Assessing the occurrence of the novel zoonotic variegated squirrel bornavirus 1 in captive squirrels in Germany —A prevalence study

The newly described zoonotic variegated squirrel bornavirus 1 (VSBV‐1) in German squirrel holdings has been associated with the death of three private owners and one zoo animal caretaker (confirmed cases). Epidemiological investigations were severely impeded by the general lack of data on holdings of the putative reservoir hosts, the family Sciuridae. To fill this lack of data for detailed epidemiological investigations of the captive squirrel population, a register of private and zoological squirrel holdings was established. The findings show a broad variety of kept species and their frequency distribution. By contacting the different stakeholders via Web‐based social groups and societies, information passed in both directions so that disease awareness could be raised and participants could be recruited for further studies. Cross‐sectional studies revealed a prevalence of VSBV‐1‐positive subpopulations of 0% (95% CI 0%–6.2%) among private squirrel collections and 1.9% (95% CI: 0%–9.9%) among zoos in Germany. The approach presented here can be transferred to other populations of non‐traditional pets, which may be equally difficult to monitor, in the case of an emerging zoonotic infectious disease.Peer Reviewe

Search for polyoma-, herpes-, and bornaviruses in squirrels of the family Sciuridae

Background Squirrels (family Sciuridae) are globally distributed members of the order Rodentia with wildlife occurrence in indigenous and non-indigenous regions (as invasive species) and frequent presence in zoological gardens and other holdings. Multiple species introductions, strong inter-species competition as well as the recent discovery of a novel zoonotic bornavirus resulted in increased research interest on squirrel pathogens. Therefore we aimed to test a variety of squirrel species for representatives of three virus families. Methods Several species of the squirrel subfamilies Sciurinae, Callosciurinae and Xerinae were tested for the presence of polyomaviruses (PyVs; family Polyomaviridae) and herpesviruses (HVs; family Herpesviridae), using generic nested polymerase chain reaction (PCR) with specificity for the PyV VP1 gene and the HV DNA polymerase (DPOL) gene, respectively. Selected animals were tested for the presence of bornaviruses (family Bornaviridae), using both a broad-range orthobornavirus- and a variegated squirrel bornavirus 1 (VSBV-1)-specific reverse transcription-quantitative PCR (RT-qPCR). Results In addition to previously detected bornavirus RNA-positive squirrels no more animals tested positive in this study, but four novel PyVs, four novel betaherpesviruses (BHVs) and six novel gammaherpesviruses (GHVs) were identified. For three PyVs, complete genomes could be amplified with long-distance PCR (LD-PCR). Splice sites of the PyV genomes were predicted in silico for large T antigen, small T antigen, and VP2 coding sequences, and experimentally confirmed in Vero and NIH/3T3 cells. Attempts to extend the HV DPOL sequences in upstream direction resulted in contiguous sequences of around 3.3 kilobase pairs for one BHV and two GHVs. Phylogenetic analysis allocated the novel squirrel PyVs to the genera Alpha- and Betapolyomavirus, the BHVs to the genus Muromegalovirus, and the GHVs to the genera Rhadinovirus and Macavirus. Conclusions This is the first report on molecular identification and sequence characterization of PyVs and HVs and the detection of bornavirus coinfections with PyVs or HVs in two squirrel species. Multiple detection of PyVs and HVs in certain squirrel species exclusively indicate their potential host association to a single squirrel species. The novel PyVs and HVs might serve for a better understanding of virus evolution in invading host species in the future

Hantavirus Brno loanvirus is highly specific to the common noctule bat (Nyctalus noctula) and widespread in Central Europe.

Bat-associated hantaviruses have been detected in Asia, Africa and Europe. Recently, a novel hantavirus (Brno loanvirus, BRNV) was identified in common noctule bats (Nyctalus noctula) in the Czech Republic, but nothing is known about its geographical range and prevalence. The objective of this study was to evaluate the distribution and host specificity of BRNV by testing bats from neighbouring countries Germany, Austria and Poland. One thousand forty-seven bats representing 21 species from Germany, 464 bats representing 18 species from Austria and 77 bats representing 12 species from Poland were screened by L segment broad-spectrum nested reverse transcription-polymerase chain reaction (RT-PCR) or by BRNV-specific real-time RT-PCR. Three common noctules from Germany, one common noctule from Austria and three common noctules from Poland were positive in the hantavirus RNA screening. Conventional RT-PCR and primer walking resulted in the amplification of partial L segment and (almost) complete S and M segment coding sequences for samples from Germany and partial L segment sequences for samples from Poland. Phylogenetic analysis of these nucleotide sequences showed highest similarity to BRNV from Czech Republic. The exclusive detection of BRNV in common noctules from different countries suggests high host specificity. The RNA detection rate in common noctules ranged between 1 of 207 (0.5%; Austria), 3 of 245 (1.2%; Germany) and 3 of 20 (15%; Poland). In conclusion, this study demonstrates a broader distribution of BRNV in common noctules in Central Europe, but at low to moderate prevalence. Additional studies are needed to prove the zoonotic potential of this hantavirus and evaluate its transmission within bat populations

