European Bat Lyssavirus Type Research Articles

Bat rabies surveillance in Finland

BackgroundIn 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.ResultsAltogether, 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.ConclusionsThese 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.

Membrane and inclusion body targeting of lyssavirus matrix proteins

Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation, differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinctintracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis.

Interspecies protein substitution to investigate the role of the lyssavirus glycoprotein

European bat lyssaviruses type 1 (EBLV-1) and type 2 (EBLV-2) circulate within bat populations throughout Europe and are capable of causing disease indistinguishable from that caused by classical rabies virus (RABV). However, the determinants of viral fitness and pathogenicity are poorly understood. Full-length genome clones based on the highly attenuated, non-neuroinvasive, RABV vaccine strain (SAD-B19) were constructed with the glycoprotein (G) of either SAD-B19 (SN), of EBLV-1 (SN-1) or EBLV-2 (SN-2). In vitro characterization of SN-1 and SN-2 in comparison to wild-type EBLVs demonstrated that the substitution of G affected the final virus titre and antigenicity. In vivo, following peripheral infection with a high viral dose (104 f.f.u.), animals infected with SN-1 had reduced survivorship relative to infection with SN, resulting in survivorship similar to animals infected with EBLV-1. The histopathological changes and antigen distribution observed for SN-1 were more representative of those observed with SN than with EBLV-1. EBLV-2 was unable to achieve a titre equivalent to that of the other viruses. Therefore, a reduced-dose experiment (103 f.f.u.) was undertaken in vivo to compare EBLV-2 and SN-2, which resulted in 100 % survivorship for all recombinant viruses (SN, SN-1 and SN-2) while clinical disease developed in mice infected with the EBLVs. These data indicate that interspecies replacement of G has an effect on virus titre in vitro, probably as a result of suboptimal G–matrix protein interactions, and influences the survival outcome following a peripheral challenge with a high virus titre in mice.

Bat Rabies Surveillance in Europe

Rabies is the oldest known zoonotic disease and was also the first recognized bat associated infection in humans. To date, four different lyssavirus species are the causative agents of rabies in European bats: the European Bat Lyssaviruses type 1 and 2 (EBLV-1, EBLV-2), the recently discovered putative new lyssavirus species Bokeloh Bat Lyssavirus (BBLV) and the West Caucasian Bat Virus (WCBV). Unlike in the new world, bat rabies cases in Europe are comparatively less frequent, possibly as a result of varying intensity of surveillance. Thus, the objective was to provide an assessment of the bat rabies surveillance data in Europe, taking both reported data to the WHO Rabies Bulletin Europe and published results into account. In Europe, 959 bat rabies cases were reported to the RBE in the time period 1977-2010 with the vast majority characterized as EBLV-1, frequently isolated in the Netherlands, North Germany, Denmark, Poland and also in parts of France and Spain. Most EBLV-2 isolates originated from the United Kingdom (UK) and the Netherlands, and EBLV-2 was also detected in Germany, Finland and Switzerland. Thus far, only one isolate of BBLV was found in Germany. Published passive bat rabies surveillance comprised testing of 28 of the 52 different European bat species for rabies. EBLV-1 was isolated exclusively from Serotine bats (Eptesicus serotinus and Eptesicus isabellinus), while EBLV-2 was detected in 14 Daubenton's bats (Myotis daubentonii) and 5 Pond bats (Myotis dasycneme). A virus from a single Natterer's bat (Myotis nattereri) was characterized as BBLV. During active surveillance, only oral swabs from 2 Daubenton's bats (EBLV-2) and from several Eptesicus bats (EBLV-1) yielded virus positive RNA. Virus neutralizing antibodies against lyssaviruses were detected in various European bat species from different countries, and its value and implications are discussed.

