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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.

The live genetically-engineered oral rabies virus (RABV) variant SPBN GASGAS induces long-lasting immunity in foxes and protection against challenge with an otherwise lethal dose of RABV field strains both after experimental oral and parenteral routes of administration. Induction of RABV-specific binding antibodies and immunoglobulin isotypes (IgM, total IgG, IgG1, IgG2) were comparable in orally and parenterally vaccinated foxes. Differences were only observed in the induction of virus-neutralizing (VNA) titers, which were significantly higher in the parenterally vaccinated group. The dynamics of rabies-specific antibodies pre- and post-challenge (365 days post vaccination) suggest the predominance of type-1 immunity protection of SPBN GASGAS. Independent of the route of administration, in the absence of IgG1 the immune response to SPBN GAGAS was mainly IgG2 driven. Interestingly, vaccination with SPBN GASGAS does not cause significant differences in inducible IFN-γ production in vaccinated animals, indicating a relatively weak cellular immune response during challenge. Notably, the parenteral application of SPBN GASGAS did not induce any adverse side effects in foxes, thus supporting safety studies of this oral rabies vaccine in various species.

Non-contact optical detection of ultrasound critically depends on the amount of light collected from the detection surface. Although it can be optimized in multiple ways for an ideal flat polished surface, industrial non-destructive testing and evaluation (NDT&E) usually requires optical detectors to be robust for unpolished material surfaces that are usually rough and curved. Confocal detectors provide the best light collection but must trade off sensitivity with depth of field. Specifically, detection efficiency increases with the numerical aperture (NA) of the detector, but the depth of field drops. Therefore, fast realignment of the detector focal point is critical for in-field applications. Here, we propose an optical distance and angle correction system (DACS) and demonstrate it in a kHz-rate laser-ultrasound inspection system. It incorporates a Sagnac interferometer on receive for the fast scanning of aircraft composites, which minimizes the required initial alignment. We show that DACS performs stably for different composite surfaces while providing ±2° angular and ±2 mm axial automatic correction with a maximum 100 ms realignment time.

Applied research is crucial in pushing the boundaries and finding a solution to the age-old problem of dog-mediated rabies. Although oral vaccination of dogs is considered to have great potential in mass dog vaccination campaigns and could have far-reaching benefits, it is perhaps the most ignored of all available tools in efforts to eliminate dog-mediated rabies, not least because of limited data on immunogenicity, efficacy, and safety of potential oral rabies vaccine candidates. In this study, the long-term immunogenicity in local Thai dogs after oral administration of the highly attenuated 3rd generation rabies virus vaccine strain SPBN GASGAS was assessed. The oral rabies vaccine was administered to dogs by either direct oral administration (n = 10) or by offering a vaccine loaded intestine bait (n = 15). The humoral immune response was then compared to three groups of dogs; a group that received a parenteral delivered inactivated rabies vaccine (n = 10), a group offered a placebo intestine bait (n = 7), and a control group (n = 4) for an observation period of 365 days. There was no significant difference in the immune response of dogs that received oral and parenteral vaccine in terms of magnitude, kinetics, and persistence of both rabies virus (RABV) neutralizing (RFFIT) and binding (ELISA) antibodies. Although the single parenteral injection of an inactivated rabies vaccine mounted a slightly higher humoral immune response than the orally delivered live vaccine, RABV specific antibodies of both types were still detectable after one year in most animals for all treatment groups and resulted in no difference in seropositivity. Characterization of rabies specific antibodies revealed two main classes of antibodies involved in the immune response of dogs vaccinated. While IgM antibodies were the first to appear, the succeeding IgG response was mainly IgG2 dominated independent of the vaccine type used. The results support the view that SPBN GASGAS induces a sustained detectable immune response in local dogs both after direct oral administration and via bait application.

The visualization of infection processes in relevant tissues and organs using microscopy methods reveals a unique link between the distribution, tropism, and abundance of pathogens and the physiological structure of the respective organ. To dissect virus replication and the host reaction in vivo at both a global and a single-cell level, conventional 2D imaging approaches can only provide limited insight. However, pathological studies of infected organ material are still mostly restricted to the immunostaining of thin sections from paraffin-embedded or frozen samples. While the 3D analysis of large tissue volumes is possible via laborious serial sectioning, a variety of problems and artifacts remain. Modern immunostaining-compatible tissue clearing techniques allow for the seamless 3D visualization of infection sites in optically cleared thick tissues sections or even entire organs. Benefiting from pure optical slicing, this approach enables the acquisition of multicolor high-volume 3D image stacks for coherent qualitative and quantitative analyses of the infection and its surrounding cellular environment. Here, we demonstrate the utility and power of this methodology by visualizing virus infections in different target tissues. For instance, we reconstructed the cellular context of rabies virus infection sites in mouse brain tissue, allowing a thorough investigation and quantitative analysis of rabies virus cell tropism. The systematic comparison of different rabies viruses with varying pathogenicity revealed a remarkable difference for highly virulent street rabies viruses and attenuated lab strains. While the virus protein expression was readily detectable at a comparable level in both neurons and non-neuronal glial cells from brains of mice infected with street rabies viruses, it was virtually absent in glial cells of lab strain-infected mice. These data provide novel and detailed insights into the pathogenesis of virus infections and substantially contribute to an improved understanding of virus–host interactions in vivo.

