Serum Neutralization Profiles of Straw-Colored Fruit Bats (Eidolon helvum) in Makurdi (Nigeria), against Four Lineages of Lagos Bat Lyssavirus.

Lagos bat lyssavirus (LBV) comprising four lineages (A, B, C and D) can potentially cause the fatal disease rabies. Although LBV-B was initially isolated in Nigeria in 1956, there is no information on LBV lineages circulating in Nigeria. This study was undertaken for the first time to measure the neutralizing antibodies against four lineages of LBVs in straw-colored fruit bats (Eidolon helvum) in Makurdi, Nigeria. Serum samples (n = 180) collected during two periods (November 2017-March 2018 and November 2018-March 2019) from terminally bled bats captured for human consumption were tested using a modified fluorescent antibody virus neutralization (mFAVN) assay. A high proportion of bat sera (74%) neutralized at least one lineage of LBV (with reciprocal titers from 9 to >420.89) and most of them neutralized LBV-A (63%), followed by LBV-D (49%), LBV-C (45%) and LBV-B (24%). The majority of positive sera (75%, n = 100) neutralized multiple LBV lineages while the remaining 25% (n = 33) neutralized only a single lineage, i.e., LBV-A (n = 23), LBV-D (n = 8) and LBV-C (n = 2). None exclusively neutralized LBV-B. The results suggest that exposure to LBV is common in E. helvum and that LBV-A (but not LBV-B) is likely to be circulating in this region of Nigeria.

Retrospective Enhanced Bat Lyssavirus Surveillance in Germany between 2018-2020.

Lyssaviruses are the causative agents for rabies, a zoonotic and fatal disease. Bats are the ancestral reservoir host for lyssaviruses, and at least three different lyssaviruses have been found in bats from Germany. Across Europe, novel lyssaviruses were identified in bats recently and occasional spillover infections in other mammals and human cases highlight their public health relevance. Here, we report the results from an enhanced passive bat rabies surveillance that encompasses samples without human contact that would not be tested under routine conditions. To this end, 1236 bat brain samples obtained between 2018 and 2020 were screened for lyssaviruses via several RT-qPCR assays. European bat lyssavirus type 1 (EBLV-1) was dominant, with 15 positives exclusively found in serotine bats (Eptesicus serotinus) from northern Germany. Additionally, when an archived set of bat samples that had tested negative for rabies by the FAT were screened in the process of assay validation, four samples tested EBLV-1 positive, including two detected in Pipistrellus pipistrellus. Subsequent phylogenetic analysis of 17 full genomes assigned all except one of these viruses to the A1 cluster of the EBLV-1a sub-lineage. Furthermore, we report here another Bokeloh bat lyssavirus (BBLV) infection in a Natterer's bat (Myotis nattereri) found in Lower Saxony, the tenth reported case of this novel bat lyssavirus.

Genetic and Antigenetic Characterization of the Novel Kotalahti Bat Lyssavirus (KBLV).

There is a growing diversity of bat-associated lyssaviruses in the Old World. In August 2017, a dead Brandt's bat (Myotis brandtii) tested positive for rabies and based on partial sequence analysis, the novel Kotalahti bat lyssavirus (KBLV) was identified. Because the bat was in an autolyzed state, isolation of KBLV was neither successful after three consecutive cell passages on cells nor in mice. Next generation sequencing (NGS) was applied using Ion Torrent ™ S5 technology coupled with target enrichment via hybridization-based capture (myBaits®) was used to sequence 99% of the genome, comprising of 11,878 nucleotides (nt). KBLV is most closely related to EBLV-2 (78.7% identity), followed by KHUV (79.0%) and BBLV (77.6%), supporting the assignment as phylogroup I lyssavirus. Interestingly, all of these lyssaviruses were also isolated from bat species of the genus Myotis, thus supporting that M. brandtii is likely the reservoir host. All information on antigenic and genetic divergence fulfil the species demarcation criteria by ICTV, so that we recommend KBLV as a novel species within the Lyssavirus genus. Next to sequence analyses, assignment to phylogroup I was functionally corroborated by cross-neutralization of G-deleted RABV, pseudotyped with KBLV-G by sera from RABV vaccinated humans. This suggests that conventional RABV vaccines also confer protection against the novel KBLV.

Phylogeny of Hungarian EBLV-1 strains using whole-genome sequence data.

