European Bat Lyssavirus Research Articles

Active sero-survey for European bat lyssavirus type-1 circulation in North African insectivorous bats

Bats have specific biological and ecological characteristics that make them suitable hosts for viruses; some of these viruses have the ability to infect humans and other domestic and wild mammals1....

Molecular and serological survey of lyssaviruses in Croatian bat populations

BackgroundRabies is the only known zoonotic disease of bat origin in Europe. The disease is caused by species belonging to the genus Lyssavirus. Five Lyssavirus species, i.e., European bat lyssavirus (EBLV)-1, EBLV-2, Bokeloh bat lyssavirus, Lleida bat lyssavirus, and West Caucasian bat virus, have been identified in European bats. More recently, a proposed sixth species, Kotalahti bat lyssavirus, was detected. Thus, in this study, active surveillance was initiated in order to obtain insights into the prevalence of lyssaviruses in Croatian bat populations and to improve our understanding of the public health threat of infected bats.ResultsIn total, 455 bats were caught throughout Continental and Mediterranean Croatia. Antibodies were found in 20 of 350 bats (5.71%, 95% confidence interval 3.73–8.66). The majority of seropositive bats were found in Trbušnjak cave (Continental Croatia, Eastern part), and most seropositive bats belonged to Myotis myotis (13/20). All oropharyngeal swabs were negative for the presence of Lyssavirus.ConclusionsThe presence of lyssaviruses in bat populations was confirmed for the first time in Croatia and Southeastern Europe. The results of this study suggest the need for further comprehensive analyses of lyssaviruses in bats in this part of Europe.

First cases of European bat lyssavirus type 1 in Iberian serotine bats: Implications for the molecular epidemiology of bat rabies in Europe

Previous studies have shown that EBLV-1 strains exclusively hosted by Eptesicus isabellinus bats in the Iberian Peninsula cluster in a specific monophyletic group that is related to the EBLV-1b lineage found in the rest of Europe. More recently, enhanced passive surveillance has allowed the detection of the first EBLV-1 strains associated to Eptesicus serotinus south of the Pyrenees. The aim of this study is the reconstruction of the EBLV-1 phylogeny and phylodynamics in the Iberian Peninsula in the context of the European continent. We have sequenced 23 EBLV-1 strains detected on nine E. serotinus and 14 E. isabellinus. Phylogenetic analyses were performed on the first 400-bp-5’ fragment of the Nucleoprotein (N) gene together with other 162 sequences from Europe. Besides, fragments of the variable region of the phosphoprotein (P) gene and the glycoprotein-polymerase (G-L) intergenic region were studied on Spanish samples. Phylogenies show that two of the new EBLV-1a strains from Iberian E. serotinus clustered together with French strains from the North of the Pyrenees, suggesting a recent expansion southwards of this subtype. The remaining seven Iberian strains from E. serotinus grouped, instead, within the cluster linked, so far, to E. isabellinus, indicating that spatial distribution prevails over species specificity in explaining rabies distribution and supporting interspecific transmission. The structure found within the Iberian Peninsula for EBLV-1b is in concordance with that described previously for E. isabellinus. Finally, we have found that the current EBLV-1 European strains could have emerged only 175 years ago according to our evolutionary dynamics analyses.

Tentative novel lyssavirus in a bat in Finland.

A tentative novel member of the genus Lyssavirus, designated as Kotalahti bat lyssavirus, was detected in a Brandt's bat (Myotis brandtii) in Finland. Based on phylogenetic analysis, the virus differs from other known lyssaviruses, being closely related to Khujand virus, Aravan virus, Bokeloh bat lyssavirus and European bat lyssavirus 2.

Molecular Epidemiology and Evolution of European Bat Lyssavirus 2.

