Glycoproteins of Predicted Amphibian and Reptile Lyssaviruses Can Mediate Infection of Mammalian and Reptile Cells.

Lyssaviruses are neurotropic rhabdoviruses thought to be restricted to mammalian hosts, and to originate from bats. The identification of lyssavirus sequences from amphibians and reptiles by metatranscriptomics thus comes as a surprise and challenges the mammalian origin of lyssaviruses. The novel sequences of the proposed American tree frog lyssavirus (ATFLV) and anole lizard lyssavirus (ALLV) reveal substantial phylogenetic distances from each other and from bat lyssaviruses, with ATFLV being the most distant. As virus isolation has not been successful yet, we have here studied the functionality of the authentic ATFLV- and ALLV-encoded glycoproteins in the context of rabies virus pseudotype particles. Cryogenic electron microscopy uncovered the incorporation of the plasmid-encoded G proteins in viral envelopes. Infection experiments revealed the infectivity of ATFLV and ALLV G-coated RABV pp for a broad spectrum of cell lines from humans, bats, and reptiles, demonstrating membrane fusion activities. As presumed, ATFLV and ALLV G RABV pp escaped neutralization by human rabies immune sera. The present findings support the existence of contagious lyssaviruses in poikilothermic animals, and reveal a broad cell tropism in vitro, similar to that of the rabies virus.

Renewed Public Health Threat from Emerging Lyssaviruses.

Pathogen discovery contributes to our knowledge of bat-borne viruses and is linked to the heightened interest globally in bats as recognised reservoirs of zoonotic agents. The transmission of lyssaviruses from bats-to-humans, domestic animals, or other wildlife species is uncommon, but interest in these pathogens remains due to their ability to cause an acute, progressive, invariably fatal encephalitis in humans. Consequently, the detection and characterisation of bat lyssaviruses continues to expand our knowledge of their phylogroup definition, viral diversity, host species association, geographical distribution, evolution, mechanisms for perpetuation, and the potential routes of transmission. Although the opportunity for lyssavirus cross-species transmission seems rare, adaptation in a new host and the possibility of onward transmission to humans requires continued investigation. Considering the limited efficacy of available rabies biologicals it is important to further our understanding of protective immunity to minimize the threat from these pathogens to public health. Hence, in addition to increased surveillance, the development of a niche pan-lyssavirus vaccine or therapeutic biologics for post-exposure prophylaxis for use against genetically divergent lyssaviruses should be an international priority as these emerging lyssaviruses remain a concern for global public health.

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.

Assessing Rabies Vaccine Protection against a Novel Lyssavirus, Kotalahti Bat Lyssavirus.

Rabies is a fatal encephalitis caused by an important group of viruses within the Lyssavirus genus. The prototype virus, rabies virus, is still the most commonly reported lyssavirus and causes approximately 59,000 human fatalities annually. The human and animal burden of the other lyssavirus species is undefined. The original reports for the novel lyssavirus, Kotalahti bat lyssavirus (KBLV), were based on the detection of viral RNA alone. In this report we describe the successful generation of a live recombinant virus, cSN-KBLV; where the full-length genome clone of RABV vaccine strain, SAD-B19, was constructed with the glycoprotein of KBLV. Subsequent in vitro characterisation of cSN-KBLV is described here. In addition, the ability of a human rabies vaccine to confer protective immunity in vivo following challenge with this recombinant virus was assessed. Naïve or vaccinated mice were infected intracerebrally with a dose of 100 focus-forming units/30 µL of cSN-KBLV; all naïve mice and 8% (n = 1/12) of the vaccinated mice succumbed to the challenge, whilst 92% (n = 11/12) of the vaccinated mice survived to the end of the experiment. This report provides strong evidence for cross-neutralisation and cross-protection of cSN-KBLV using purified Vero cell rabies vaccine.

Phenotypic Divergence of P Proteins of Australian Bat Lyssavirus Lineages Circulating in Microbats and Flying Foxes.

