Identification of rhabdoviral sequences in oropharyngeal swabs from German and Danish bats

BACKGROUND: In the frame of active lyssavirus surveillance in bats, oropharyngeal swabs from German (N = 2297) and Danish (N = 134) insectivorous bats were investigated using a newly developed generic pan-lyssavirus real-time reverse transcriptase PCR (RT-qPCR). FINDINGS: In total, 15 RT-qPCR positive swabs were detected. Remarkably, sequencing of positive samples did not confirm the presence of bat associated lyssaviruses but revealed nine distinct novel rhabdovirus-related sequences. CONCLUSIONS: Several novel rhabdovirus-related sequences were detected both in German and Danish insectivorous bats. The results also prove that the novel generic pan-lyssavirus RT-qPCR offers a very broad detection range that allows the collection of further valuable data concerning the broad and complex diversity within the family Rhabdoviridae

Newly defined ATP-binding cassette subfamily B member 5 positive dermal mesenchymal stem cells promote healing of chronic iron-overload wounds via secretion of interleukin-1 receptor antagonist

In this study, we report the beneficial effects of a newly identified dermal cell subpopulation expressing the ATP‐binding cassette subfamily B member 5 (ABCB5) for the therapy of nonhealing wounds. Local administration of dermal ABCB5+‐derived mesenchymal stem cells (MSCs) attenuated macrophage‐dominated inflammation and thereby accelerated healing of full‐thickness excisional wounds in the iron‐overload mouse model mimicking the nonhealing state of human venous leg ulcers. The observed beneficial effects were due to interleukin‐1 receptor antagonist (IL‐1RA) secreted by ABCB5+‐derived MSCs, which dampened inflammation and shifted the prevalence of unrestrained proinflammatory M1 macrophages toward repair promoting anti‐inflammatory M2 macrophages at the wound site. The beneficial anti‐inflammatory effect of IL‐1RA released from ABCB5+‐derived MSCs on human wound macrophages was conserved in humanized NOD‐scid IL2rγ null mice. In conclusion, human dermal ABCB5+ cells represent a novel, easily accessible, and marker‐enriched source of MSCs, which holds substantial promise to successfully treat chronic nonhealing wounds in humans

Monogenic variants in dystonia: an exome-wide sequencing study

Background Dystonia is a clinically and genetically heterogeneous condition that occurs in isolation (isolated dystonia), in combination with other movement disorders (combined dystonia), or in the context of multisymptomatic phenotypes (isolated or combined dystonia with other neurological involvement). However, our understanding of its aetiology is still incomplete. We aimed to elucidate the monogenic causes for the major clinical categories of dystonia. Methods For this exome-wide sequencing study, study participants were identified at 33 movement-disorder and neuropaediatric specialty centres in Austria, Czech Republic, France, Germany, Poland, Slovakia, and Switzerland. Each individual with dystonia was diagnosed in accordance with the dystonia consensus definition. Index cases were eligible for this study if they had no previous genetic diagnosis and no indication of an acquired cause of their illness. The second criterion was not applied to a subset of participants with a working clinical diagnosis of dystonic cerebral palsy. Genomic DNA was extracted from blood of participants and whole-exome sequenced. To find causative variants in known disorder-associated genes, all variants were filtered, and unreported variants were classified according to American College of Medical Genetics and Genomics guidelines. All considered variants were reviewed in expert round-table sessions to validate their clinical significance. Variants that survived filtering and interpretation procedures were defined as diagnostic variants. In the cases that went undiagnosed, candidate dystonia-causing genes were prioritised in a stepwise workflow. Findings We sequenced the exomes of 764 individuals with dystonia and 346 healthy parents who were recruited between June 1, 2015, and July 31, 2019. We identified causative or probable causative variants in 135 (19%) of 728 families, involving 78 distinct monogenic disorders. We observed a larger proportion of individuals with diagnostic variants in those with dystonia (either isolated or combined) with coexisting non-movement disorder-related neurological symptoms (100 [45%] of 222;excepting cases with evidence of perinatal brain injury) than in those with combined (19 [19%] of 98) or isolated (16 [4%] of 388) dystonia. Across all categories of dystonia, 104 (65%) of the 160 detected variants affected genes which are associated with neurodevelopmental disorders. We found diagnostic variants in 11 genes not previously linked to dystonia, and propose a predictive clinical score that could guide the implementation of exome sequencing in routine diagnostics. In cases without perinatal sentinel events, genomic alterations contributed substantively to the diagnosis of dystonic cerebral palsy. In 15 families, we delineated 12 candidate genes. These include IMPDH2, encoding a key purine biosynthetic enzyme, for which robust evidence existed for its involvement in a neurodevelopmental disorder with dystonia. We identified six variants in IMPDH2, collected from four independent cohorts, that were predicted to be deleterious de-novo variants and expected to result in deregulation of purine metabolism. Interpretation In this study, we have determined the role of monogenic variants across the range of dystonic disorders, providing guidance for the introduction of personalised care strategies and fostering follow-up pathophysiological explorations