Bat rabies cases in Europe are mainly attributed to two lyssaviruses, namely European Bat Lyssavirus 1 (EBLV-1) and European Bat Lyssavirus 2 (EBLV-2). Prior to the death of a bat worker in Finland in 1985, very few bat rabies cases were reported. Enhanced surveillance in the two subsequent years (1986–1987) identified 263 cases (more than a fifth of all reported cases to date). Between 1977 and 2016, 1183 cases of bat rabies were reported, with the vast majority (>97%) being attributed to EBLV-1. In contrast, there have been only 39 suspected cases of EBLV-2, of which 34 have been confirmed by virus typing and presently restricted to just two bat species; Myotis daubentonii and Myotis dasycneme. The limited number of EBLV-2 cases in Europe prompted the establishment of a network of European reference laboratories to collate all available viruses and data. Despite the relatively low number of EBLV-2 cases, a large amount of anomalous data has been published in the scientific literature, which we have here reviewed and clarified. In this review, 29 EBLV-2 full genome sequences have been analysed to further our understanding of the diversity and molecular evolution of EBLV-2 in Europe. Analysis of the 29 complete EBLV-2 genome sequences clearly corroborated geographical relationships with all EBLV-2 sequences clustering at the country level irrespective of the gene studied. Further geographical clustering was also observed at a local level. There are high levels of homogeneity within the EBLV-2 species with nucleotide identities ranging from 95.5–100% and amino acid identities between 98.7% and 100%, despite the widespread distribution of the isolates both geographically and chronologically. The mean substitution rate for EBLV-2 across the five concatenated genes was 1.65 × 10−5, and evolutionary clock analysis confirms the slow evolution of EBLV-2 both between and within countries in Europe. This is further supported by the first detailed EBLV-2 intra-roost genomic analysis whereby a relatively high sequence homogeneity was found across the genomes of three EBLV-2 isolates obtained several years apart (2007, 2008, and 2014) from M. daubentonii at the same site (Stokesay Castle, Shropshire, UK).

Longitudinal survey of two serotine bat (Eptesicus serotinus) maternity colonies exposed to EBLV-1 (European Bat Lyssavirus type 1): Assessment of survival and serological status variations using capture-recapture models.

This study describes two longitudinal serological surveys of European Bat Lyssavirus type 1 (EBLV-1) antibodies in serotine bat (Eptesicus serotinus) maternity colonies located in the North-East of France. This species is currently considered as the main EBLV-1 reservoir. Multievent capture-recapture models were used to determine the factors influencing bat rabies transmission as this method accounts for imperfect detection and uncertainty in disease states. Considering the period of study, analyses revealed that survival and recapture probabilities were not affected by the serological status of individuals, confirming the capacity of bats to be exposed to lyssaviruses without dying. Five bats have been found with EBLV-1 RNA in the saliva at the start of the study, suggesting they were caught during virus excretion period. Among these bats, one was interestingly recaptured one year later and harbored a seropositive status. Along the survey, some others bats have been observed to both seroconvert (i.e. move from a negative to a positive serological status) and serorevert (i.e. move from a positive to a negative serological status). Peak of seroprevalence reached 34% and 70% in site A and B respectively. On one of the 2 sites, global decrease of seroprevalence was observed all along the study period nuanced by oscillation intervals of approximately 2–3 years supporting the oscillation infection dynamics hypothesized during a previous EBLV-1 study in a Myotis myotis colony. Seroprevalence were affected by significantly higher seroprevalence in summer than in spring. The maximum time observed between successive positive serological statuses of a bat demonstrated the potential persistence of neutralizing antibodies for at least 4 years. At last, EBLV-1 serological status transitions have been shown driven by age category with higher seroreversion frequencies in adults than in juvenile. Juveniles and female adults seemed indeed acting as distinct drivers of the rabies virus dynamics, hypothesis have been addressed but their exact role in the EBLV-1 transmission still need to be specified.

