the most relevant reservoirs and/or vectors. It is widely accepted that insectivorous bats are not important vectors of RABV in Argentina despite the great diversity of bat species and the extensive Argentinean territory.
Methods
We studied the positivity rate of RABV detection in different areas of the country, and the antigenic and genetic diversity of 99 rabies virus (RABV) strains obtained from 14 species of insectivorous bats collected in Argentina between 1991 and 2008.
Results
Based on the analysis of bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats ranged from 3.1 to 5.4%, depending on the geographic location. The findings were distributed among an extensive area of the Argentinean territory. The 99 strains of insectivorous bat-related sequences were divided into six distinct lineages associated with Tadarida brasiliensis, Myotis spp, Eptesicus spp, Histiotus montanus, Lasiurus blosseviilli and Lasiurus cinereus. Comparison with RABV sequences obtained from insectivorous bats of the Americas revealed co-circulation of similar genetic variants in several countries. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples.
Conclusions
This study demonstrates the presence of several independent enzootics of rabies in insectivorous bats of Argentina. This information is relevant to identify potential areas at risk for human and animal infection.
Author Summary
In Argentina, successful vaccination and control of terrestrial rabies in the 1980s revealed the importance of the aerial route in RABV transmission. Current distribution of cases shows a predominance of rabies by hematophagous bats in the Northern regions where rabies is a major public health concern; in contrast, in Central and Southern regions where rabies is not a major public health concern, little surveillance is performed. Based on the analysis of insectivorous bats received for RABV analysis by the National Rabies system of surveillance, the positivity rate of RABV in insectivorous bats in these regions ranged from 3.1 to 5.4%. This rate is comparable to other nations such as the United States (9–10%) where insectivorous bats are an important cause of concern for RABV surveillance systems. Antigenic and genetic analysis of a wide collection of rabies strains shows the presence of multiple endemic cycles associated with six bat insectivorous species distributed among an extensive area of the Argentinean territory and several countries of the Americas. Finally, inter-species transmission, mostly related with Lasiurus species, was demonstrated in 11.8% of the samples. Increased public education about the relationship between insectivorous bats and rabies are essential to avoid human cases and potential spread to terrestrial mammals.
doi:10.1371/journal.pntd.0001635
PMCID: PMC3348165  PMID: 22590657
22.  A Panel of Monoclonal Antibodies Targeting the Rabies Virus Phosphoprotein Identifies a Highly Variable Epitope of Value for Sensitive Strain Discrimination 
Journal of Clinical Microbiology  2000;38(4):1397-1403.
A recombinant rabies virus phosphoprotein fusion product (GST-P) was used to generate a series of monoclonal antibodies (MAbs) with anti-P reactivity. Competitive binding assays classified 27 of these MAbs into four groups (I to IV), and 24 of them were deemed to recognize linear epitopes, as judged by their reaction in immunoblots. The linear epitope recognized in each case was mapped by using two series of N- and C-terminally deleted recombinant phosphoproteins. Assessment of the reactivities of representative MAbs to a variety of lyssavirus isolates by an indirect fluorescent antibody test indicated that group I MAbs, which recognized a highly conserved N-terminal epitope, were broadly cross-reactive with all lyssaviruses assayed, while group III MAbs, which reacted with a site overlapping that of group I MAbs, exhibited variable reactivities and group IV MAbs reacted with most isolates of genotypes 1, 6, and 7 only. In contrast, group II MAbs, which recognized an epitope located within a highly divergent central portion of the protein, were exquisitely strain specific. These anti-P MAbs are potentially useful tools for lyssavirus identification and discrimination.
PMCID: PMC86452  PMID: 10747114
23.  Bat-transmitted Human Rabies Outbreaks, Brazilian Amazon 
Emerging Infectious Diseases  2006;12(8):1197-1202.
