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.     

First isolation of EBLV-2 in Germany

In Europe, rabies in bats is caused by European Bat Lyssavirus (EBLV) type 1 (EBLV-1) or type 2 (EBLV-2) which form two distinct genotypes (gt 5 and 6) within the genus Lyssavirus of the family of Rhadoviridae. Spill-over infections of EBLV in humans have caused fatal rabies encephalitis and highlighted the relevance of this wildlife disease for public health. The vast majority of the 831 European bat rabies cases reported between 1977 and 2006 were identified as EBLV-1. Only few virus isolates originating from Switzerland, The Netherlands and the United Kingdom were characterized as EBLV-2. Here we report the first EBLV-2 case detected in Germany in a Daubenton's bat (Myotis daubentonii) in August 2007. The bat showed clinical signs of disorders of the central nervous system and subsequently tested positive for rabies. The virus was isolated and characterized as EBLV-2 based on its antigen pattern and by nucleotide sequencing. Phylogenetic analysis indicated an association to EBLV-2 isolates from Switzerland which correlates with the origin of the bat close to the Swiss border.     

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.     

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.     

Active surveillance of bat rabies in France: a 5-year study (2004-2009)

Active surveillance of bats in France started in 2004 with an analysis of 18 of the 45 bat species reported in Europe. Rabies antibodies were detected in six indigenous species, mainly in Eptesicus serotinus and Myotis myotis, suggesting previous contact with the EBLV-1 rabies virus. Nineteen of the 177 tested bats were shown serologically positive in seven sites, particularly in central and south-western France. Neither infectious viral particles nor viral genomes were detected in 173 and 308 tested oral swabs, respectively. The presence of neutralising antibodies in female bats (18.6%) was significantly higher than in males (5.6%).     

北京1株犬源狂犬病病毒全基因组序列测定及分析

本试验旨在对北京1株犬源狂犬病病毒株(BJ2012ZW株)在全基因组水平上进行分子进化研究,比较与全国流行株及疫苗株之间的差异.试验采集犬脑组织以直接免疫荧光技术和内基氏小体检查方法进行检测,以RT-PCR扩增病毒核酸覆盖全基因组,对产物测序后进行遗传学分析.结果显示,BJ2012ZW株狂犬病病毒属于基因1型,与目前中国的主要流行株全序列同源性为83.9%~99.7%.与同样分离自北京的毒株BJ2011E和CNM1101C的核苷酸全序列同源性最高(99.7%),进化关系近.G蛋白主要抗原位点分析结果表明,BJ2012ZW株与国内疫苗株相比有部分抗原位点发生了替换.BJ2012ZW株属于中国目前的流行株,与目前国内所使用的疫苗株存在一定的差异.     

Perspectives on molecular detection methods of lyssaviruses

Lyssaviruses belong to the family Rhabdoviridae within the order Mononegavirales. Their genomes consist of a negative stranded linear RNA. Although dog transmitted rabies causes the majority of the estimated 55 000 worldwide fatalities per year, bat lyssaviruses have also caused human cases. Based on a broad range of different host species, their geographical distribution and the evolutionary age lyssaviruses display a high degree of genetic variability. Thus the development of molecular diagnostics for the reliable detection and identification of members of the genus Lyssavirus (pan-lyssavirus assay) is a challenge, but is valuable for the fight against rabies worldwide. In this study different published PCR-systems or primers for the detection of a wide range of lyssaviruses were tested as real-time RT-PCRs with different one-step and two-step assays. Comparison of different two-step assays by varying the RT-chemistry revealed differences in sensitivity. Nevertheless, most of the tested one-step systems provided an improved performance, including a reduced assay time and a reduced risk of cross-contaminations, when compared to the optimized two-step assay. Finally, we also provide an overview of additional state-of-the-art molecular methods to detect and differentiate lyssavirus species in general.     

