Isolation of a European bat lyssavirus type 2 from a Daubenton's bat in the United Kingdom

European bat lyssavirus type 2 (EBLV-2) has been isolated once previously from a bat in the UK in June 1996. In September 2002, a Daubenton's bat (Myotis daubentonii) found in Lancashire developed abnormal behaviour, including unprovoked aggression, while it was in captivity. Brain samples from the bat were tested for virus of the Lyssavirus genus, which includes EBLV-2 (genotype 6), and classical rabies virus (genotype 1). A positive fluorescent antibody test confirmed that it was infected with a lyssavirus, and PCR and genomic sequencing identified the virus as an EBLV-2a. Phylogenetic comparisons with all the published sequences from genotype 6 showed that it was closely related to the previous isolate of EBLV-2 in the UK and suggested links to isolates from bats in The Netherlands. The isolation of EBLV-2 from a bat found on the west coast of England provides evidence that this virus may be present within the UK Daubenton's bat population at a low prevalence level.     

Detection of European bat lyssavirus 2 (EBLV-2) in a Daubenton's bat (Myotis daubentonii) from Magdeburg, Germany

In Europe bat rabies in Daubenton's bats (Myotisdaubentonii) and in Pond bats (Myotis dasycneme) caused by the European bat lyssavirus 2 (EBLV-2) has been confirmed in less than 20 cases to date. Here we report the second encounter of this virus species in Germany. A Daubenton's bat found grounded in the zoological garden in Magdeburg died shortly after. In the frame of a retrospective study the bat carcass was eventually transferred to the national reference laboratory for rabies at the Friedrich-Loeffler-Institute for rabies diagnosis. Lyssavirus was isolated and characterized as EBLV-2.     

Susceptibility of sheep to European bat lyssavirus type-1 and -2 infection: a clinical pathogenesis study

European bat lyssaviruses (EBLVs) have been known to cross the species barrier from their native bat host to other terrestrial mammals. In this study, we have confirmed EBLV-1 and EBLV-2 susceptibility in sheep (Ovis ammon) following intracranial and peripheral (intramuscular) inoculation. Notably, mild clinical disease was observed in those exposed to virus via the intramuscular route. Following the intramuscular challenge, 75% of the animals infected with EBLV-1 and 100% of those that were challenged with EBLV-2 developed clinical signs of rabies and then recovered during the 94-day observation period. Disease pathogenesis also varied substantially between the two viruses. Infection with EBLV-1 resulted in peracute clinical signs, which are suggestive of motor neuron involvement. Antibody induction was observed and substantial inflammatrory infiltrate in the brain. In contrast, more antigen was detected in the EBLV-2-infected sheep brains but less inflammatory infiltrate and no virus neutralising antibody was evident. The latter involved a more protracted disease that was behaviour orientated. A high infectious dose was required to establish EBLV infection under experimental conditions (> or =5.0 logs/ml) but the infectious dose in field cases remains unknown. These data confirm that sheep are susceptible to infection with EBLV but that there is variability in pathogenesis including neuroinvasiveness that varies with the route of infection. This study suggests that inter-species animal-to-animal transmission of a bat variant of rabies virus to a terrestrial mammal host may be limited, and may not always result in fatal encephalitis.     

Genetic analysis of European bat lyssavirus type 1 isolates from France

European bat lyssavirus type 1a (EBLV-1a) was first identified in central France from a serotine bat (Eptesicus serotinus) collected at the end of 2002. Rabies was diagnosed by reference rabies diagnosis methods and molecular tools. Phylogenetic analysis of 14 viral isolates obtained from French bats infected with EBLV-1 between 1989 and the end of 2002 against 47 nucleoprotein sequences showed a north-west to east distribution of EBLV-1a virus and a south to north distribution of EBLV-1b virus, isolates of which could be divided into two groups: group 1 in north-eastern France and group 2 in central and north-western France.     

