Lyssavirus P-protein selectively targets STAT3-STAT1 heterodimers to modulate cytokine signalling.

Many viruses target signal transducer and activator of transcription (STAT) 1 to antagonise antiviral interferon signalling, but targeting of STAT3, a pleiotropic molecule that mediates signalling by diverse cytokines, is poorly understood. Here, using lyssavirus infection, quantitative live cell imaging, innate immune signalling and protein interaction assays, and complementation/depletion of STAT expression, we show that STAT3 antagonism is conserved among P-proteins of diverse pathogenic lyssaviruses and correlates with pathogenesis. Importantly, P-protein targeting of STAT3 involves a highly selective mechanism whereby P-protein antagonises cytokine-activated STAT3-STAT1 heterodimers, but not STAT3 homodimers. RT-qPCR and reporter gene assays indicate that this results in specific modulation of interleukin-6-dependent pathways, effecting differential antagonism of target genes. These data provide novel insights into mechanisms by which viruses can modulate cellular function to support infection through discriminatory targeting of immune signalling complexes. The findings also highlight the potential application of selective interferon-antagonists as tools to delineate signalling by particular STAT complexes, significant not only to pathogen-host interactions but also cell physiology, development and cancer.

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.

Antigenic site changes in the rabies virus glycoprotein dictates functionality and neutralizing capability against divergent lyssaviruses.

Lyssavirus infection has a near 100 % case fatality rate following the onset of clinical disease, and current rabies vaccines confer protection against all reported phylogroup I lyssaviruses. However, there is little or no protection against more divergent lyssaviruses and so investigation into epitopes within the glycoprotein (G) that dictate a neutralizing response against divergent lyssaviruses is warranted. Importantly, the facilities required to work with these pathogens, including wild-type and mutated forms of different lyssaviruses, are scarcely available and, as such, this type of study is inherently difficult to perform. The relevance of proposed immunogenic antigenic sites within the lyssavirus glycoprotein was assessed by swapping sites between phylogroup-I and -II glycoproteins. Demonstrable intra- but limited inter-phylogroup cross-neutralization was observed. Pseudotype viruses (PTVs) presenting a phylogroup-I glycoprotein containing phylogroup-II antigenic sites (I, II III or IV) were neutralized by antibodies raised against phylogroup-II PTV with the site II (IIb, aa 34-42 and IIa, aa 198-200)-swapped PTVs being efficiently neutralized, whilst site IV-swapped PTV was poorly neutralized. Specific antibodies raised against PTV-containing antigenic site swaps between phylogroup-I and -II glycoproteins neutralized phylogroup-I PTVs efficiently, indicating an immunodominance of antigenic site II. Live lyssaviruses containing antigenic site-swapped glycoproteins were generated and indicated that specific residues within the lyssavirus glycoprotein dictate functionality and enable differential neutralizing antibody responses to lyssaviruses.

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.

Bat and pig IFN-induced transmembrane protein 3 restrict cell entry by influenza virus and lyssaviruses.

IFN-induced transmembrane protein 3 (IFITM3) is a restriction factor that blocks cytosolic entry of numerous viruses that utilize acidic endosomal entry pathways. In humans and mice, IFITM3 limits influenza-induced morbidity and mortality. Although many IFITM3-sensitive viruses are zoonotic, whether IFITMs function as antiviral restriction factors in mammalian species other than humans and mice is unknown. Here, IFITM3 orthologues in the microbat (Myotis myotis) and pig (Sus scrofa domesticus) were identified using rapid amplification of cDNA ends. Amino acid residues known to be important for IFITM3 function were conserved in the pig and microbat orthologues. Ectopically expressed pig and microbat IFITM3 co-localized with transferrin (early endosomes) and CD63 (late endosomes/multivesicular bodies). Pig and microbat IFITM3 restricted cell entry mediated by multiple influenza haemagglutinin subtypes and lyssavirus glycoproteins. Expression of pig or microbat IFITM3 in A549 cells reduced influenza virus yields and nucleoprotein expression. Conversely, small interfering RNA knockdown of IFITM3 in pig NPTr cells and primary microbat cells enhanced virus replication, demonstrating that these genes are functional in their species of origin at endogenous levels. In summary, we showed that IFITMs function as potent broad-spectrum antiviral effectors in two mammals - pigs and bats - identified as major reservoirs for emerging viruses.