Trends in national surveillance for rabies among bats in the United States (1993-2000)

OBJECTIVE: To describe surveillance trends and epidemiologic features of rabies in bats in the United States, focusing on 3 bat species primarily associated with variants of the rabies virus that affect humans. DESIGN: Retrospective study. ANIMALS: 31,380 bats. PROCEDURE: Data on rabies for bats identified to species and reported by state laboratories from 1993 to 2000 were analyzed, focusing on silver-haired, eastern pipistrelle, and Brazilian free-tailed bats. Categoric variables were derived from other provided information. RESULTS: Data were reported from 37 states during the study interval; complete species-specific data were not reported by any state for the entire interval. Bats primarily associated with rabies virus variants affecting humans were more likely to yield positive test results for rabies (22.7%), compared with all other bats (5.5%) in most seasons and from most regions of the United States. However, certain other bat species had higher percentages of positive results. Risk of positive results was highest in the fall and highest among bats originating in the southwestern United States. CONCLUSIONS AND CLINICAL RELEVANCE: Increased risk of rabies among certain groups of bat species was consistently found across seasons and most geographic regions of the United States. Results were in general agreement with those of previous studies conducted within smaller geographic regions. There are ongoing efforts to improve surveillance of rabies in bats, although surveillance is incomplete in some regions.     

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.     

Determination and molecular analysis of the complete genome sequence of two wild-type rabies viruses isolated from a haematophagous bat and a frugivorous bat in Brazil

The complete genome sequences of two Brazilian wild-type rabies viruses (RABV), a BR-DR1 isolate from a haematophagous bat (Desmodus rotundus) and a BR-AL1 isolate from a frugivorous bat (Artibeus lituratus), were determined. The genomes of the BR-DR1 and BR-AL1 had 11,923 and 11,922 nt, respectively, and both encoded the five standard genes of rhabdoviruses. The complete nucleotide sequence identity between the BR-DR1 and BR-AL1 isolates was 97%. The BR-DR1 and BR-AL1 isolates had some conserved functional sites revealed by the fixed isolates, whereas both isolates had unique amino acid substitutions in the antigenic region IV of the nucleocapsid gene. Therefore, it is speculated that both isolates were nearly identical in virologic character. According to our phylogenetic analysis based on the complete genomes, both isolates belonged to genotype 1, and to the previously defined "vampire bat-related RABV lineage" which consisted of mainly D. rotundus- and A. lituratus-isolates; however, a branch pattern with high bootstrap values suggested that BR-DR1 was more closely related to the 9001FRA isolate, which was collected from a dog bitten by a bat in French Guiana, than to BR-AL1. This result suggests that the vampire bat-related RABV lineage includes Brazilian vampire bat and Brazilian frugivorous bat RABV and is further divided into Brazilian vampire bat and Brazilian frugivorous bat RABV sub-lineages. The phylogenetic analysis based on the complete genomes was valuable in discriminating among very closely related isolates.     

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.     

Bat rabies in alberta 1979-1982

The infection rate among eight species of bats submitted for rabies diagnosis in Alberta during 1979-82 was 4.6%. Prevalence of rabies was greatest (24%) for hoary bats Lasiurus cinereus, while the big brown bat Eptesicus fuscus was the species in which rabies was most commonly diagnosed, and the species submitted most frequently for rabies diagnosis was the little brown bat Myotis lucifugus. The rabies infection rate among male hoary bats was significantly greater than in either sex of all other submitted species. The frequency of rabies diagnosis in hoary bats submitted during 1979-82 was also significantly higher than in those submitted between 1971 and 1978. There has been a significant decrease in the rabies prevalence or infection rate of little brown bats since 1971-78.     

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.     

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.     

Increased detection of rabies virus in bats in Ceará State (Northeast Brazil) after implementation of a passive surveillance programme