Bats are reservoirs of many pathogenic viruses, including the lyssaviruses rabies virus (RABV) and Australian bat lyssavirus (ABLV). Lyssavirus strains are closely associated with particular host reservoir species, with evidence of specific adaptation. Associated phenotypic changes remain poorly understood but are likely to involve phosphoprotein (P protein), a key mediator of the intracellular virus-host interface. Here, we examine the phenotype of P protein of ABLV, which circulates as two defined lineages associated with frugivorous and insectivorous bats, providing the opportunity to compare proteins of viruses adapted to divergent bat species. We report that key functions of P protein in the antagonism of interferon/signal transducers and activators of transcription 1 (STAT1) signaling and the capacity of P protein to undergo nuclear trafficking differ between lineages. Molecular mapping indicates that these differences are functionally distinct and appear to involve modulatory effects on regulatory regions or structural impact rather than changes to defined interaction sequences. This results in partial but significant phenotypic divergence, consistent with "fine-tuning" to host biology, and with potentially distinct properties in the virus-host interface between bat families that represent key zoonotic reservoirs.

Lagos Bat Virus, an Under-Reported Rabies-Related Lyssavirus.

Lagos bat virus (LBV), one of the 17 accepted viral species of the Lyssavirus genus, was the first rabies-related virus described in 1956. This virus is endemic to the African continent and is rarely encountered. There are currently four lineages, although the observed genetic diversity exceeds existing lyssavirus species demarcation criteria. Several exposures to rabid bats infected with LBV have been reported; however, no known human cases have been reported to date. This review provides the history of LBV and summarizes previous knowledge as well as new detections. Genetic diversity, pathogenesis and prevention are re-evaluated and discussed.

Full-Genome Sequences and Phylogenetic Analysis of Archived Danish European Bat Lyssavirus 1 (EBLV-1) Emphasize a Higher Genetic Resolution and Spatial Segregation for Sublineage 1a.

European bat lyssavirus type 1 (EBLV-1) is the causative agent for almost all reported rabies cases found in European bats. In recent years, increasing numbers of available EBLV-1 full genomes and their phylogenetic analyses helped to further elucidate the distribution and genetic characteristics of EBLV-1 and its two subtypes, namely EBLV-1a and EBLV-1b. Nonetheless, the absence of full-genome sequences from regions with known detections of EBLV-1 still limit the understanding of the phylogeographic relations between viruses from different European regions. In this study, a set of 21 archived Danish EBLV-1 samples from the years 1985 to 2009 was processed for the acquisition of full-genome sequences using a high-throughput sequencing approach. Subsequent phylogenetic analysis encompassing all available EBLV-1 full genomes from databases revealed the Danish sequences belong to the EBLV-1a subtype and further highlighted the distinct, close phylogenetic relationship of Danish, Dutch and German isolates in this region. In addition, the formation of five putative groups nearly exclusively formed by Danish isolates and the overall increased resolution of the EBLV-1a branch indicate a higher genetic diversity and spatial segregation for this sublineage than was previously known. These results emphasize the importance of phylogenetic analyses of full-genome sequences of lyssaviruses for genetic geography.

Isolation and Characterization of Cross-Reactive Human Monoclonal Antibodies That Potently Neutralize Australian Bat Lyssavirus Variants and Other Phylogroup 1 Lyssaviruses.

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections consists of wound cleansing, administration of the rabies vaccine and injection of rabies immunoglobulin (RIG) proximal to the wound. Despite the efficacy of PEP, the inaccessibility of human RIG (HRIG) in the developing world and the high immunogenicity of equine RIG (ERIG) has led to consideration of human monoclonal antibodies (hmAbs) as a passive immunization option that offers enhanced safety and specificity. Using a recombinant vesicular stomatitis virus (rVSV) expressing the glycoprotein (G) protein of ABLVs and phage display, we identified two hmAbs, A6 and F11, which completely neutralize ABLVs/ABLVp, and RABV at concentrations ranging from 0.39 and 6.25 µg/mL and 0.19 and 0.39 µg/mL respectively. A6 and F11 recognize overlapping epitopes in the lyssavirus G protein, effectively neutralizing phylogroup 1 lyssaviruses, while having little effect on phylogroup 2 and non-grouped diverse lyssaviruses. These results suggest that A6 and F11 could be effective therapeutic and diagnostic tools for phylogroup 1 lyssavirus infections.