We describe 2 bat-transmitted outbreaks in remote, rural areas of Portel and Viseu Municipalities, Pará State, northern Brazil. Central nervous system specimens were taken after patients' deaths and underwent immunofluorescent assay and histopathologic examination for rabies antigens; also, specimens were injected intracerebrally into suckling mice in an attempt to isolate the virus. Strains obtained were antigenically and genetically characterized. Twenty-one persons died due to paralytic rabies in the 2 municipalities. Ten rabies virus strains were isolated from human specimens; 2 other cases were diagnosed by histopathologic examination. Isolates were antigenically characterized as Desmodus rotundus variant 3 (AgV3). DNA sequencing of 6 strains showed that they were genetically close to D. rotundus–related strains isolated in Brazil. The genetic results were similar to those obtained by using monoclonal antibodies and support the conclusion that the isolates studied belong to the same rabies cycle, the virus variants found in the vampire bat D. rotundus.
doi:10.3201/1208.050929
PMCID: PMC3291204  PMID: 16965697
human rabies virus, bat transmission, antigenic and genetic characterization; Brazilian Amazon
24.  A universal real-time assay for the detection of Lyssaviruses 
Journal of Virological Methods  2011;177(1-24):87-93.
Highlights
► Universal real-time PCR primer pair demonstrated to hybridize to and detect each of the known Lyssaviruses (including Rabies virus) with greater sensitivity than a standard pan-Lyssavirus hemi-nested RT-PCR typically used. ► Target sequences of bat derived virus species unavailable for analysis (Aravan-, Khujand-, Irkut-, West Caucasian bat- and Shimoni bat virus) were synthesized to produce oligonucleotides and the synthetic DNA was used as a target for primer hybridization.
Rabies virus (RABV) is enzootic throughout most of the world. It is now widely accepted that RABV had its origins in bats. Ten of the 11 Lyssavirus species recognised, including RABV, have been isolated from bats. There is, however, a lack of understanding regarding both the ecology and host reservoirs of Lyssaviruses. A real-time PCR assay for the detection of all Lyssaviruses using universal primers would be beneficial for Lyssavirus surveillance. It was shown that using SYBR® Green, a universal real-time PCR primer pair previously demonstrated to detect European bat Lyssaviruses 1 and 2, and RABV, was able to detect reverse transcribed RNA for each of the seven virus species available to us. Target sequences of bat derived virus species unavailable for analysis were synthesized to produce oligonucleotides. Lagos Bat-, Duvenhage- and Mokola virus full nucleoprotein gene clones enabled a limit of 5–50 plasmid copies to be detected. Five copies of each of the synthetic DNA oligonucleotides of Aravan-, Khujand-, Irkut-, West Caucasian bat- and Shimoni bat virus were detected. The single universal primer pair was therefore able to detect each of the most divergent known Lyssaviruses with great sensitivity.
doi:10.1016/j.jviromet.2011.07.002
PMCID: PMC3191275  PMID: 21777619
Lyssavirus; Rabies; Bat; SYBR Green; Real-time PCR; Synthetic DNA
25.  Ecology and Geography of Transmission of Two Bat-Borne Rabies Lineages in Chile 
Rabies was known to humans as a disease thousands of years ago. In America, insectivorous bats are natural reservoirs of rabies virus. The bat species Tadarida brasiliensis and Lasiurus cinereus, with their respective, host-specific rabies virus variants AgV4 and AgV6, are the principal rabies reservoirs in Chile. However, little is known about the roles of bat species in the ecology and geographic distribution of the virus. This contribution aims to address a series of questions regarding the ecology of rabies transmission in Chile. Analyzing records from 1985–2011 at the Instituto de Salud Pública de Chile (ISP) and using ecological niche modeling, we address these questions to help in understanding rabies-bat ecological dynamics in South America. We found ecological niche identity between both hosts and both viral variants, indicating that niches of all actors in the system are undifferentiated, although the viruses do not necessarily occupy the full geographic distributions of their hosts. Bat species and rabies viruses share similar niches, and our models had significant predictive power even across unsampled regions; results thus suggest that outbreaks may occur under consistent, stable, and predictable circumstances.
Author Summary
The situation of rabies in America has been changing: rabies in dogs has decreased considerably, but bats are increasingly documented as natural reservoirs of other rabies variants. A significant gap exists in understanding of bat-borne rabies in Latin America. We identified bat species known to be connected with enzootic rabies with different antigenic variants in Chile, and compiled large-scale data sets by which to test for ecological niche differences among virus lineages and bat hosts. Our results begin to characterize important ecological factors affecting rabies distribution; modeling rabies in Chile allows comparisons across different latitudes and diverse landscapes. We found that rabies virus strains are found in similar environments, regardless of the bat host involved. This research improves understanding of bat-borne rabies dynamics, and important step towards preventing and controlling this and other emergent diseases linked to bats.
doi:10.1371/journal.pntd.0002577
PMCID: PMC3861194  PMID: 24349592

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