European bat lyssavirus 1 (EBLV-1) is a widespread lyssavirus across Europe, whose epizootic cycle is linked to a few bat species. Occasionally, EBLV-1 infection may occur in domestic animals and humans. EBLV-1 can be classified into two subtypes, where subtype EBLV-1a shows a wide geographic distribution between France and Russia whereas subtype EBLV-1b is distributed between Spain and Poland. In this study, we determined the genome sequence of two recent EBLV-1a strains detected in Hungary and analysed their adaptive evolution and phylodynamics. The data set that included 100 EBLV-1 genome sequences identified positive selection at selected sites in genes coding for viral proteins (N, codon 18; P, 141 and 155; G, 244 and 488; L, 168, 980, 1597 and 1754). A major genetic clade containing EBLV-1a isolates from Hungary, Slovakia, Denmark and Poland was estimated to have diverged during the 19th century whereas the divergence of the most recent ancestor of Hungarian and Slovakian isolates dates back to 1950 (time span, 1930 to 1970). Phylogeographic analysis of the EBLV-1a genomic sequences demonstrated strong evidence of viral dispersal from Poland to Hungary. This new information indicates that additional migratory flyways may help the virus spread, a finding that supplements the general theory on a west-to-east dispersal of EBLV-1a strains. Long-distance migrant bats may mediate the dispersal of EBLV-1 strains across Europe; however, structured surveillance and extended genome sequencing would be needed to better understand the epizootiology of EBLV-1 infections in Europe.

Identification of a reptile lyssavirus in Anolis allogus provided novel insights into lyssavirus evolution.

Lyssaviruses (genus Lyssavirus) are negative-strand RNA viruses belonging to the family Rhabdoviridae. Although a lyssa-like virus (frog lyssa-like virus 1 [FLLV-1]), which is distantly related to lyssaviruses, was recently identified in frogs, a large phylogenetic gap exists between those viruses, and thus the evolution of lyssaviruses is unclear. In this study, we detected a lyssa-like virus from publicly available RNA-seq data obtained using the brain and skin of Anolis allogus (Spanish flag anole), which was designated anole lyssa-like virus 1 (ALLV-1), and determined its complete coding sequence. Via mapping analysis, we demonstrated that ALLV-1 was actively replicating in the original brain and skin samples. Phylogenetic analyses revealed that ALLV-1 is more closely related to lyssaviruses than FLLV-1. Overall, the topology of the tree is compatible with that of hosts, suggesting the long-term co-divergence of lyssa-like and lyssaviruses and vertebrates. The ψ region, which is a long 3' untranslated region of unknown origin present in the G mRNA of lyssaviruses (approximately 400-700 nucleotides), is also present in the genome of ALLV-1, but it is much shorter (approximately 180 nucleotides) than those of lyssaviruses. Interestingly, FLLV-1 lacks the ψ region, suggesting that the ψ region was acquired after the divergence of the FLLV-1 and ALLV-1/lyssavirus lineages. To the best of our knowledge, this is the first report to identify a lyssa-like virus in reptiles, and thus, our findings provide novel insights into the evolution of lyssaviruses.

Active surveillance for antibodies confirms circulation of lyssaviruses in Palearctic bats.

BACKGROUND: Palearctic bats host a diversity of lyssaviruses, though not the classical rabies virus (RABV). As surveillance for bat rabies over the Palearctic area covering Central and Eastern Europe and Siberian regions of Russia has been irregular, we lack data on geographic and seasonal patterns of the infection. RESULTS: To address this, we undertook serological testing, using non-lethally sampled blood, on 1027 bats of 25 species in Bulgaria, the Czech Republic, Poland, Russia and Slovenia between 2014 and 2018. The indirect enzyme-linked immunosorbent assay (ELISA) detected rabies virus anti-glycoprotein antibodies in 33 bats, giving an overall seroprevalence of 3.2%. Bat species exceeding the seroconversion threshold included Myotis blythii, Myotis gracilis, Myotis petax, Myotis myotis, Murina hilgendorfi, Rhinolophus ferrumequinum and Vespertilio murinus. While Myotis species (84.8%) and adult females (48.5%) dominated in seropositive bats, juveniles of both sexes showed no difference in seroprevalence. Higher numbers tested positive when sampled during the active season (10.5%), as compared with the hibernation period (0.9%). Bat rabies seroprevalence was significantly higher in natural habitats (4.0%) compared with synanthropic roosts (1.2%). Importantly, in 2018, we recorded 73.1% seroprevalence in a cave containing a M. blythii maternity colony in the Altai Krai of Russia. CONCLUSIONS: Identification of such "hotspots" of non-RABV lyssavirus circulation not only provides important information for public health protection, it can also guide research activities aimed at more in-depth bat rabies studies.

Circumstances of Human-Bat interactions and risk of lyssavirus transmission in metropolitan France.