Distribution of zoonotic variegated squirrel bornavirus 1 in naturally infected variegated and Prevost’s squirrels

Recently, the zoonotic capacity of the newly discovered variegated squirrel bornavirus 1 (VSBV-1) was confirmed in humans with a lethal encephalitis. Transmission to humans occurred by variegated and Prevost’s squirrels as presumed reservoir hosts but possible ways of virus shedding and the route of infection still need to be elucidated. Thus, the tissue distribution of VSBV-1 antigen and RNA was investigated in detail via immunohistochemistry (IHC) in six variegated and eight Prevost’s squirrels and by in situ hybridisation (ISH) in one Prevost’s squirrel, respectively. VSBV-1 antigen and RNA positive cells were most numerous in the nervous system and were also found in nearly all tissues and different cell types indicating a broad organ and cell tropism of VSBV-1. Presence of VSBV-1 in several organs might indicate potential virus shedding via various routes and implies the risk of intra- and interspecies transmission, respectively

Antibodies against viral nucleo-, phospho-, and X protein contribute to serological diagnosis of fatal Borna disease virus 1 infections

Borna disease virus 1 (BoDV-1) causes rare but often fatal encephalitis in humans. Late diagnosis prohibits an experimental therapeutic approach. Here, we report a recent case of fatal BoDV-1 infection diagnosed on day 12 after hospitalization by detection of BoDV-1 RNA in the cerebrospinal fluid. In a retrospective analysis, we detect BoDV-1 RNA 1 day after hospital admission when the cell count in the cerebrospinal fluid is still normal. We develop a new ELISA using recombinant BoDV-1 nucleoprotein, phosphoprotein, and accessory protein X to detect seroconversion on day 12. Antibody responses are also shown in seven previously confirmed cases. The individual BoDV-1 antibody profiles show variability, but the usage of three different BoDV-1 antigens results in a more sensitive diagnostic tool. Our findings demonstrate that early detection of BoDV-1 RNA in cerebrospinal fluid and the presence of antibodies against at least two different viral antigens contribute to BoDV-1 diagnosis. Physicians in endemic regions should consider BoDV-1 infection in cases of unclear encephalopathy and initiate appropriate diagnostics at an early stage

Variegated squirrel bornavirus 1 in squirrels, Germany and the Netherlands

We screened squirrels in Germany and the Netherlands for the novel zoonotic variegated squirrel bornavirus 1 (VSBV-1). The detection of VSBV-1 in 11 squirrels indicates a considerable risk for transmission to humans handling those animals. Therefore, squirrels in contact with humans should routinely be tested for VSBV-1

Encephalitides induced by lyssa-, borna- and astroviruses: molecular detection and characterization