Anti-lyssaviral activity of interferons κ and ω from the serotine bat, Eptesicus serotinus.

UNLABELLED: Interferons (IFNs) are cytokines produced by host cells in response to the infection with pathogens. By binding to the corresponding receptors, IFNs trigger different pathways to block intracellular replication and growth of pathogens and to impede the infection of surrounding cells. Due to their key role in host defense against viral infections, as well as for clinical therapies, the IFN responses and regulation mechanisms are well studied. However, studies of type I IFNs have mainly focused on alpha interferon (IFN-α) and IFN-ß subtypes. Knowledge of IFN-κ and IFN-ω is limited. Moreover, most studies are performed in humans or mouse models but not in the original host of zoonotic pathogens. Bats are important reservoirs and transmitters of zoonotic viruses such as lyssaviruses. A few studies have shown an antiviral activity of IFNs in fruit bats. However, the function of type I IFNs against lyssaviruses in bats has not been studied yet. Here, IFN-κ and IFN-ω genes from the European serotine bat, Eptesicus serotinus, were cloned and functionally characterized. E. serotinus IFN-κ and IFN-ω genes are intronless and well conserved between microchiropteran species. The promoter regions of both genes contain essential regulatory elements for transcription factors. In vitro studies indicated a strong activation of IFN signaling by recombinant IFN-ω, whereas IFN-κ displayed weaker activation. Noticeably, both IFNs inhibit to different extents the replication of different lyssaviruses in susceptible bat cell lines. The present study provides functional data on the innate host defense against lyssaviruses in endangered European bats. IMPORTANCE: We describe here for the first time the molecular and functional characterization of two type I interferons (IFN-κ and -ω) from European serotine bat (Eptesicus serotinus). The importance of this study is mainly based on the fact that very limited information about the early innate immune response against bat lyssaviruses in their natural host serotine bats is yet available. Generally, whereas the antiviral activity of other type I interferons is well studied, the functional involvement of IFN-κ and -ω has not yet been investigated.

Antigenic and genetic characterization of a divergent African virus, Ikoma lyssavirus.

In 2009, a novel lyssavirus (subsequently named Ikoma lyssavirus, IKOV) was detected in the brain of an African civet (Civettictis civetta) with clinical rabies in the Serengeti National Park of Tanzania. The degree of nucleotide divergence between the genome of IKOV and those of other lyssaviruses predicted antigenic distinction from, and lack of protection provided by, available rabies vaccines. In addition, the index case was considered likely to be an incidental spillover event, and therefore the true reservoir of IKOV remained to be identified. The advent of sensitive molecular techniques has led to a rapid increase in the discovery of novel viruses. Detecting viral sequence alone, however, only allows for prediction of phenotypic characteristics and not their measurement. In the present study we describe the in vitro and in vivo characterization of IKOV, demonstrating that it is (1) pathogenic by peripheral inoculation in an animal model, (2) antigenically distinct from current rabies vaccine strains and (3) poorly neutralized by sera from humans and animals immunized against rabies. In a laboratory mouse model, no protection was elicited by a licensed rabies vaccine. We also investigated the role of bats as reservoirs of IKOV. We found no evidence for infection among 483 individuals of at least 13 bat species sampled across sites in the Serengeti and Southern Kenya.

Comparative studies on the genetic, antigenic and pathogenic characteristics of Bokeloh bat lyssavirus.