Molecular Epidemiology of Bat Lyssaviruses in Europe

Bat rabies cases in Europe are principally attributed to two lyssaviruses, namely European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2). Between 1977 and 2011, 961 cases of bat rabies were reported to Rabies Bulletin Europe, with the vast majority (>97%) being attributed to EBLV-1. There have been 25 suspected cases of EBLV-2, of which 22 have been confirmed. In addition, two single isolations of unique lyssaviruses from European insectivorous bats were reported in south-west Russia in 2002 (West Caucasian bat virus) and in Germany in 2010 (Bokeloh bat lyssavirus). In this review, we present phylogenetic analyses of the EBLV-1 and EBLV-2 using partial nucleoprotein (N) gene sequences. In particular, we have analysed all EBLV-2 cases for which viral sequences (N gene, 400 nucleotides) are available (n = 21). Oropharyngeal swabs collected from two healthy Myotis daubentonii during active surveillance programmes in Scotland and Switzerland also yielded viral RNA (EBLV-2). Despite the relatively low number of EBLV-2 cases, a surprisingly large amount of anomalous data has been published in the scientific literature and Genbank, which we have collated and clarified. For both viruses, geographical relationships are clearly defined on the phylogenetic analysis. Whilst there is no clear chronological clustering for either virus, there is some evidence for host specific relationships, particularly for EBLV-1 where more host variation has been observed. Further genomic regions must be studied, in particular for EBLV-1 isolates from Spain and the EBLV-2 isolates to provide support for the existence of sublineages.

Effect of Media Warnings on Rabies Postexposure Prophylaxis, France

Effect of Media Warnings on Rabies Postexposure Prophylaxis, France

Bat population genetics andLyssaviruspresence in Great Britain

Most lyssaviruses appear to have bat species as reservoir hosts. In Europe, of around 800 reported cases in bats, most were of European bat lyssavirus type 1 (EBLV-1) in Eptesicus serotinus (where the bat species was identified). About 20 cases of EBLV-2 were recorded, and these were in Myotis daubentonii and M. dasycneme. Through a passive surveillance scheme, Britain reports about one case a year of EBLV-2, but no cases of the more prevalent EBLV-1. An analysis of E. serotinus and M. daubentonii bat genetics in Britain reveals more structure in the former population than in the latter. Here we briefly review these differences, ask if this correlates with dispersal and movement patterns and use the results to suggest an hypothesis that EBLV-2 is more common than EBLV-1 in the UK, as genetic data suggest greater movement and regular immigration from Europe of M. daubentonii. We further suggest that this genetic approach is useful to anticipate the spread of exotic diseases in bats in any region of the world.

Generation of recombinant European bat lyssavirus type 1 and inter-genotypic compatibility of lyssavirus genotype 1 and 5 antigenome promoters

Bat lyssaviruses (Fam. Rhabdoviridae) represent a source for the infection of terrestial mammals and the development of rabies disease. Molecular differences in the replication of bat and non-bat lyssaviruses and their contribution to pathogenicity, however, are unknown. One reason for this is the lack of reverse genetics systems for bat-restricted lyssaviruses. To investigate bat lyssavirus replication and host adaptation, we developed a reverse genetics system for European bat lyssavirus type 1 (EBLV-1; genotype 5). This was achieved by co-transfection of HEK-293T cells with a full-length EBLV-1 genome cDNA and expression plasmids for EBLV-1 proteins, resulting in recombinant EBLV-1 (rEBLV-1). Replication of rEBLV-1 was comparable to that of parental virus, showing that rEBLV-1 is a valid tool to investigate EBLV-1 replication functions. In a first approach, we tested whether the terminal promoter sequences of EBLV-1 are genotype-specific. Although genotype 1 (rabies virus) minigenomes were successfully amplified by EBLV-1 helper virus, in the context of the complete virus, only the antigenome promoter (AGP) sequence of EBLV-1 was replaceable, as indicated by comparable replication of rEBLV-1 and the chimeric virus. These analyses demonstrate that the terminal AGPs of genotype 1 and genotype 5 lyssaviruses are compatible with those of the heterologous genotype.

Reassessing the risk from rabies: A continuing threat to the UK?

The threat of re-introduction of rabies virus into the United Kingdom exists on several levels. Firstly, importation of live animals into the UK continues to challenge the regulations in place to ensure that the virus does not enter the country. Secondly, the indigenous bat population is known to carry a virus genetically related to rabies virus, the European bat lyssavirus (EBLV). Molecular characterization of this virus has further characterized it as being EBLV type 2, genetically distinct from a similar virus that has caused several human deaths across Europe. Finally, a lack of awareness of the threat of rabies and related viruses to travelers visiting endemic areas also constitutes a re-introduction threat to the UK population. This review will address the most recent cases of lyssavirus infection, in both humans and animals, either contracted within the UK or from abroad. We highlight the current diagnostic necessity for testing indigenous and foreign cases and comment on current UK government policy in light of a European call to harmonise rabies legislation across Europe.