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.

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

No abstract available.

Rabies in the African Civet: An Incidental Host for Lyssaviruses?

In South Africa, canid rabies virus (RABV) infection is maintained in domestic and wildlife species. The identification of rabies in African civets raised the question of whether this wildlife carnivore is a potential reservoir host of RABVs of direct and ancestral dog origin (dog-maintained and dog-derived origins) with an independent cycle of transmission. Genetic analyses of African civet nucleoprotein sequences for 23 African civet RABVs and historically published sequences demonstrated that RABVs from African civets have two origins related to dog and mongoose rabies enzootics. The data support observations of the interaction of civets with domestic dogs and wildlife mongooses, mostly in Northern South Africa and North-East Zimbabwe. Within each host species clade, African civet RABVs group exclusively together, implying intra-species virus transfer occurs readily. The canid RABV clade appears to support virus transfer more readily between hosts than mongoose RABVs. Furthermore, these data probably indicate short transmission chains with conspecifics that may be related to transient rabies maintenance in African civets. Hence, it is important to continue monitoring the emergence of lyssaviruses in this host. Observations from this study are supported by ongoing and independent similar cases, in which bat-eared foxes and black-backed jackal species maintain independent rabies cycles of what were once dog-maintained RABVs.

Serological Survey of Lyssaviruses in Polish Bats in the Frame of Passive Rabies Surveillance Using an Enzyme-Linked Immunosorbent Assay.

Background: Bats are known to host a number of nonpathogenic viruses, as well as highly pathogenic viruses causing fatal diseases like rabies. Serological surveys as part of active and passive bat rabies surveillance mainly use seroneutralization assays, demonstrating the presence of lyssavirus-specific antibodies in a variety of European bats, particularly against European bat lyssaviruses type 1 (EBLV-1). Here, we present the first serological survey in European bats of this kind during which European bats from Poland collected in the frame of passive rabies surveillance between 2012 and 2018, as well as Serotine bats (Eptesicus serotinus) and North American Big Brown bats (Eptesicus fuscus) from previous experimental studies, were tested using a commercial ELISA kit for the detection of anti-lyssavirus antibodies. Results: Lyssavirus-specific antibodies were detected in 35 (30.4%) out of 115 Polish bats of both sexes, representing nine out of 13 identified bat species endemic mainly to Central Southern Europe and Western Asia, i.e., Eptesicus serotinus, Nyctalus noctula, Myotis daubentonii, Plecotus auritus, Vespertillo murinus,Pipistrellus pipistrellus, Pipistrellus pipilstrellus/Pipistrellus pygmaeus, Myotis brandtii, and Barbastella barbastellus. Seroprevalence was highest in bat species of Nyctalus noctula, Eptesicus serotinus, Plecotus auritus, and Myotis daubentonii. More than 60% of the ELISA seropositive bats originated from the voivodeships of Silesia, Lower-Silesian, Warmian-Mazurian, and Mazowian. Rabies-specific antibodies were also found in Eptesicus fuscus bats from North America. Conclusions: The study demonstrates the principal application of the BioPro Rabies ELISA Ab Kit for the detection of anti-lyssavirus specific antibodies in body fluids and serum samples of bats. However, results may only be reliable for North American bats, whereas interpretation of results for European bats per se is difficult because proper validation of the test is hampered by the protected status of these species.

Current Rabies Vaccines Do Not Confer Protective Immunity against Divergent Lyssaviruses Circulating in Europe.

The use of the rabies vaccine for post-exposure prophylaxis started as early as 1885, revealing a safe and efficient tool to prevent human rabies cases. Preventive vaccination is the basis for the control of canine-mediated rabies, which has already been eliminated from extensive parts of the world, including Europe. Plans to eliminate canine-mediated human rabies by 2030 have been agreed upon by international organisations. However, rabies vaccines are not efficacious against some divergent lyssaviruses. The presence in European indigenous bats of recently described lyssaviruses, which are not neutralised by antibody responses to existing vaccines, as well as the declaration of an imported case of an African lyssavirus, which also escapes vaccine-derived protection, leaves the European health authorities unable to provide efficacious protective vaccines to some potential situations of human exposure. All these circumstances highlight the need for a universal pan-lyssavirus rabies vaccine, able to prevent human rabies in all circumstances.