Bokeloh bat lyssavirus (BBLV), a novel lyssavirus, was isolated from a Natterer's bat (Myotis nattererii), a chiropteran species with a widespread and abundant distribution across Europe. As a novel lyssavirus, the risks of BBLV to animal and human health are unknown and as such characterization both in vitro and in vivo was required to assess pathogenicity and vaccine protection. Full genome sequence analysis and antigenic cartography demonstrated that the German BBLV isolates are most closely related to European bat lyssavirus type 2 (EBLV-2) and Khujand virus and can be characterized within phylogroup I. In vivo characterization demonstrated that BBLV was pathogenic in mice when inoculated peripherally causing clinical signs typical for rabies encephalitis, with higher pathogenicity observed in juvenile mice. A limited vaccination-challenge experiment in mice was conducted and suggested that current vaccines would afford some protection against BBLV although further studies are warranted to determine a serological cut-off for protection.

RelAp43, a member of the NF-κB family involved in innate immune response against Lyssavirus infection.

NF-κB transcription factors are crucial for many cellular processes. NF-κB is activated by viral infections to induce expression of antiviral cytokines. Here, we identified a novel member of the human NF-κB family, denoted RelAp43, the nucleotide sequence of which contains several exons as well as an intron of the RelA gene. RelAp43 is expressed in all cell lines and tissues tested and exhibits all the properties of a NF-κB protein. Although its sequence does not include a transactivation domain, identifying it as a class I member of the NF-κB family, it is able to potentiate RelA-mediated transactivation and stabilize dimers comprising p50. Furthermore, RelAp43 stimulates the expression of HIAP1, IRF1, and IFN-ß - three genes involved in cell immunity against viral infection. It is also targeted by the matrix protein of lyssaviruses, the agents of rabies, resulting in an inhibition of the NF-κB pathway. Taken together, our data provide the description of a novel functional member of the NF-κB family, which plays a key role in the induction of anti-viral innate immune response.

Cross-Neutralization Activities of Antibodies against 18 Lyssavirus Glycoproteins.

Some lyssaviruses, including the rabies virus (RABV), cause lethal neurological symptoms in humans. However, the efficacy of commercial vaccines has only been evaluated against RABV. To assess cross-reactivity among lyssaviruses, including RABV, sera from rabbits inoculated with human and animal RABV vaccines and polyclonal antibodies from rabbits immunized with expression plasmids of the glycoproteins of all 18 lyssaviruses were prepared, and cross-reactivity was evaluated via virus-neutralization tests using Duvenhage lyssavirus (DUVV), European bat lyssavirus-1 (EBLV-1), Mokola lyssavirus (MOKV), Lagos bat lyssavirus (LBV), and RABV. The sera from rabbits inoculated with RABV vaccines showed cross-reactivity with EBLV-1 and DUVV, both belonging to phylogroup I. However, reactivity with MOKV and LBV in phylogroup II was notably limited or below the detection level. Next, we compared the cross-reactivity of the polyclonal antibodies against all lyssavirus glycoproteins. Polyclonal antibodies had high virus-neutralization titers against the same phylogroup but not different phylogroups. Our findings indicate that a new vaccine should be developed for pre- and post-exposure prophylaxis against lyssaviral infections.

[Postexposure prophylaxis after bite of a broad-winged bat with evidence of European bat lyssavirus 1 (EBLV-1)]. / Postexpositionsprophylaxe nach Biss einer Breitflügelfledermaus mit Nachweis des Europäischen Fledermaus-Lyssavirus 1 (EBLV-1).

Germany has been considered free of terrestrial rabies since 2008 as a result of intensive vaccination and surveillance efforts but reservoirs of the lyssaviruses EBLV­1 and EBLV­2 persist in bat colonies and thus pose a potential risk of infection. We report on a patient who suffered a bat bite in an urban setting in which European bat lyssavirus 1 (EBLV-1) was detected in the euthanized bat. We performed active and passive postexposure prophylaxis (PEP). This case study illustrates the ongoing risk of rabies infection due to close bat contacts in Germany and is intended to sensitize primary care physicians to take such exposure events seriously and to perform a regular PEP including administration of rabies immunoglobulin.