First encounter of European bat lyssavirus type 2 (EBLV-2) in a bat in Finland

In Finland, rabies in bats was suspected for the first time in 1985 when a bat researcher, who had multiple bat bites, died in Helsinki. The virus isolated from the researcher proved to be antigenically related to rabies viruses previously detected in German bats. Later, the virus was typed as EBLV-2b. Despite an epidemiological study in bats 1986 and subsequent rabies surveillance, rabies in bats was not detected in Finland until the first case in a Daubenton's bat (Myotis daubentonii) was confirmed in August 2009. The bat was paralysed, occasionally crying, and biting when approached; it subsequently tested positive for rabies. The virus was genetically typed as EBLV-2. This is the northernmost case of bat rabies ever detected in Europe. Phylogenetic analyses showed that the EBLV-2b isolate from the human case in 1985 and the isolate from the bat in 2009 were genetically closely related, demonstrating that EBLV-2 may have been circulating in Finland for many years.

Repeated detection of European bat lyssavirus type 2 in dead bats found at a single roost site in the UK

In August 2007, European bat lyssavirus type 2 (EBLV-2) was isolated from a Daubenton’s bat found at Stokesay Castle. In September 2008, another bat from the same vicinity of Stokesay Castle also tested positive for EBLV-2. This is the first occurrence of repeated detection of EBLV-2 from a single site. Here, we report the detection of low levels of viral RNA in various bat organs by qRT-PCR and detection of viral antigen by immunohistochemistry. We also report sequence data from both cases and compare data with those derived from other EBLV-2 isolations in the UK.

TARGETED SURVEILLANCE FOR EUROPEAN BAT LYSSAVIRUSES IN ENGLISH BATS (2003–06)

In 2003-06, targeted (active) surveillance for European bat lyssaviruses (EBLVs) was undertaken throughout England, focusing on two species most likely to host these viruses, Myotis daubentonii and Eptesicus serotinus. Blood was sampled for the detection of EBLV-specific neutralizing antibodies and oropharyngeal swabs were taken for the detection of viral RNA or infectious virus in saliva. Between 2003 and 2006, 273 E. serotinus and 363 M. daubentonii blood samples were tested by the EBLV-1 or EBLV-2 specific modified fluorescent antibody neutralization test. The EBLV-2 antibody prevalence estimate was 1.0-4.1% (95% confidence interval [CI]; mean=2.2%) for M. daubentonii. European bat lyssavirus type 1-specific antibodies were detected only in a single E. serotinus. Other nontarget species (n=5) were sampled in small numbers (n=24), with no EBLV-specific antibody detected. No viral RNA or live virus was detected in any of the oropharyngeal swabs analyzed. Host RNA was detected from 83% of the oropharyngeal swabs analyzed (total swabs 2003-06: n=766). These data show that EBLV-2 is present in M. daubentonii in England. In contrast, there is insufficient evidence to suggest that EBLV-1 is present in E. serotinus in England, although further research is warranted.

Antibodies induced by vaccination with purified chick embryo cell culture vaccine (PCECV) cross-neutralize non-classical bat lyssavirus strains

Tissue-culture vaccines like purified chick embryo cell vaccine (PCECV) have been shown to provide protection against classical rabies virus (RABV) via pre-exposure or post-exposure prophylaxis. A cross-neutralization study was conducted using a panel of 100 human sera, to determine, to what extent after vaccination with PCECV protection exists against non-classical bat lyssavirus strains like European bat lyssavirus (EBLV) type 1 and 2 and Australian bat lyssavirus (ABLV). Virus neutralizing antibody (VNA) concentrations against the rabies virus variants CVS-11, ABLV, EBLV-1 and EBLV-2 were determined by using a modified rapid fluorescent focus inhibition test. For ABLV and EBLV-2, the comparison to CVS-11 revealed almost identical results (100% adequate VNA concentrations ≥0.5 IU/mL; correlation coefficient r 2 = 0.69 and 0.77, respectively), while for EBLV-1 more scattering was observed (97% adequate VNA concentrations; r 2 = 0.50). In conclusion, vaccination with PCECV produces adequate VNA concentrations against classical RABV as well as non-classical lyssavirus strains ABLV, EBLV-1, and EBLV-2.