Collective phenomena are studied in a range of contexts-from controlling locust plagues to efficiently evacuating stadiums-but the central question remains: how can a large number of independent individuals form a seemingly perfectly coordinated whole? Previous attempts to answer this question have reduced the individuals to featureless particles, assumed particular interactions between them and studied the resulting collective dynamics. While this approach has provided useful insights, it cannot guarantee that the assumed individual-level behaviour is accurate, and, moreover, does not address its origin-that is, the question of why individuals would respond in one way or another. We propose a new approach to studying collective behaviour, based on the concept of learning agents: individuals endowed with explicitly modelled sensory capabilities, an internal mechanism for deciding how to respond to the sensory input and rules for modifying these responses based on past experience. This detailed modelling of individuals favours a more natural choice of parameters than in typical swarm models, which minimises the risk of spurious dependences or overfitting. Most notably, learning agents need not be programmed with particular responses, but can instead develop these autonomously, allowing for models with fewer implicit assumptions. We illustrate these points with the example of marching locusts, showing how learning agents can account for the phenomenon of density-dependent alignment. Our results suggest that learning agent-based models are a powerful tool for studying a broader class of problems involving collective behaviour and animal agency in general.

To evaluate recent developments regarding the epidemiological situation of pseudorabies virus (PRV) infections in wild boar populations in Germany, nationwide serological monitoring was conducted between 2010 and 2015. During this period, a total of 108,748 sera from wild boars were tested for the presence of PRV-specific antibodies using commercial enzyme-linked immunosorbent assays. The overall PRV seroprevalence was estimated at 12.09% for Germany. A significant increase in seroprevalence was observed in recent years indicating both a further spatial spread and strong disease dynamics. For spatiotemporal analysis, data from 1985 to 2009 from previous studies were incorporated. The analysis revealed that PRV infections in wild boar were endemic in all German federal states; the affected area covers at least 48.5% of the German territory. There were marked differences in seroprevalence at district levels as well as in the relative risk (RR) of infection of wild boar throughout Germany. We identified several smaller clusters and one large region, where the RR was two to four times higher as compared to the remaining areas under investigation. Based on the present monitoring intensity and outcome, we provide recommendations with respect to future monitoring efforts concerning PRV infections in wild boar in Germany.

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.

Although conventional immunohistochemistry for neurotropic rabies virus (RABV) usually shows high preference for neurons, non-neuronal cells are also potential targets, and abortive astrocyte infection is considered a main trigger of innate immunity in the CNS. While in vitro studies indicated differences between field and less virulent lab-adapted RABVs, a systematic, quantitative comparison of astrocyte tropism in vivo is lacking. Here, solvent-based tissue clearing was used to measure RABV cell tropism in infected brains. Immunofluorescence analysis of 1 mm-thick tissue slices enabled 3D-segmentation and quantification of astrocyte and neuron infection frequencies. Comparison of three highly virulent field virus clones from fox, dog, and raccoon with three lab-adapted strains revealed remarkable differences in the ability to infect astrocytes in vivo. While all viruses and infection routes led to neuron infection frequencies between 7−19%, striking differences appeared for astrocytes. Whereas astrocyte infection by field viruses was detected independent of the inoculation route (8−27%), only one lab-adapted strain infected astrocytes route-dependently [0% after intramuscular (i.m.) and 13% after intracerebral (i.c.) inoculation]. Two lab-adapted vaccine viruses lacked astrocyte infection altogether (0%, i.c. and i.m.). This suggests a model in which the ability to establish productive astrocyte infection in vivo functionally distinguishes field and attenuated lab RABV strains.

Natural control of phytopathogenic microorganisms is assumed as a priority function of the commensal plant microbiota. In this study, the suitability of fluorescent pseudomonads in the phyllosphere of crop plants as natural control agents was evaluated. Under field conditions, ears of winter wheat were found to be colonized with high consistency and at a high density by pseudomonads at the late milk dough stage. Isolates of these bacteria were evaluated for their potential to protect the plants from phytopathogenic Alternaria and Fusarium fungi. More Pseudomonas isolates were antagonistically active against alternaria than against fusaria in the dual culture test. The alternaria responded species-specifically and more sensitively to bacterial antagonism than the strain-specific reacting fusaria. A total of 110 randomly selected Pseudomonas isolates were screened for genes involved in the biosynthesis of the antibiotics 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid, pyoluteorin, and pyrrolnitrin. The key gene for production of the phloroglucinol was found in none of these isolates. At least one of the genes, encoding the biosynthesis of the other antibiotics was detected in 81% of the isolates tested. However, the antagonistic effect found in the dual culture assay was not necessarily associated with the presence of these antibiotic genes. Wheat grains as natural substrate were inoculated with selected antagonistic Pseudomonas isolates and Alternaria and Fusarium strains, respectively. The fungal growth was only slightly delayed, but the mycotoxin production was significantly reduced in most of these approaches. In conclusion, the distribution of phytopathogenic fungi of the genera Alternaria and Fusarium in the field is unlikely to be inhibited by naturally occurring pseudomonads, also because the bacterial antagonists were not evenly distributed in the field. However, pseudomonads can reduce the production of Alternaria and Fusarium mycotoxins in wheat grains and thus have the potential to improve the crop quality.

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.