Spill-over of European bat lyssavirus type 1 into a stone marten (Martes foina) in Germany

European bat lyssavirus type 1 (EBLV-1, genotype 5) is known to endemically circulate in insectivorous bat populations in Germany. In August 2001, a rabies suspect stone marten (Martes foina) was found in the city of Burg (Saxony-Anhalt, Germany) and was sent to the regional veterinary laboratory for routine rabies diagnosis. Whereas brain samples repeatedly tested negative in the fluorescent antibody test for classical rabies virus (genotype 1), the mouse inoculation test and the rabies tissue culture inoculation test yielded positive results. Rabies viral RNA was also detected in the stone marten brain sample both by nested and heminested RT-PCR specific for the nucleoprotein gene and for the nucleoprotein phosphoprotein junction of rabies virus. The amplification products were sequenced to genotype the isolate. Sequence data obtained from the first-round RT-PCR products were analysed and the suspect stone marten isolate was confirmed as a rabies related virus (EBLV-1a). Phylogenetic comparison with sequences from recent genotype five isolates from Germany and Denmark showed that it was closely related to a previous isolate of EBLV-1 from a serotine bat in Saxony-Anhalt obtained in the same year in an area adjacent to the place where the EBLV-1 infected stone marten was found. Both EBLV-1 isolates share a 99.5% identity. This is the first report of an EBLV-1a spill-over from an insectivorous bat into wildlife in Europe.     

Spill‐over of European Bat Lyssavirus Type 1 into a Stone Marten (Martes foina) in Germany

European bat lyssavirus type 1 (EBLV‐1, genotype 5) is known to endemically circulate in insectivorous bat populations in Germany. In August 2001, a rabies suspect stone marten (Martes foina) was found in the city of Burg (Saxony‐Anhalt, Germany) and was sent to the regional veterinary laboratory for routine rabies diagnosis. Whereas brain samples repeatedly tested negative in the fluorescent antibody test for classical rabies virus (genotype 1), the mouse inoculation test and the rabies tissue culture inoculation test yielded positive results. Rabies viral RNA was also detected in the stone marten brain sample both by nested and heminested RT‐PCR specific for the nucleoprotein gene and for the nucleoprotein phosphoprotein junction of rabies virus. The amplification products were sequenced to genotype the isolate. Sequence data obtained from the first‐round RT‐PCR products were analysed and the suspect stone marten isolate was confirmed as a rabies related virus (EBLV‐1a). Phylogenetic comparison with sequences from recent genotype five isolates from Germany and Denmark showed that it was closely related to a previous isolate of EBLV‐1 from a serotine bat in Saxony‐Anhalt obtained in the same year in an area adjacent to the place where the EBLV‐1 infected stone marten was found. Both EBLV‐1 isolates share a 99.5% identity. This is the first report of an EBLV‐1a spill‐over from an insectivorous bat into wildlife in Europe.     

Identification of fox rabies by a monoclonal antibody directed against nucleocapsid of a street rabies virus

Hybridomas were prepared by fusion of spleen cells from BALB/c mice immunized with dog rabies isolates from Nigeria with P3x63Ag8 myeloma cells. More than 69 hybridomas secreted antinucleocapsid (antiNC) antibodies when tested with homologous viruses by indirect immunofluorescence. One hybridoma (Z144-88) was found which secreted antiNC antibody that reacted negatively with fox rabies isolates from the Federal Republic of Germany, Switzerland and France and with rabies-related viruses and European bat isolates. It reacted positively with other strains/isolates of rabies virus. It is possible to use this antiNC monoclonal antibody (mab) for the investigation of fox rabies outbreaks in Europe.     

Susceptibility of ferrets (Mustela putorius furo) to experimentally induced rabies with European Bat Lyssaviruses (EBLV)

Twenty ferrets (Mustela putorius furo) were inoculated by intramuscular (i.m.) injection with European Bat Lyssaviruses (EBLV) type-1 and 2 using 10(4.0) foci-forming units (FFU) EBLV-2 (n = 6), 10(4.0) FFU EBLV-1 (n = 7) and 10(6.0) FFU EBLV-1 (n = 7). Furthermore, 15 mice received 10(2.5) FFU EBLV-2 (n = 5), 10(2.5) FFU EBLV-1 (n = 5) and 10(4.5) FFU EBLV-1 (n = 5) by i.m. inoculation. All ferrets and mice receiving the higher dose of EBLV-1 succumbed to infection. In contrast, only three of seven ferrets and two of five mice inoculated experimentally with the lower EBLV-1 dose died. By comparison, all of the EBLV-2 infected ferrets and four of five mice survived infection. All 20 infected ferrets seroconverted. Using sensitive molecular tools, the virus was detected in different tissues, but it could not be found in any saliva samples taken during the 84-day observation period.     