Detection of High Levels of European Bat Lyssavirus Type‐1 Viral RNA in the Thyroid Gland of Experimentally‐Infected Eptesicus fuscus Bats

Two common bat lyssavirus species have been identified in many European countries: European bat lyssavirus type-1 and -2 (EBLV-1 and EBLV-2). Only limited knowledge on the susceptibility of the natural EBLV-hosts, insectivorous bats, to lyssavirus infection is available. Our study was undertaken to evaluate the susceptibility and pathology associated with an EBLV-1 infection in Eptesicus fuscus following different routes of virus inoculation including intracranial (n = 6), intramuscular (n = 14), oral (n = 7) and intranasal (n = 7). Blood and saliva samples were collected from all bats on a monthly basis. Four bats inoculated intracranially developed rabies with a mean of 11 days to death, whilst seven bats inoculated intramuscularly developed rabies, with an extended incubation period prior to death. We did not observe any mortality in the oral (p.o.) or intranasal (i.n.) groups and both groups had detectable levels of virus neutralizing antibodies (data not shown). Virus shedding was demonstrated in the saliva by virus isolation and the detection of viral RNA in ill bats, particularly immediately prior to the development of disease. In addition, the presence of virus and viral RNA was detected in the thyroid gland in bats challenged experimentally with EBLV-1, which exceeded that detected in all other extra-neural tissue. The significance of detecting EBLV-1 in the thyroid gland of rabid bats is not well understood. We speculate that the infection of the thyroid gland may cause subacute thyroiditis, a transient form of thyroiditis causing hyperthyroidism, resulting in changes in adrenocortical activity that could lead to hormonal dysfunction, thereby distinguishing the clinical presentation of rabies in the rabid host.

Experimental infection of serotine bats (Eptesicus serotinus) with European bat lyssavirus type 1a

The serotine bat (Eptesicus serotinus) accounts for the vast majority of bat rabies cases in Europe and is considered the main reservoir for European bat lyssavirus type 1 (EBLV-1, genotype 5). However, so far the disease has not been investigated in its native host under experimental conditions. To assess viral virulence, dissemination and probable means of transmission, captive bats were infected experimentally with an EBLV-1a virus isolated from a naturally infected conspecific from Germany. Twenty-nine wild caught bats were divided into five groups and inoculated by intracranial (i.c.), intramuscular (i.m.) or subcutaneous (s.c.) injection or by intranasal (i.n.) inoculation to mimic the various potential routes of infection. One group of bats was maintained as uninfected controls. Mortality was highest in the i.c.-infected animals, followed by the s.c. and i.m. groups. Incubation periods varied from 7 to 26 days depending on the route of infection. Rabies did not develop in the i.n. group or in the negative-control group. None of the infected bats seroconverted. Viral antigen was detected in more than 50% of the taste buds of an i.c.-infected animal. Shedding of viable virus was measured by virus isolation in cell culture for one bat from the s.c. group at 13 and 14 days post-inoculation, i.e. 7 days before death. In conclusion, it is postulated that s.c. inoculation, in nature caused by bites, may be an efficient way of transmitting EBLV-1 among free-living serotine bats.

Experimental infection of Foxes with European bat Lyssaviruses type-1 and 2

BackgroundSince 1954, there have been in excess of 800 cases of rabies as a result of European Bat Lyssaviruses types 1 and 2 (EBLV-1, EBLV-2) infection, mainly in Serotine and Myotis bats respectively. These viruses have rarely been reported to infect humans and terrestrial mammals, as the only exceptions are sheep in Denmark, a stone marten in Germany and a cat in France. The purpose of this study was to investigate the susceptibility of foxes to EBLVs using silver foxes (Vulpes vulpes) as a model.ResultsOur experimental studies have shown that the susceptibility of foxes to EBLVs is low by the intramuscular (IM) route, however, animals were sensitive to intracranial (IC) inoculation. Mortality was 100% for both EBLV-1 (~4.5 logs) and EBLV-2 (~3.0 logs) delivered by the IC route. Virus dissemination and inflammatory infiltrate in the brain were demonstrated but virus specific neutralising antibody (VNA) was limited (log(ED50) = 0.24–2.23 and 0.95–2.39 respectively for specific EBLV-1 and EBLV-2). Foxes were also susceptible, at a low level, to peripheral (IM) infection (~3.0 logs) with EBLV-1 but not EBLV-2. Three out of 21 (14.3%) foxes developed clinical signs between 14 and 24 days post-EBLV-1 infection. None of the animals given EBLV-2 developed clinical disease.ConclusionThese data suggest that the chance of a EBLV spill-over from bat to fox is low, but with a greater probability for EBLV-1 than for EBLV-2 and that foxes seem to be able to clear the virus before it reaches the brain and cause a lethal infection.