Untersuchungen zum Vorkommen der Fledermaustollwut in Deutschland

Das Wissen über fledermausassoziierte Lyssaviren in Hinblick auf die Diversität, Abundanz, geographische Verbreitung, Wirtsspezifität, Pathogenität und mögliche Übertragungswege ist lückenhaft. In Europa wird zur Überwachung der Fledermaustollwut die Untersuchung von moribunden oder toten Tieren (passive Surveillance) und/oder die Beprobung von freilebenden Fledermauspopulationen (aktive Surveillance) empfohlen. Ziel der vorliegenden Arbeit war es, den derzeitigen Kenntnisstand zum Vorkommen von fledermausassoziierten Lyssaviren in Deutschland zu erweitern und mit Daten anderer europäischer Länder zu vergleichen. Die Untersuchungen stützten sich dabei auf drei Teilprojekte: 1) Die initiale Statuserhebung und Analyse der Fledermaustollwut-Surveillance in Europa hat gezeigt, dass trotz internationaler Empfehlungen die Tollwut-Überwachung bei Fledermäusen uneinheitlich durchgeführt wird. Diese Unterschiede sind unter anderem die Folge (i) fehlender Zusammenarbeit zwischen Fledermausbiologen und Veterinär- sowie Gesundheitsbehörden, (ii) fehlender Netzwerke von Fledermaussachverständigen, (iii) länderspezifischer Regelungen, aber auch (iv) fehlenden Bewusstseins sowie Kenntnisstandes für die Fledermaustollwut in der Bevölkerung. 2) In Deutschland wurde zusätzlich zur Tollwut-Routinediagnostik eine intensivierte passive Tollwut-Surveillance (retrospektive Studie, 1998 – 2013) durchgeführt, bei der 5478 Tiere aus insgesamt 21 einheimischen Arten akquiriert und auf das Vorliegen einer Lyssavirusinfektion untersucht wurden. Insgesamt konnten 52 EBLV-1 Infektionen (E. serotinus (n=49), P. pipistrellus, P. nathusii, Pl. auritus) sowie drei EBLV-2 Infektionen (M. daubentonii) diagnostiziert werden. Die Untersuchungen verdeutlichen, dass diese retrospektive Studie im Vergleich zur Routinediagnostik entscheidende Vorteile in Bezug auf den Stichprobenumfang, das Artenspektrum sowie die Fehlerfreiheit von artbezogenen biologischen und epidemiologischen Daten und somit entscheidende Voraussetzungen für eine gezielte Risikobewertung einer potentiellen Gesundheitsgefährdung des Menschen durch fledermausassoziierte Lyssaviren bietet. 3) Im Rahmen der aktiven Tollwut-Surveillance (1998 – 2012) erfolgte an 42 Standorten in Deutschland die Beprobung von Fledermauspopulationen. Es wurden 4546 Maultupfer- und 1226 Serumproben von 18 Fledermausspezies untersucht. EBLV-1-spezifische RNA wurde in Maultupfern von fünf Breitflügelfledermäusen, einer Fransen- und einer Mopsfledermaus detektiert. In dieser Arbeit konnte erstmalig EBLV-1 aus einer RT-PCR-positiven Maultupferprobe von einer scheinbar gesunden Breitflügelfledermaus isoliert werden. Bei der serologischen Testung von Serumproben gegen EBLV-1 wurden virus-neutralisierende Antikörper in acht verschiedenen Spezies festgestellt, wobei hauptsächlich Seren von Breitflügelfledermäusen höhere Titer aufwiesen. Ein Vergleich von Ergebnissen verschiedener Sero-Surveillance-Studien ist durch das Fehlen standardisierter Testverfahren und durch kreuzneutralisierende Antikörper gegenüber Lyssaviren gleicher Phylogruppen kaum möglich. Die Daten der aktiven Surveillance liefern im Gegensatz zur passiven Surveillance nur begrenzte Erkenntnisse zum Vorkommen, der Prävalenz und Dynamik von Fledermaustollwut in einheimischen Fledermauspopulationen. Die Form der intensivierten passiven Surveillance sollte daher als Standard für eine zukünftige Surveillance der Fledermaustollwut in Deutschland und anderen europäischen Ländern betrachtet werden. Darüber hinaus wurde bei natürlich infizierten Fledermäusen (E. serotinus, M. daubentonii, P. nathusii) die Virusverteilung und -last in Geweben verschiedener Organe unter dem Aspekt möglicher Ausscheidungswege untersucht. Virus-spezifische RNA wurde in allen untersuchten Organen nachgewiesen; bedingt durch die neurotropen Eigenschaften der Lyssaviren wurde die höchste Viruslast im Gehirn festgestellt. Signifikant hohe Viruslasten waren zudem in der Speichel¬drüse nachweisbar. Zusätzlich zur Speicheldrüse scheint die Zunge ein Organ zu sein, in dem Virusreplikation und -ausscheidung stattfindet, da in verschiedenen zellulären Strukturen des Zungengewebes Lyssavirusantigen bzw. virale RNA histologisch nachgewiesen wurden. Die Ausscheidung und Übertragung von fledermausassoziierten Lyssaviren über den Harntrakt oder die