Efficacy of rabies vaccines in dogs and cats and protection in a mouse model against European bat lyssavirus type 2

BackgroundRabies is preventable by pre- and/or post-exposure prophylaxis consisting of series of rabies vaccinations and in some cases the use of immunoglobulins. The success of vaccination can be estimated either by measuring virus neutralising antibodies or by challenge experiment. Vaccines based on rabies virus offer cross-protection against other lyssaviruses closely related to rabies virus. The aim was to assess the success of rabies vaccination measured by the antibody response in dogs (n = 10,071) and cats (n = 722), as well as to investigate the factors influencing the response to vaccination when animals failed to reach a rabies antibody titre of ≥ 0.5 IU/ml. Another aim was to assess the level of protection afforded by a commercial veterinary rabies vaccine against intracerebral challenge in mice with European bat lyssavirus type 2 (EBLV-2) and classical rabies virus (RABV), and to compare this with the protection offered by a vaccine for humans.ResultsA significantly higher proportion of dogs (10.7%, 95% confidence interval CI 10.1–11.3) than cats (3.5%; 95% CI 2.3–5.0) had a vaccination antibody titre of < 0.5 IU/ml. In dogs, vaccination with certain vaccines, vaccination over 6 months prior the time of antibody determination and vaccination of dogs with a size of > 60 cm or larger resulted in a higher risk of failing to reach an antibody level of at least 0.5 IU/ml. When challenged with EBLV-2 and RABV, 80 and 100% of mice vaccinated with the veterinary rabies vaccine survived, respectively. When mice were vaccinated with the human rabies vaccine and challenged with EBLV-2, 75–80% survived, depending on the booster. All vaccinated mice developed sufficient to high titres of virus-neutralising antibodies (VNA) against RABV 21–22 days post-vaccination, ranging from 0.5 to 128 IU/ml. However, there was significant difference between antibody titres after vaccinating once in comparison to vaccinating twice (P < 0.05).ConclusionsThere was a significant difference between dogs and cats in their ability to reach a post vaccination antibody titre of ≥ 0.5 IU/ml. Mice vaccinated with RABV-based rabies vaccines were partly cross-protected against EBLV-2, but there was no clear correlation between VNA titres and cross-protection against EBLV-2. Measurement of the RABV VNA titre can only be seen as a partial tool to estimate the cross-protection against other lyssaviruses. Booster vaccination is recommended for dogs and cats if exposed to infected bats.

First detection of European bat lyssavirus type 2 (EBLV-2) in Norway

BackgroundIn Europe, bat rabies is primarily attributed to European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2) which are both strongly host-specific. Approximately thirty cases of infection with EBLV-2 in Daubenton’s bats (Myotis daubentonii) and pond bats (M. dasycneme) have been reported. Two human cases of rabies caused by EBLV-2 have also been confirmed during the last thirty years, while natural spill-over to other non-flying mammals has never been reported. Rabies has never been diagnosed in mainland Norway previously.Case presentationIn late September 2015, a subadult male Daubenton’s bat was found in a poor condition 800 m above sea level in the southern part of Norway. The bat was brought to the national Bat Care Centre where it eventually displayed signs of neurological disease and died after two days. EBLV-2 was detected in brain tissues by polymerase chain reaction (PCR) followed by sequencing of a part of the nucleoprotein gene, and lyssavirus was isolated in neuroblastoma cells.ConclusionsThe detection of EBLV-2 in a bat in Norway broadens the knowledge on the occurrence of this zoonotic agent. Since Norway is considered free of rabies, adequate information to the general public regarding the possibility of human cases of bat-associated rabies should be given. No extensive surveillance of lyssavirus infections in bats has been conducted in the country, and a passive surveillance network to assess rabies prevalence and bat epidemiology is highly desired.

Serological Evidence of Lyssaviruses among Bats on Southwestern Indian Ocean Islands.