Comparative Pathological Study of the Murine Brain after Experimental Infection with Classical Rabies Virus and European Bat Lyssaviruses

European bat lyssaviruses (EBLVs) types 1 (EBLV-1) and 2 (EBLV-2) cause rabies in terrestrial species, but the pathological changes associated with neuroinvasion have yet to be fully elucidated. Swiss OF-1 mice were inoculated peripherally with strain RV61 (classical rabies virus), RV1423 (EBLV-1) or RV1332 (EBLV-2) to compare the nature and extent of histopathological changes produced. Inoculated animals developed varying degrees of non-suppurative encephalitis, and lyssavirus infection was confirmed by the detection of viral antigen. The lesions produced, which included perivascular cuffs and gliosis, were more severe after RV1423 or RV1332 infection than after RV61 infection. Perivascular cuffs were mainly localized to caudal brain regions, irrespective of the infecting strain; after RV1332 infection, however, they were particularly abundant, being composed of large numbers of inflammatory cells. T cells were the predominant lymphocytic component of the inflammatory infiltrate in both the Virchow-Robin space and the brain parenchyma. Viral antigen, which was widespread throughout the brain, was apparently unrelated to the degree of cuffing. The study suggested that there was increased immune activation after inoculation with strain RV1423 or RV1332, particularly the latter, but that this did not affect the final outcome.

Reexamination of Human Rabies Case with Long Incubation, Australia

Reexamination of Human Rabies Case with Long Incubation, Australia

Experimental study of European bat lyssavirus type-2 infection in Daubenton's bats (Myotis daubentonii)

European bat lyssavirus type 2 (EBLV-2) can be transmitted from Daubenton's bats to humans and cause rabies. EBLV-2 has been repeatedly isolated from Daubenton's bats in the UK but appears to be present at a low level within the native bat population. This has prompted us to investigate the disease in its natural host under experimental conditions, to assess its virulence, dissemination and likely means of transmission between insectivorous bats. With the exception of direct intracranial inoculation, only one of seven Daubenton's bats inoculated by subdermal inoculation became infected with EBLV-2. Both intramuscular and intranasal inoculation failed to infect the bats. No animal inoculated with EBLV-2 seroconverted during the study period. During infection, virus excretion in saliva (both viral RNA and live virus) was confirmed up to 3 days before the development of rabies. Disease was manifested as a gradual loss of weight prior to the development of paralysis and then death. The highest levels of virus were measured in the brain, with much lower levels of viral genomic RNA detected in the tongue, salivary glands, kidney, lung and heart. These observations are similar to those made in naturally infected Daubenton's bats and this is the first documented report of isolation of EBLV-2 in bat saliva. We conclude that EBLV-2 is most likely transmitted in saliva by a shallow bite.

A reassessment of the emergence time of European bat lyssavirus type 1

The previous study of the evolutionary rates of European bat lyssavirus type 1 (EBLV-1) used a strict molecular clock to estimate substitution rates of the nucleoprotein gene and in turn times of the most recent common ancestor (tMRCA) of the entire genotype and the two major EBLV-1 lineages (EBLV-1A and EBLV-1B). The results of that study suggested that the evolutionary rate of EBLV-1 was one of the lowest recorded for RNA viruses and that genetic diversity of EBLV-1 arose 500–750 years ago. Here I have shown that the use of a relaxed molecular clock (allowing branch rates to vary within a phylogeny) shows that these previous estimates should be revised. The relaxed clock provides a significantly better fit to all datasets. The substitution rate of EBLV-1B is compatible to that expected given previous estimates for the N gene of rabies virus whilst rate estimations for EBLV-1A appear to be confounded by substantial rate variation within the phylogeny. The relaxed clock substitution rate for EBLV-1 (1.1 × 10 −4) is higher than had been estimated previously, and closer to that expected for the N gene. Moreover, tMRCA estimates for EBLV-1 are substantially reduced using the relaxed molecular clock (70–300 years) although the differing dynamics of EBLV-1A and EBLV-1B confound the confidence in this estimate. Current diversity of both EBLV-1A and EBLV-1B appears to have emerged within the last 100 years. Reconstruction of the population histories suggests that EBLV-1B may be emerging whilst the signal derived from the EBLV-1A phylogeny may be dampened by clade-specific dynamics.