Atemwege ist wissenschaftlich umstritten und konnte in dieser Studie immunhistochemisch nicht bestätigt werden.The knowledge about the diversity, abundance and occurrence, number of reservoir hosts, pathogenesis and transmission of bat associated lyssaviruses is still incomplete. In Europe, bat rabies surveillance is based on testing of moribund or dead bats (passive surveillance) and by sampling of free-living indigenous bat populations (active surveillance). The objective of this study was to obtain further information and data about the occurrence and epidemiology of bat-associated lyssaviruses in Germany and to compare them with data from other European countries. This study was based on three individual projects. 1) An initial analyses of officially reported or published bat rabies surveillance data indicated that despite international recommendations the level of bat rabies surveillance in Europe is very heterogeneous. This is likely a result of (i) missing collaboration between bat biologists and veterinary- as well as public health authorities, (ii) lack of functioning networks of bat biologists, (iii) country-specific regulations as regards the protection of bats and (iv) a lack of disease awareness among the general public. 2) In Germany, additionally to routine bat rabies surveillance an enhanced passive surveillance study (retrospective study, 1998 – 2013) was initiated in collaboration with different institutions, authorities and bat biologists. Of 5478 bats comprising 21 species screened for lyssavirus infections 55 bats were positive. European bat lyssavirus 2 (EBLV-2) was isolated from three Daubenton´s bats (M. daubentonii). In contrast, EBLV-1 infections were not only detected in the Serotine bat (E. serotinus n=49) but also in three other indigenous bat species. Based on the experience gained in this project, this enhanced passive surveillance approach has crucial advantages compared to rabies routine diagnosis with regard to sample size, range and correct identification of bat species and other denominator data and hence, provides better insights into the true epidemiological situation. Therefore, we propose that enhanced passive bat rabies surveillance is a useful complement to routine rabies diagnosis to enable a better and more focussed risk assessment of bat lyssaviruses for public health in Germany. 3) During active bat rabies surveillance (1998 – 2012) a total of 4546 oro-pharyngeal swabs and 1226 serum samples of 18 free-living bat species from 42 locations in Germany were obtained. Lyssavirus-specific RNA was found in five oro-pharyngeal swabs from Serotine bats and also in single samples of M. nattereri and B. barbastellus. At an European level, in our survey we were the first to detect and isolate EBLV-1 from an oro-pharyngeal swab of an apparently healthy Serotine bat. Virus-neutralizing antibodies were detected in eight different bat species with highest titres against EBLV-1 detected in sera from Serotine bats. The serological results are difficult to interpret. In general, a comparison of data with other sero-surveillance studies is hampered by a lack of validated test methods including differing cut-offs and test viruses used, and by cross-neutralization of lyssaviruses within phylogroups. Compared to passive surveillance, active bat rabies surveillance provides only limited additional information on the distribution, prevalence and epidemiology of bat lyssaviruses in Europe. Therefore, enhanced passive surveillance should be the standard approach for future bat rabies surveillance in Germany and Europe. Furthermore, to elucidate potential routes of virus excretion virus dissemination and viral load within naturally infected bats (E. serotinus, M. daubentonii, P. nathusii) were investigated by different molecular techniques. The highest viral loads were found in brain tissue followed by salivary glands. However, specific lyssavirus RNA was also detected in a variety of other organs. The tongue appears to be another prominent site for virus replication and possible shedding as immunohistochemistry confirmed the presence of lyssaviruses in various cells of the tongue. These findings indicate that excretion and shedding of viable virus occurs mainly via salivary glands associated with the oral cavity and that other routes of virus excretion, e.g. via urinary tract or respiratory system, are highly unlikely