First Isolation of a Rabid Bat Infected with European Bat Lyssavirus in Luxembourg

Rabid bats are regularly reported in Europe, especially in countries that have implemented a bat surveillance network. In May 2013, bat rabies was evidenced for the first time in Luxembourg (southern city of Differdange). The rabies virus, an EBLV‐1b strain, was diagnosed in a serotine bat that bit a 29‐year‐old male person while he was asleep. The man received rapidly a post‐exposure RABV treatment and was put under strict medical supervision.     

Lleida Bat Lyssavirus isolation in Miniopterus schreibersii in France

Bat rabies cases are attributed in Europe to five different Lyssavirus species of 16 recognized Lyssavirus species causing rabies. One of the most genetically divergent Lyssavirus spp. has been detected in a dead Miniopterus schreibersii bat in France. Brain samples were found positive for the presence of antigen, infectious virus and viral RNA by classical virological methods and molecular methods respectively. The complete genome sequence was determined by next‐generation sequencing. The analysis of the complete genome sequence confirmed the presence of Lleida bat lyssavirus (LLEBV) in bats in France with 99.7% of nucleotide identity with the Spanish LLEBV strain (KY006983).     

Faeces as a novel material to estimate lyssavirus prevalence in bat populations

Rabies is caused by infection with a lyssavirus. Bat rabies is of concern for both public health and bat conservation. The current method for lyssavirus prevalence studies in bat populations is by oral swabbing, which is invasive for the bats, dangerous for handlers, time‐consuming and expensive. In many situations, such sampling is not feasible, and hence, our understanding of epidemiology of bat rabies is limited. Faeces are usually easy to collect from bat colonies without disturbing the bats and thus could be a practical and feasible material for lyssavirus prevalence studies. To further explore this idea, we performed virological analysis on faecal pellets and oral swabs of seven serotine bats (Eptesicus serotinus) that were positive for European bat 1 lyssavirus in the brain. We also performed immunohistochemical and virological analyses on digestive tract samples of these bats to determine potential sources of lyssavirus in the faeces. We found that lyssavirus detection by RT‐qPCR was nearly as sensitive in faecal pellets (6/7 bats positive, 86%) as in oral swabs (7/7 bats positive, 100%). The likely source of lyssavirus in the faeces was virus excreted into the oral cavity from the salivary glands (5/6 bats positive by immunohistochemistry and RT‐qPCR) or tongue (3/4 bats positive by immunohistochemistry) and swallowed with saliva. Virus could not be isolated from any of the seven faecal pellets, suggesting the lyssavirus detected in faeces is not infectious. Lyssavirus detection in the majority of faecal pellets of infected bats shows that this novel material should be further explored for lyssavirus prevalence studies in bats.     

From rabies to rabies-related viruses

Antigenic differences between rabies virus strains characterized with monoclonal antibodies presently define at least four serotypes within the Lyssavirus genus of the Rhabdoviridae family: classical rabies virus strains (serotype 1), Lagos bat virus (serotype 2), Mokola virus (serotype 3) and Duvenhage virus (serotype 4). The wide distribution of rabies-related virus strains (serotypes 2, 3 and 4) and above all, the weak protection conferred by rabies vaccines against some of them (principally Mokola virus) necessitates the development of new specific vaccines. We first determined the complete nucleotide sequence of a rabies virus strain of serotype 1 (Pasteur virus) and characterized the structure of the viral genes and their regulatory sequences. We then extended this study to the Mokola virus genome. Five non-overlapping open reading frames were found in both viruses and had similar sizes and positions in both. Similarities were also found in the mRNA start and stop sequences and at the genomic extremities. Comparison of both genomes helps to analyze the basis of the particular antigenicity of these two serotypes. The sequence homology in the region coding for the viral glycoprotein was only 58% between the two viruses, compared with 94% between different rabies virus strains within serotype 1. This comparison, extended to other unsegmented negative strand RNA viruses, gives new insight into the understanding of rhabdoviruses and paramyxoviruses. Furthermore, molecular cloning provides a rationale for the genetic engineering of a future vaccine.     