Since the elimination of dog and terrestrial wild animal rabies, bat exposures remain the only source of autochthonous lyssavirus transmission to humans in Western Europe. European bats have already been found infected with several lyssaviruses, and human-bat interactions represent a risk of viral transmission and fatal encephalitis for humans. In this study, we aim to better characterize exposures to bats in metropolitan France from 2003 to 2016 and to identify circumstances associated with exposures to lyssavirus-positive bats. Two complementary sources of data were analysed: 1/ data associated with bats responsible for human exposure received for Lyssavirus testing by the French National Reference Centre for Rabies (NRCR); and 2/ data pertaining to individuals seeking medical care through the French Anti-Rabies Clinics network after contact with a bat. From 2003 to 2016, 425 bats originating from metropolitan France were submitted to the NRCR and 16 (4%) were found positive with a lyssavirus (EBLV-1b was diagnosed in 9 bats, EBLV-1a in 6 and BBLV in one specimen). The two factors associated with bat positivity in our study were the female sex and the bat belonging to the E. serotinus species. During the same study period, 1718 individuals sought care at an Anti-Rabies Clinic after exposure to a bat resulting in an estimated incidence of human-bat interactions of 1.96 per 106 person-years. The two most frequent circumstances of exposure were handling or bites. Interactions mostly involved one adult human being and one live and non-sick-looking bat. Our study provides new insights about circumstances of human-bat interactions and may be helpful to target prevention interventions to improve the awareness of the population of the risk of lyssavirus transmission.

Human rabies associated with domestic cat exposures in South Africa, 1983-2018.

No abstract available.

Seroprevalence of three paramyxoviruses; Hendra virus, Tioman virus, Cedar virus and a rhabdovirus, Australian bat lyssavirus, in a range expanding fruit bat, the Grey-headed flying fox (Pteropus poliocephalus).

Habitat-mediated global change is driving shifts in species' distributions which can alter the spatial risks associated with emerging zoonotic pathogens. Many emerging infectious pathogens are transmitted by highly mobile species, including bats, which can act as spill-over hosts for pathogenic viruses. Over three years, we investigated the seroepidemiology of paramyxoviruses and Australian bat lyssavirus in a range-expanding fruit bat, the Grey-headed flying fox (Pteropus poliocephalus), in a new camp in Adelaide, South Australia. Over six, biannual, sampling sessions, we quantified median florescent intensity (MFI) antibody levels for four viruses for a total of 297 individual bats using a multiplex Luminex binding assay. Where appropriate, florescence thresholds were determined using finite mixture modelling to classify bats' serological status. Overall, apparent seroprevalence of antibodies directed at Hendra, Cedar and Tioman virus antigens was 43.2%, 26.6% and 95.7%, respectively. We used hurdle models to explore correlates of seropositivity and antibody levels when seropositive. Increased body condition was significantly associated with Hendra seropositivity (Odds ratio = 3.67; p = 0.002) and Hendra virus levels were significantly higher in pregnant females (p = 0.002). While most bats were seropositive for Tioman virus, antibody levels for this virus were significantly higher in adults (p < 0.001). Unexpectedly, all sera were negative for Australian bat lyssavirus. Temporal variation in antibody levels suggests that antibodies to Hendra virus and Tioman virus may wax and wane on a seasonal basis. These findings suggest a common exposure to Hendra virus and other paramyxoviruses in this flying fox camp in South Australia.

An unprecedented cluster of Australian bat lyssavirus in Pteropus conspicillatus indicates pre-flight flying fox pups are at risk of mass infection.

In November 2017, two groups of P. conspicillatus pups from separate locations in Far North Queensland presented with neurological signs consistent with Australian bat lyssavirus (ABLV) infection. These pups (n = 11) died over an 11-day period and were submitted to a government laboratory for testing where ABLV infection was confirmed. Over the next several weeks, additional ABLV cases in flying foxes in Queensland were also detected. Brain tissue from ABLV-infected flying foxes during this period, as well as archived brain tissue, was selected for next-generation sequencing. Phylogenetic analysis suggests that the two groups of pups were each infected from single sources. They were likely exposed while in crèche at night as their dams foraged. This study identifies crèche-age pups at a potentially heightened risk for mass ABLV infection.

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.

Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan.

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are natural reservoirs of most lyssaviruses, a lyssavirus surveillance program of bats has been conducted in Taiwan since 2008 to understand the ecology of these viruses in bats. In this program, non-governmental bat conservation organizations and local animal disease control centers cooperated to collect dead bats or bats dying of weakness or illness. Brain tissues of bats were obtained through necropsy and subjected to direct fluorescent antibody test (FAT) and reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus antigens and nucleic acids. For the FAT, at least two different rabies diagnosis conjugates are recommended. For the RT-PCR, two sets of primers (JW12/N165-146, N113F/N304R) are used to amplify a partial sequence of the lyssavirus nucleoprotein gene. This surveillance program monitors lyssaviruses and other zoonotic agents in bats. Taiwan bat lyssavirus is found in two cases of the Japanese pipistrelle (Pipistrellus abramus) in 2016-2017. These findings should inform the public, health professionals, and scientists of the potential risks of contacting bats and other wildlife.