The intensification of dog, cat and livestock vaccination campaigns significantly reduced rabies cases in humans and domestic animals in Ceará State, Brazil. However, sylvatic animals—bats (order Chiroptera), wild canids, raccoons and non‐human primates— remain as reservoirs for the virus. Our hypothesis is that surveillance and monitoring of rabies virus in bats, especially passive surveillance, is of fundamental importance, besides the implementation of health education and strengthening of surveillance actions in humans exposed to aggressions. Thus, we assessed the occurrence of rabies virus in animals focusing on bats, before and after launching of the Sylvatic Rabies Surveillance Program in 2010. Surveillance data from the 184 municipalities of Ceará State were analysed, collected during the periods 2003–2010 (active surveillance) and 2011–2016 (passive surveillance), respectively. A total of 13,543 mammalian samples were received for rabies diagnosis from 2003 to 2016. Of these, 10,960 were from dogs or cats (80.9%), 1,180 from bats (8.7%), 806 from other sylvatic animals (foxes, marmosets, raccoons; 6.0%) and 597 from herbivores (cattle, goats, sheep, equines, pigs; 4.4%). A total of 588 (4.3%) samples were positive for rabies. About 8.4% (99/1,180) of the bat samples were infected with rabies virus, 92 (92.9%) of these were from non‐haematophagous bat species and 7 (7.1%) from haematophagous species. The number of bat samples received and infection rates increased considerably, after a shift from active surveillance (9/355 [2.5%] samples positive), to passive surveillance (90/825 [10.9%] samples positive). Surveillance of rabies virus in bats is fundamental for human and domestic animal health in Ceará State. Bats have to be considered as targets in surveillance and control programmes. Virus lineages should be characterized to increase knowledge on transmission dynamics of sylvatic rabies virus to domestic animals and the human population, and to provide additional evidence for planning and implementation of improved control measures.     

Characteristics of bat rabies in Alberta.

Rabies in bats was monitored in Alberta from 1971 to 1978 Big brown bats replaced silver-haired bats as the species most frequently reported rabid during these years. Rabies infection was comparatively high among little brown bats in central Alberta in 1973 and has subsequently declined. Only one rabid little brown bat was discovered in southern Alberta which is populated by a different subspecies. Outbreaks of rabies in little brown and big brown bat colonies tended to be brief events. Observations of free-ranging bats with probable furious rabies suggested that bats do not generally identify humans as targets for attack. Independent trends in infection rates suggested that spread of rabies is primarily intraspecific but there is evidence that migratory bats play a role in introduction and maintenance of rabies in northern temperate bat communities. The dynamics of bat rabies in Alberta are discussed.     

Experimental infection of Artibeus intermedius with a vampire bat rabies virus

Experimental infection of Artibeus intermedius, the great fruit-eating bat, was performed with vampire bat rabies isolates. Bats (n = 35) were captured in the wild and quarantined prior to experimental infection. No rabies antibodies were detected by rapid fluorescent focus inhibition test (RFFIT) prior to infection. Three doses of rabies virus (RV) and three different routes of infection were used. One out of 35 bats died without showing any clinical signs at day 14 and was positive for rabies. None of the 34 other bats showed clinical signs for rabies, but high antibody titers were detected post-inoculation, suggesting either innate immune response to the vampire bat rabies virus or possible pre-exposure to RV and inoculation leading to a booster effect. Rabies virus was detected by hemi-nested RT-PCR (hnRT-PCR) in the brain (n = 3), stomach (n = 1) of bats that were negative by immunofluorescence and that survived rabies infection. The bat that died on day 14 was positive by hnRT-PCR on the brain, heart and liver. These results suggest that either previous non-lethal exposure to RV or natural low susceptibility to vampire bat viruses somehow protected Artibeus intermedius from clinical rabies infection leading to a marginal lethality effect on this bats species population in the wild.     

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.     

Re‐emergence of rabies virus maintained by canid populations in Paraguay

Paraguay has registered no human cases of rabies since 2004, and the last case in dogs, reported in 2009, was due to a variant maintained in the common vampire bat “Desmodus rotundus”. In 2014, a dog was diagnosed as positive for rabies with aggression towards a boy and all required measures of control were successfully adopted. Epidemiological investigation revealed that the dog was not vaccinated and had been attacked by a crab‐eating fox, “zorro” (Cerdocyon thous). The sample was diagnosed by the Official Veterinary Service of the Country and sent to the Center on Rabies Research from the University of São Paulo, Brazil, for antigenic and genetic characterization. A second sample from a dog positive for rabies in the same region in 2015 and 11 samples from a rabies outbreak from Asuncion in 1996 were also characterized. The antigenic profile of the samples, AgV2, was compatible with one of the variants maintained by dogs in Latin America. In genetic characterization, the samples segregated in the canine (domestic and wild species)‐related group in an independent subgroup that also included samples from Argentina. These results and the epidemiology of the case indicate that even with the control of rabies in domestic animals, the virus can still circulate in wildlife and may be transmitted to domestic animals and humans, demonstrating the importance of continuous and improved surveillance and control of rabies, including in wild species, to prevent outbreaks in controlled areas.