Bat Bites and Rabies PEP in the Croatian Reference Centre for Rabies 1995-2020.

Seroprevalence of lyssaviruses in certain bat species has been proven in the Republic of Croatia, but there have been no confirmed positive bat brain isolates or human fatalities associated with bat injuries/bites. The study included a retrospective analysis of bat injuries/bites, post-exposure prophylaxis (PEP) and geographic distribution of bat injuries in persons examined at the Zagreb Antirabies Clinic, the Croatian Reference Centre for Rabies. In the period 1995-2020, we examined a total of 21,910 patients due to animal injuries, of which 71 cases were bat-related (0.32%). Of the above number of patients, 4574 received rabies PEP (20.87%). However, for bat injuries, the proportion of patients receiving PEP was significantly higher: 66 out of 71 patients (92.95%). Of these, 33 received only the rabies vaccine, while the other 33 patients received the vaccine with human rabies immunoglobulin (HRIG). In five cases, PEP was not administered, as there was no indication for treatment. Thirty-five of the injured patients were biologists or biology students (49.29%). The bat species was confirmed in only one of the exposure cases. This was a serotine bat (Eptesicus serotinus), a known carrier of Lyssavirus hamburg. The results showed that the bat bites were rather sporadic compared to other human injuries caused by animal bites. All bat injuries should be treated as if they were caused by a rabid animal, and according to WHO recommendations. People who come into contact with bats should be strongly advised to be vaccinated against rabies. Entering bat habitats should be done with caution and in accordance with current recommendations, and nationwide surveillance should be carried out by competent institutions and in close collaboration between bat experts, epidemiologists and rabies experts.

Interspecies protein substitution to investigate the role of the lyssavirus glycoprotein.

European bat lyssaviruses type 1 (EBLV-1) and type 2 (EBLV-2) circulate within bat populations throughout Europe and are capable of causing disease indistinguishable from that caused by classical rabies virus (RABV). However, the determinants of viral fitness and pathogenicity are poorly understood. Full-length genome clones based on the highly attenuated, non-neuroinvasive, RABV vaccine strain (SAD-B19) were constructed with the glycoprotein (G) of either SAD-B19 (SN), of EBLV-1 (SN-1) or EBLV-2 (SN-2). In vitro characterization of SN-1 and SN-2 in comparison to wild-type EBLVs demonstrated that the substitution of G affected the final virus titre and antigenicity. In vivo, following peripheral infection with a high viral dose (10(4) f.f.u.), animals infected with SN-1 had reduced survivorship relative to infection with SN, resulting in survivorship similar to animals infected with EBLV-1. The histopathological changes and antigen distribution observed for SN-1 were more representative of those observed with SN than with EBLV-1. EBLV-2 was unable to achieve a titre equivalent to that of the other viruses. Therefore, a reduced-dose experiment (10(3) f.f.u.) was undertaken in vivo to compare EBLV-2 and SN-2, which resulted in 100 % survivorship for all recombinant viruses (SN, SN-1 and SN-2) while clinical disease developed in mice infected with the EBLVs. These data indicate that interspecies replacement of G has an effect on virus titre in vitro, probably as a result of suboptimal G-matrix protein interactions, and influences the survival outcome following a peripheral challenge with a high virus titre in mice.

Conservation of a unique mechanism of immune evasion across the Lyssavirus genus.

The evasion of host innate immunity by Rabies virus, the prototype of the genus Lyssavirus, depends on a unique mechanism of selective targeting of interferon-activated STAT proteins by the viral phosphoprotein (P-protein). However, the immune evasion strategies of other lyssaviruses, including several lethal human pathogens, are unresolved. Here, we show that this mechanism is conserved between the most distantly related members of the genus, providing important insights into the pathogenesis and potential therapeutic targeting of lyssaviruses.

Novel lyssavirus in Natterer's bat, Germany.