An inter-laboratory comparison to evaluate the technical performance of rabies diagnosis lateral flow assays.

As in previous years, the European Union Reference Laboratory (EURL) for rabies organised in 2018 an Inter-laboratory trial (ILT) on rabies diagnosis. Contrarily to past years, the 2018 ILT did not aim to evaluate the performance of participating laboratories, but the technical performance of new rapid tests. Two lateral Flow Assays (LFA), namely the Anigen® and the CDIA™ Rabies Virus Antigen Rapid Test" (commercialized by Bionote and Creative Diagnostics Cie respectively), were evaluated together with the Fluorescent Antibody Test (FAT). One panel of virus samples (including RABV as well as EBLV1a, EBLV-1b, and EBLV2 strains) was sent to participating laboratories to compare results obtained with these different techniques. The study revealed that the FAT provided a good agreement toward expected results for both negative/positive samples (99.1%). The Anigen® test produced similar results to the FAT, with only one false negative result (0.5%) reported by all participants and a concordance of 100% for all but one sample demonstrating a good inter-laboratory reproducibility of the Anigen® batch. The CDIA™ test produced reproducible results for Rabies Virus (RABV) samples only. However, it hardly detected the Bokeloh Bat Lyssavirus (BBLV) and the European Bat Lyssaviruses types 1b and 2 (EBLV-1b and EBLV-2) in most laboratories resulting in a moderate inter-laboratory concordance (58.4%-82.7%) for these lyssaviruses. The two LFAs provided reliable and reproducible results on all RABV samples (100%) but lead to heterogeneous performances with other lyssaviruses leading to different levels of diagnostic/analytical sensitivity, specificity. The study confirmed that LFAs should be used with caution and that their validation are of upmost importance before any use in laboratories.

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).

Bokeloh bat lyssavirus isolation in a Natterer's bat, Poland.

In recent years, Bokeloh bat lyssavirus (BBLV), a member of the novel lyssavirus genus Bokeloh bat lyssavirus in the family Rhabdoviridae, has been detected in Germany (five cases) and France (two cases). Here, we report the isolation of BBLV in a Natterer's bat (Myotis nattereri) in Poland. The bat brain tested positive for rabies using classical diagnostics tests (FAT and RTCIT) and then subsequently confirmed by molecular techniques. Viral RNA was found in all peripheral organs tested, and the highest viral loads were detected in brain, the salivary gland and bladder. Phylogenetic analysis performed on complete viral genome sequences revealed the closest homology to representatives of BBLV lineage B, isolated previously in southern Germany. This case provides further evidence that BBLV is widespread in Europe.

Molecular and serological survey of lyssaviruses in Croatian bat populations.

BACKGROUND: Rabies 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. RESULTS: In 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 Trbusnjak 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. CONCLUSIONS: The 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.

Evidence of Australian bat lyssavirus infection in diverse Australian bat taxa.

Historically, Australia was considered free of rabies and rabieslike viruses. Thus, the identification of Australian bat lyssavirus (ABLV) in 1996 in a debilitated bat found by a member of the public precipitated both public health consternation and a revision of lyssavirus taxonomy. Subsequent observational studies sought to elaborate the occurrence and frequency of ABLV infection in Australian bats. This paper describes the taxonomic diversity of bat species showing evidence of ABLV infection to better inform public health considerations. Blood and/or brain samples were collected from two cohorts of bats (wild-caught and diagnostic submissions) from four Australian states or territories between April 1996 and October 2002. Fresh brain impression smears were tested for ABLV antigen using fluorescein-labelled anti-rabies monoclonal globulin (CENTOCOR) in a direct fluorescent antibody test; sera were tested for the presence of neutralising antibodies using a rapid fluorescent focus inhibition test. A total of 3,217 samples from 2,633 bats were collected and screened: brain samples from 1,461 wild-caught bats and 1,086 submitted bats from at least 16 genera and seven families, and blood samples from 656 wild-caught bats and 14 submitted bats from 14 genera and seven families. Evidence of ABLV infection was found in five of the six families of bats occurring in Australia, and in three of the four Australian states/territories surveyed, supporting the historic presence of the virus in Australia. While the infection prevalence in the wild-caught cohort is evidently low, the significantly higher infection prevalence in rescued bats in urban settings represents a clear and present public health significance because of the higher risk of human exposure.