Encephalitis is a severe inflammatory disease of the brain which often has a fatal outcome or can lead to subsequent damages. In around two-thirds of all human encephalitis cases, the causative agent is, despite improved diagnostics, unknown today. Aim of this work was the development, improvement and validation of diagnostic methods, improvement of sampling strategies and the development of optimized systems for characterization of three viruses causing viral encephalitis. The main burden of RABV lies in developing countries, were standard diagnostic tools are often not realizable. Therefore, simple and rapid diagnostic tests for the use under resource limited settings, so called point-of-care tests (POCT), are favorable. Commercially available lateral flow device (LFD) based immunodiagnostic tests were analyzed and failed in terms of sensitivity compared to the standard FAT and RT-qPCR (Paper I). Therefore, molecular RABV alternative targeting genome tests were developed and combined with rapid nucleic acid extraction methods. The new HighSpeed RT-qPCR and RPA assays together with magnetic bead based automated or manual extraction methods delivered a specificity between 100% and 97.2% and a limit of detection of 10 or 1,000 genome copies per reaction, respectively and seem suitable as novel POCT (Paper II). Recently, a novel zoonotic VSBV-1, responsible for fatal encephalitis of three squirrel breeders, was detected. For further investigations of this new virus, methods for an in-vivo sampling approach of squirrels were established. They were useful to identify animals harboring this dangerous virus, and new sequence data could be obtained from the VSBV-1 positive animals. Until now, 3.5% of all investigated squirrels were VSBV-1 RNA positive and two subfamilies (Sciurinae and Callosciurinae) are affected. The pathogen occurs not only in Germany, but also squirrel holdings and zoological gardens in the Netherlands and Croatia were tested positive, indicating a serious human health threat of this virus (Paper III and IV). With the help of a metagenomic approach, astroviruses were detected to be associated to encephalitis in cattle and sheep. These viruses were detected in a cow in Germany (Paper V), and in brain samples from two sheep in the United Kingdom (Paper VI). In both cases, the sequences generated by high-throughput-sequencing (HTS) were confirmed by specific RT-qPCRs, which could be used for subsequent screening approaches. Together, methods for the detection of three different encephalitis viruses were developed, validated and applied for different sample material.Die Enzephalitis ist eine entzündliche Erkrankung des Gehirns, die oftmals tödlich verläuft oder Folgeschäden hervorruft. Trotz verbesserter diagnostischer Methoden wird auch gegenwärtig bei zwei Drittel aller Enzephalitiden die Ursache nicht ermittelt. Ziel dieser Arbeit war es, für drei mit Enzephalitis im Zusammenhang stehende Viren Nachweisverfahren zu entwickeln, zu verbessern und zu validieren, Beprobungsmethoden zu verbessern und Systems für die Viruscharakterisierung zu optimieren. Die Standardmethoden der Tollwut-Diagnostik kommen in Entwicklungsländern oftmals nicht zum Einsatz. Abhilfe könnten so genannte „point-of-care tests (POCT)“ schaffen, welche für den schnellen und einfachen Feldeinsatz konzipiert sind. Ein Beispiel sind lateral-flow-device (LFD) basierte immundiagnostische Schnelltests. Diese wurden in einer Studie hinsichtlich ihrer Performance untersucht, wobei sich erhebliche Schwächen in der Sensitivität zeigten (Paper I). Daher wurden molekularbiologische POCT entwickelt und zusammen mit angepassten Magnetpartikel-basierten Schnellextraktionsverfahren getestet. Die entwickelte High-Speed RT-PCR und die recombinase polymerase amplification (RPA) lieferten eine Nachweisempfindlichkeit von 10 bzw. 1.000 Genomkopien pro Reaktion und eine Spezifität zwischen 100% und 97,2% (Paper II). Als weiteres Virus wurde das kürzlich neu entdeckte VSBV-1 untersucht, welches für die tödliche Enzephalitis dreier Bunthörnchenzüchter verantwortlich ist. Um die Verbreitung dieses Virus innerhalb der Hörnchenpopulation untersuchen zu können, wurden zwei in-vivo-Beprobungsmethode etabliert. Unter Zuhilfenahme dieser Methoden konnten mehrere VSBV-1-Infektionen in Hörnchen (aus den Unterfamilien Sciurinae und Callosciurinae) in Privathaltungen und Zoologischen Gärten in Deutschland, den Niederlanden und Kroatien identifiziert werden (ca. 3,5% aller beprobten Hörnchen). Dies weist auf eine ernstzunehmende Gefahr der Übertragung des Virus auf Menschen hin (Paper III und IV). Des Weiteren wurden mithilfe von high-throughput sequencing (HTS) und Metagenomanalyse zwei neue Astroviren identifiziert, die einen Neurotropismus anstatt der bekannten gastrointestinalen Pathogenese aufweisen. Dies betraf einerseits eine Kuh (Paper V) und andererseits zwei Schafen (Paper VI). Zur Charakterisierung wurden spezifische RT-qPCR Systeme sowie eine in-situ Hybridisierung entwickelt und eingesetzt. Zusammenfassend konnten neue Methoden zur Detektion und Charakterisierung von drei unterschiedlichen Enzephalitis-Viren entwickelt, validiert und für unterschiedliches Probenmaterial angewendet werden