A virus isolated from a Natterer's bat (Myotis nattererii) in Germany was differentiated from other lyssaviruses on the basis of the reaction pattern of a panel of monoclonal antibodies. Phylogenetic analysis supported the assumption that the isolated virus, Bokeloh bat lyssavirus, may represent a new member of the genus Lyssavirus.

MHC class II DRB diversity in raccoons (Procyon lotor) reveals associations with raccoon rabies virus (Lyssavirus).

In North America, the raccoon rabies virus (RRV) is an endemic wildlife disease which causes acute encephalopathies and is a strong selective force on raccoons (Procyon lotor), with estimates of ∼85% of the population succumbing to the disease when epizootic. RRV is regarded as a lethal disease if untreated; therefore, no evolutionary response would be expected of raccoon populations. However, variable immune responses to RRV have been observed in raccoons indicating a potential for evolutionary adaptation. Studies of variation within the immunologically important major histocompatibility complex (MHC) have revealed relationships between MHC alleles and diseases in humans and other wildlife species. This enhances our understanding of how hosts and pathogens adapt and co-evolve. In this study, we used RRV as a model system to study host-pathogen interaction in raccoons from a challenge study and from four wild populations that differ in exposure times and viral lineages. We investigated the potential role of Prlo-DRB polymorphism in relation to susceptibility/resistance to RRV in 113 RRV positive and 143 RRV negative raccoons. Six alleles were found to be associated with RRV negative status and five alleles with RRV positive animals. We found variable patterns of MHC associations given the relative number of selective RRV sweeps in the studied regions and correlations between MHC diversity and RRV lineages. The allelic associations established provide insight into how the genetic variation of raccoons may affect the disease outcome and this can be used to examine similar associations between other rabies variants and their hosts.

Quantifying antigenic relationships among the lyssaviruses.

All lyssaviruses cause fatal encephalitis in mammals. There is sufficient antigenic variation within the genus to cause variable vaccine efficacy, but this variation is difficult to characterize quantitatively: sequence analysis cannot yet provide detailed antigenic information, and antigenic neutralization data have been refractory to high-resolution robust interpretation. Here, we address these issues by using state-of-the-art antigenic analyses to generate a high-resolution antigenic map of a global panel of 25 lyssaviruses. We compared the calculated antigenic distances with viral glycoprotein ectodomain sequence data. Although 67% of antigenic variation was predictable from the glycoprotein amino acid sequence, there are in some cases substantial differences between genetic and antigenic distances, thus highlighting the risk of inferring antigenic relationships solely from sequence data at this time. These differences included epidemiologically important antigenic differences between vaccine strains and wild-type rabies viruses. Further, we quantitatively assessed the antigenic relationships measured by using rabbit, mouse, and human sera, validating the use of nonhuman experimental animals as a model for determining antigenic variation in humans. The use of passive immune globulin is a crucial component of rabies postexposure prophylaxis, and here we also show that it is possible to predict the reactivity of immune globulin against divergent lyssaviruses.

Prevalence of rabies antibodies in street and household dogs in Chandigarh, India.

The present study reports the first survey for the detection of antirabies antibodies in street and household dogs in India. We aimed to check the efficacy of control programs for the disease in the Union territory of Chandigarh. The serum samples were collected from 100 street and 50 household dogs and tested for the presence of antirabies antibodies by ELISA. As per WHO criteria, a titre of >0.5 IU/ml of antirabies antibody in serum samples was taken as the protective level. Protective antirabies antibody titre was found only in 1% of the street dogs and 16% of the pet dogs. The awareness among the pet dog owners about the antirabies vaccination schedule was low as 18% did not know the vaccination status of the dog and 66% had got the initial immunization done with only three doses and annual boosters were not given. A National Rabies Elimination Program needs to be launched as a collaborative venture by both medical and veterinary practitioners to curb this deadly disease. Also periodic surveys to test the status of antirabies IgG among dogs need to be carried out to ascertain the attainment of WHO protective levels.

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.

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.