We provide serological evidence of lyssavirus circulation among bats on southwestern Indian Ocean (SWIO) islands. A total of 572 bats belonging to 22 species were collected on Anjouan, Mayotte, La Réunion, Mauritius, Mahé and Madagascar and screened by the Rapid Fluorescent Focus Inhibition Test for the presence of neutralising antibodies against the two main rabies related lyssaviruses circulating on the African continent: Duvenhage lyssavirus (DUVV) and Lagos bat lyssavirus (LBV), representing phylogroups I and II, respectively. A total of 97 and 42 sera were able to neutralise DUVV and LBV, respectively. No serum neutralised both DUVV and LBV but most DUVV-seropositive bats (n = 32/220) also neutralised European bat lyssavirus 1 (EBLV-1) but not Rabies lyssavirus (RABV), the prototypic lyssavirus of phylogroup I. These results highlight that lyssaviruses belonging to phylogroups I and II circulate in regional bat populations and that the putative phylogroup I lyssavirus is antigenically closer to DUVV and EBLV-1 than to RABV. Variation between bat species, roost sites and bioclimatic regions were observed. All brain samples tested by RT-PCR specific for lyssavirus RNA were negative.

Lyssavirus-reactive antibodies in Swedish bats

IntroductionTo study the presence of European bat lyssavirus (EBLV) infections in bat reservoirs in Sweden, active surveillance was performed during the summers from 2008 to 2013.Material and methodsBat specimens were collected at >20 bat colonies in the central, southeastern, and southern parts of Sweden. In total, blood and saliva of 452 bats were examined by a virus neutralization test and by reverse transcription polymerase chain reactions (RT-PCRs).Results and discussionEBLV neutralizing antibodies were detected in 14 Daubenton's bats (Myotis daubentonii), all trapped in Skåne or Småland (south and southeast of Sweden). The result was not unexpected since EBLV has been shown to be present in many neighboring countries, for example, Denmark, Finland, Germany, and Norway. However, Sweden has been regarded free of rabies in terrestrial mammals since 1896. Although very rare, spillover of EBLV into other animals and humans have occurred, and the risk of EBLV infection to other species including humans should not be ignored. This is the first report of lyssavirus infection in Swedish bats.

Evolutionary trends of European bat lyssavirus type 2 including genetic characterization of Finnish strains of human and bat origin 24 years apart.

Among other Lyssaviruses, Daubenton’s and pond-bat-related European bat lyssavirus type 2 (EBLV-2) can cause human rabies. To investigate the diversity and evolutionary trends of EBLV-2, complete genome sequences of two Finnish isolates were analysed. One originated from a human case in 1985, and the other originated from a bat in 2009. The overall nucleotide and deduced amino acid sequence identity of the two Finnish isolates were high, as well as the similarity to fully sequenced EBLV-2 strains originating from the UK and the Netherlands. In phylogenetic analysis, the EBLV-2 strains formed a monophyletic group that was separate from other bat-type lyssaviruses, with significant support. EBLV-2 shared the most recent common ancestry with Bokeloh bat lyssavirus (BBLV) and Khujan virus (KHUV). EBLV-2 showed limited diversity compared to RABV and appears to be well adapted to its host bat species. The slow tempo of viral evolution was evident in the estimations of divergence times for EBLV-2: the current diversity was estimated to have built up during the last 2000 years, and EBLV-2 diverged from KHUV about 8000 years ago. In a phylogenetic tree of partial N gene sequences, the Finnish EBLV-2 strains clustered with strains from Central Europe, supporting the hypothesis that EBLV-2 circulating in Finland might have a Central European origin. The Finnish EBLV-2 strains and a Swiss strain were estimated to have diverged from other EBLV-2 strains during the last 1000 years, and the two Finnish strains appear to have evolved from a common ancestor during the last 200 years.

Population genetic structure of serotine bats (Eptesicus serotinus) across Europe and implications for the potential spread of bat rabies (European bat lyssavirus EBLV-1).