Major antigenic groups of rabies virus in Canada determined by anti-nucleocapsid monoclonal antibodies

A total of 123 rabies virus isolates from various geographical areas in Canada were characterized by a panel of 43 anti-nucleocapsid monoclonal antibodies. Four major antigenic groups are found in terrestrial mammals: "Canadian Arctic" from Ontario, Quebec and the Northwest Territories; "south-eastern Georgian Bay" from Ontario; "south mid-central skunk" from Alberta, Saskatchewan and Manitoba; and "Brook's, Alberta skunk" from a restricted area in Alberta. Bat isolates can be divided into 4 major antigenic groups: "B-1" in Eptesicus fuscus from Ontario; "B-2" in a variety of bat species from British Columbia eastward into Ontario; "B-3" in Myotis spp. from Ontario and New Brunswick; and "B-4" in E. fuscus from Alberta and Saskatchewan. A single case of bat to horse transmission of rabies virus is recorded. These street isolates are compared with isolates of fixed virus. Epidemiological aspects are discussed.     

Geographical distribution of vampire bat-related cattle rabies in Brazil

Seventy-seven rabies virus (RV) isolates originating from Brazilian cattle were genetically characterized. Partial nucleoprotein gene sequences of these isolates were phylogenetically and geographically analyzed. Cattle isolates, which clustered with the vampire bat-related RV group, were further subdivided into nine genetic subgroups. These subgroups were distributed widely in lowland regions, with some subgroups separated from each other by mountain ranges. In addition, separation of the groups in mountainous regions was correlated with altitude. These results indicate that cattle rabies is derived from several regionally-defined variants, which suggests that its geographical distribution is related to that of the vampire bat population.     

The rabies viruses of bats

In the 1930s rabies was shown to affect blood-, insect- and fruit-eating bats. We have prepared anti-nucleocapsid monoclonal antibodies (MAbs) using Mokola and bat (Lagos, Duvenhage and Denmark) rabies viruses as immunogens. With these MAbs we have examined rabies viruses from vampire, insectivorous and frugivorous bats from the Americas, Africa, Europe and the Soviet Union and have compared them with isolates from terrestrial species including man. As well as confirming the findings of others with viruses of African and American bat origin, the results revealed the presence of a second biotype in European bats and demonstrated the presence of serotype 1 as well as serotype 4 viruses in bats of the Soviet Union.     

Rabies-free status of Switzerland following 30 years of rabies in foxes

The European fox rabies epizootic starting in 1939 at the eastern border of Poland reached Switzerland on March 3, 1967. Rabies spread over large parts of the country until 1977, the year it caused three human deaths. In 1978 the first field trial world-wide for the oral immunization of foxes against rabies was conducted in Switzerland. Initially, the expansion of the vaccination area led to a rapid reduction in rabies cases. However, the 1990s were characterized by a recrudescence of rabies in spite of regular oral immunization of foxes. The last endemic case of rabies was diagnosed in 1996 after an adaptation of the vaccination strategy. A total of 17,109 rabies cases, of which 73% in foxes and 14% in domestic animals were diagnosed, leading to an estimated number of some 25,000 postexposure treatments in humans. To eliminate rabies, a total of 2.8 million baits containing a modified live virus were distributed--mostly by hand--in the field.     

A case study of rabies diagnosis from formalin-fixed brain material

Rabies is caused by several Lyssavirus species, a group of negative sense RNA viruses. Although rabies is preventable, it is often neglected particularly in developing countries in the face of many competing public and veterinary health priorities. Epidemiological information based on laboratory-based surveillance data is critical to adequately strategise control and prevention plans. In this regard the fluorescent antibody test for rabies virus antigen in brain tissues is still considered the basic requirement for laboratory confirmation of animal cases. Occasionally brain tissues from suspected rabid animals are still submitted in formalin, although this has been discouraged for a number of years. Immunohistochemical testing or a modified fluorescent antibody technique can be performed on such samples. However, this method is cumbersome and cannot distinguish between different Lyssavirus species. Owing to RNA degradation in formalin-fixed tissues, conventional RT-PCR methodologies have also been proven to be unreliable. This report is concerned with a rabies case in a domestic dog from an area in South Africa where rabies is not common. Typing of the virus involved was therefore important, but the only available sample was submitted as a formalin-fixed specimen. A real-time RT-PCR method was therefore applied and it was possible to confirm rabies and obtain phylogenetic information that indicated a close relationship between this virus and the canid rabies virus variants from another province (KwaZulu-Natal) in South Africa.