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.

Isolation, antigenicity and immunogenicity of Lleida bat lyssavirus.

The lyssaviruses are an important group of viruses that cause a fatal encephalitis termed rabies. The prototypic lyssavirus, rabies virus, is predicted to cause more than 60 000 human fatalities annually. The burden of disease for the other lyssaviruses is undefined. The original reports for the recently described highly divergent Lleida bat lyssavirus were based on the detection of virus sequence alone. The successful isolation of live Lleida bat lyssavirus from the carcass of the original bat and in vitro characterization of this novel lyssavirus are described here. In addition, the ability of a human rabies vaccine to confer protective immunity following challenge with this divergent lyssavirus was assessed. Two different doses of Lleida bat lyssavirus were used to challenge vaccinated or naïve mice: a high dose of 100 focus-forming units (f.f.u.) 30 µl-1 and a 100-fold dilution of this dose, 1 f.f.u. 30 µl-1. Although all naïve control mice succumbed to the 100 f.f.u. 30 µl-1 challenge, 42 % (n=5/12) of those infected intracerebrally with 1 f.f.u. 30 µl-1 survived the challenge. In the high-challenge-dose group, 42 % of the vaccinated mice survived the challenge (n=5/12), whilst at the lower challenge dose, 33 % (n=4/12) survived to the end of the experiment. Interestingly, a high proportion of mice demonstrated a measurable virus-neutralizing antibody response, demonstrating that neutralizing antibody titres do not necessarily correlate with the outcome of infection via the intracerebral route. Assessing the ability of existing rabies vaccines to protect against novel divergent lyssaviruses is important for the development of future public health strategies.

Lyssavirus in Japanese Pipistrelle, Taiwan.

A putative new lyssavirus was found in 2 Japanese pipistrelles (Pipistrellus abramus) in Taiwan in 2016 and 2017. The concatenated coding regions of the virus showed 62.9%-75.1% nucleotide identities to the other 16 species of lyssavirus, suggesting that it may be representative of a new species of this virus.

Pathogenesis of bat rabies in a natural reservoir: Comparative susceptibility of the straw-colored fruit bat (Eidolon helvum) to three strains of Lagos bat virus.

Rabies is a fatal neurologic disease caused by lyssavirus infection. People are infected through contact with infected animals. The relative increase of human rabies acquired from bats calls for a better understanding of lyssavirus infections in their natural hosts. So far, there is no experimental model that mimics natural lyssavirus infection in the reservoir bat species. Lagos bat virus is a lyssavirus that is endemic in straw-colored fruit bats (Eidolon helvum) in Africa. Here we compared the susceptibility of these bats to three strains of Lagos bat virus (from Senegal, Nigeria, and Ghana) by intracranial inoculation. To allow comparison between strains, we ensured the same titer of virus was inoculated in the same location of the brain of each bat. All bats (n = 3 per strain) were infected, and developed neurological signs, and fatal meningoencephalitis with lyssavirus antigen expression in neurons. There were three main differences among the groups. First, time to death was substantially shorter in the Senegal and Ghana groups (4 to 6 days) than in the Nigeria group (8 days). Second, each virus strain produced a distinct clinical syndrome. Third, the spread of virus to peripheral tissues, tested by hemi-nested reverse transcriptase PCR, was frequent (3 of 3 bats) and widespread (8 to 10 tissues positive of 11 tissues examined) in the Ghana group, was frequent and less widespread in the Senegal group (3/3 bats, 3 to 6 tissues positive), and was rare and restricted in the Nigeria group (1/3 bats, 2 tissues positive). Centrifugal spread of virus from brain to tissue of excretion in the oral cavity is required to enable lyssavirus transmission. Therefore, the Senegal and Ghana strains seem most suitable for further pathogenesis, and for transmission, studies in the straw-colored fruit bat.

Molecular Epidemiology and Evolution of European Bat Lyssavirus 2.

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