Understanding of the movements of species at multiple scales is essential to appreciate patterns of population connectivity and in some cases, the potential for pathogen transmission. The serotine bat (Eptesicus serotinus) is a common and widely distributed species in Europe where it frequently harbours European bat lyssavirus type 1 (EBLV-1), a virus causing rabies and transmissible to humans. In the United Kingdom, it is rare, with a distribution restricted to south of the country and so far the virus has never been found there. We investigated the genetic structure and gene flow of E. serotinus across the England and continental Europe. Greater genetic structuring was found in England compared with continental Europe. Nuclear data suggest a single population on the continent, although further work with more intensive sampling is required to confirm this, while mitochondrial sequences indicate an east-west substructure. In contrast, three distinct populations were found in England using microsatellite markers, and mitochondrial diversity was very low. Evidence of nuclear admixture indicated strong male-mediated gene flow among populations. Differences in connectivity could contribute to the high viral prevalence on the continent in contrast with the United Kingdom. Although the English Channel was previously thought to restrict gene flow, our data indicate relatively frequent movement from the continent to England highlighting the potential for movement of EBLV-1 into the United Kingdom.

Complete Genomic Sequence of European Bat Lyssavirus 1, Isolated from Eptesicus isabellinus in Spain

All members of the lyssavirus genus cause the disease rabies. European bat lyssavirus 1 (EBLV-1) viruses are divided genetically into three groups according to geographic location and host reservoir. We report here the first genome sequence for an EBLV-1 isolated from Eptesiscus isabellinus in the Iberian Peninsula, Spain.

On the Classification of Lyssaviruses

On the Classification of Lyssaviruses The genus Lyssavirus belongs to Rhabdoviridae and includes viruses causing the fatal encephalitis rabies. Previous phylogenetic analyses of lyssaviruses were mainly based on partial sequence of the nucleoprotein or concatenated gene sequences. With the recent identification of new species and availability of their complete genome sequences, a revision of the taxonomy and classification of Lyssavirus is desirable to understand the phylogenetic relationships within Lyssavirus. We propose here a new classification for Lyssavirus through comparison of complete genome or nucleoprotein gene sequence comprising the currently identified 15 lyssaviruses. According to this proposal, the updated classification of Lyssavirus should include four phylogroups: phylogroup I (Rabies virus, Duvenhage virus, European bat lyssavirus types 1 and 2, Australian bat lyssavirus, Khujand virus, Bokeloh bat lyssavirus, Irkut virus and Aravan virus), phylogroup II (Lagos bat virus, Mokola virus and Shimoni bat virus), phylogroup III (West Caucasian bat virus) and phylogroup IV (Ikoma lyssavirus and Lleida bat lyssavirus).

Cross-platform evaluation of commercial real-time SYBR green RT-PCR kits for sensitive and rapid detection of European bat Lyssavirus type 1.

This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R 2 values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes.

Roosting Habits of Daubenton's Bat (Myotis daubentonii) during Reproduction Differs between Adjacent River Valleys

The use of different roost types by Daubenton's bats (Myotis daubentonii) during reproduction was investigated in two adjacent river valleys in northeastern Scotland. Forty-six individuals from six colonies were radiotracked during the summers of 2004–2006. The frequency of roost switching varied with reproductive status, and was lowest in lactating females and highest in non-reproductive females, which changed roosts on average once every 5.0 and 1.5 days, respectively. Although Daubenton's bats regularly switched roosts, strong faithfulness to the roosting area was apparent regardless of whether they formed maternity colonies in trees or buildings. Although most roosts found at both study areas were in trees, lactating females and juveniles in one valley roosted only in trees whereas in the other they roosted exclusively in buildings, in which ambient temperatures were significantly higher. The implications of roosting habits for the transmission of European bat lyssavirus are discussed.

Cross-neutralization of antibodies induced by vaccination with Purified Chick Embryo Cell Vaccine (PCECV) against different Lyssavirus species