Optical near-fields of triangular nanostructures

We compare simulations of optical near-fields of single triangular nanostructures with experimental results from a near-field ablation technique on a periodic arrangement of triangles. We find good agreement of the lateral near-field distributions; nevertheless their dependency on the polarization of the incident light differs by 90°. Upon increasing the lateral distances of the nanotriangle arrangement in the experiment, the polarization dependence agrees with the simulation. We conclude that this at first sight unexpected behaviour stems from the coupling of near-fields by scattered surface waves and their interaction with the incoming beam

Demographic and clinical characteristics of the patient sample.

<p><i>Note.</i> Groups: SA =  subacute, CH =  chronic, CG =  control group. Pt =  patient; M/F =  male/female. NIHSS: National Institutes of Health Stroke Scale. Stroke etiology: i =  ischemic, h =  hemorrhagic stroke. V&TDS: visual and tactile double stimulation. CAV screen: CAV visual field screening. CAV-ET: CAV extinction test. NET Score: for subtests 1 to 8 and for the whole test battery. Mean (M) and standard deviation (SD) given for patients and healthy controls.</p

Lesion patterns of individual patients.

<p>Patient numbers correspond to patient IDs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082892#pone-0082892-t001" target="_blank">Table 1</a> (patients #2-10). No MRI was available for patient #1. Right side of the brain corresponds to right hemisphere.</p

Schematical drawing of the CM.

<p>Schematical drawing of the CM.</p

Star Cancellation Test: Percentage of detected stars, split into test-system, experimental group and visual field.

<p>The figure shows means and standard errors. Best possible performance (100%) in NET was 27 stars, in CM 20 stars per side. CM: Circle-Monitor, NET: Neglect-Test.</p

Variables included in the discriminant analysis.

<p><i>Note.</i> CM: Circle Monitor. NET: Neglect Test. Explained variance: explained variance by the given predictors. Stars (*) indicate significant predictors in the discriminant analyses.</p