Background: Rabies is a neglected zoonotic disease caused by viruses belonging to the genus lyssavirus. In endemic countries of Asia and Africa, where the majority of the estimated 60,000 human rabies deaths occur, it is mainly caused by the classical rabies virus (RABV) transmitted by dogs. Over the last decade new species within the genus lyssavirus have been identified. Meanwhile 15 (proposed or classified) species exist, including Australian bat lyssavirus (ABLV), European bat lyssavirus (EBLV-1 and -2), Duvenhage virus (DUVV), as well as Lagos bat virus (LBV) and Mokola virus (MOKV) and recently identified novel species like Bokeloh bat lyssavirus (BBLV), Ikoma bat lyssavirus (IKOV) or Lleida bat lyssavirus (LLBV). The majority of these lyssavirus species are found in bat reservoirs and some have caused human infection and deaths. Previous work has demonstrated that Purified Chick Embryo Cell Rabies Vaccine (PCECV) not only induces immune responses against classical RABV, but also elicits cross-neutralizing antibodies against ABLV, EBLV-1 and EBLV-2.Material & Methods: Using the same serum samples as in our previous study, this study extension investigated cross-neutralizing activities of serum antibodies measured by rapid fluorescent focus inhibition test (RFFIT) against selected other non-classical lyssavirus species of interest, namely DUVV and BBLV, as well as MOKV and LBV.Results: Antibodies developed after vaccination with PCECV have neutralizing capability against BBLV and DUVV in the same range as against ABLV and EBLV-1 and -2. As expected, for the phylogenetically more distant species LBV no cross-neutralizing activity was found. Interestingly, 15 of 94 serum samples (16%) with a positive neutralizing antibody titer against RABV displayed specific cross-neutralizing activity (65-fold lower than against RABV) against one specific MOKV strain (Ethiopia isolate), which was not seen against a different strain (Nigeria isolate).Conclusion: Cross-neutralizing activities partly correlate with the phylogenetic distance of the virus species. Cross-neutralizing activities against the species BBLV and DUVV of phylogroup 1 were demonstrated, in line with previous results of cross-neutralizing activities against ABLV and EBLV-1 and -2. Potential partial cross-neutralizing activities against more distant lyssavirus species like selected MOKV strains need further research.

Seroprevalence dynamics of European bat lyssavirus type 1 in a multispecies bat colony.

We report an active surveillance study of the occurrence of specific antibodies to European Bat Lyssavirus Type 1 (EBLV-1) in bat species, scarcely studied hitherto, that share the same refuge. From 2004 to 2012, 406 sera were obtained from nine bat species. Blood samples were subjected to a modified fluorescent antibody virus neutralization test to determine the antibody titer. EBLV-1-neutralizing antibodies were detected in six of the nine species analyzed (Pipistrellus pipistrellus, P. kuhlii, Hypsugo savii, Plecotus austriacus, Eptesicus serotinus and Tadarida teniotis). Among all bats sampled, female seroprevalence (20.21%, 95% CI: 14.78%–26.57%) was not significantly higher than the seroprevalence in males (15.02%, 95% CI: 10.51%–20.54%). The results showed that the inter-annual variation in the number of seropositive bats in T. teniotis and P. austriacus showed a peak in 2007 (>70% of EBLV-1 prevalence). However, significant differences were observed in the temporal patterns of the seroprevalence modeling of T. teniotis and P. austriacus. The behavioral ecology of these species involved could explain the different annual fluctuations in EBLV-1 seroprevalence.

Comparative studies on the genetic, antigenic and pathogenic characteristics of Bokeloh bat lyssavirus.

Bokeloh bat lyssavirus (BBLV), a novel lyssavirus, was isolated from a Natterer’s bat (Myotis nattererii), a chiropteran species with a widespread and abundant distribution across Europe. As a novel lyssavirus, the risks of BBLV to animal and human health are unknown and as such characterization both in vitro and in vivo was required to assess pathogenicity and vaccine protection. Full genome sequence analysis and antigenic cartography demonstrated that the German BBLV isolates are most closely related to European bat lyssavirus type 2 (EBLV-2) and Khujand virus and can be characterized within phylogroup I. In vivo characterization demonstrated that BBLV was pathogenic in mice when inoculated peripherally causing clinical signs typical for rabies encephalitis, with higher pathogenicity observed in juvenile mice. A limited vaccination-challenge experiment in mice was conducted and suggested that current vaccines would afford some protection against BBLV although further studies are warranted to determine a serological cut-off for protection.

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.