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.

ownership or trading routes of exotic animals are scarce and usually not available to the competent veterinary authorities (Karesh et al., 2005). Our epidemiological approach deals with a novel zoonotic pathogen of largely unknown features in a neglected niche of human-animal interface. Facing globalization and human population growth, this becomes of increasing importance within our modern one health comprehension (Marano et al., 2007;Webster et al., 2016).
The variegated squirrel bornavirus 1 (VSBV-1) is a novel zoonotic pathogen and forms its own species, the Mammalian 2 orthobornavirus, in the family Bornaviridae (Amarasinghe et al. (2019). In at least four cases, it caused progressive fatal encephalitis in infected human patients in Germany (Hoffmann et al., 2015;. All known cases of VSBV-1-associated human disease were linked to the husbandry of various exotic squirrel species. When captive squirrels were examined, virus was found not only in neuronal tissue, but also in various organs including those with excretional and secretional functions without causing any clinical signs (Schlottau, Jenckel, et al., 2017). Accordingly, the squirrels are thought to be the reservoir host for this zoonotic virus . In European holdings, the Central American variegated squirrels (Sciurus variegatoides Ogilby) and the South-East Asian Prevost's squirrels (Callosciurus prevostii Desmarest) were predominantly affected.
In addition, a Finlayson squirrel (Callosciurus finlaysoni Horsfield), a Swinhoe's striped squirrel (Tamiops swinhoei Milne-Edwards) and a red-tailed squirrel (Sciurus granatensis Humboldt) were found positive Schlottau, Jenckel, et al., 2017). These five species are members of two different subfamilies of the family Sciuridae, the Sciurinae and the Callosciurinae, and they originate in the tropics and subtropics of two different continents, America and Asia, respectively (Steppan et al., 2004). Therefore, the origin of the virus is uncertain. Besides an extra-European origin, a 'non-squirrel' regional virus reservoir of European origin is discussed, as described for the Borna disease virus 1, which is hosted by the bicoloured white-toothed shrew (Crocidura leucodon Hermann) (Durrwald et al., 2014). Indigenous Eurasian squirrels (Sciurus vulgaris Linnaeus) or free-ranging neozootic populations of Eastern grey squirrels (Sciurus carolinensis Gmelin) and Pallas's squirrels (Callosciurus erythraeus Pallas) in Europe were so far not found infected with VSBV-1 (Schlottau, Jenckel, et al., 2017) (Schulze et al., 2020).
In the aftermath of the detection of VSBV-1, epidemiological investigations among private breeders and owners of squirrels were undertaken in Germany. A core network of affected owners and of captive tropical squirrels was assembled (Tappe et al., 2019), and the testing of a growing number of squirrels revealed more infected animals. However, a systematic approach to sampling was not possible. As the screened holdings were selected by a certain degree of contact between the owners and included predominantly similar species, the VSBV-1 prevalence among captive squirrels in Germany could not be reliably estimated.
Accordingly, an approach was chosen, which is similar to those recommended for hard-to-survey populations (Tourangeau, 2014).
Objectives were the determination of the size of the population at risk and the estimation of the prevalence of VSBV-1 within this target population as the most important parameters. To this end, a register of squirrel owners in Germany was implemented, which elucidates several features of squirrel husbandries in the country. On this basis, we conducted a cross-sectional study to assess the prevalence of VSBV-1 in captive squirrels in Germany in private holdings and zoos.

| Registration process
Captive animals of all species of the family Sciuridae were regarded as the population at risk due to the unknown interspecific susceptibility for VSBV-1. Two major populations were identified: squirrels in private hands and those kept in zoological gardens. Considerations for separate investigations were the limited numbers of zoos versus the potentially not completely recordable private holdings, different levels of accessibility of owners, their compliance and the documentation of animal movements. Furthermore, public and occupational health concerns were considered.
To assess the size and characteristics of the population at risk in the private husbandry sector, the squirrel owners were identified as the best source of information. To address this community, an online approach was chosen for a simple and fast access to a broad spectrum of potential participants. Possible sources of bias in a Web survey are the coverage of Internet use and the sensitivity for self-selection (Bethlehem, 2010). Although the coverage of Internet use in Germany is over 90% (German Federal Statistical Office, 2018), the possible under-coverage in the target population was addressed by a parallel offline strategy. Different online platforms of breeders and owners of squirrels and other small mammals were identified and explored. If they had been active within the last six months prior to the launch of the online survey in January 2018, general information on VSBV-1, the project and the planned study, and the rationale for participation in the online survey were shared. The same information was promoted in specialized German

Impacts
• This survey resulted in the first and most comprehensive data set on captive squirrels in zoos and private holdings in Germany.
• With the owners' support, insight into characteristics of squirrel holdings was gained, whereas at the same time, a communication channel for public health information was established.
• Subsequent cross-sectional studies revealed a prevalence of 0% (95% CI 0%-6.2%) positive subpopulations among private squirrel collections and 1.9% (95% CI: 0%-9.9%) among zoos in Germany. magazines for squirrel owners or enthusiasts of rodents and other small mammals (Allendorf et al., 2017 and by visiting meetings of small mammal breeder societies and exotic animal markets. The online post included a link to a questionnaire for self-registration of the squirrel owners. Offline information on the pathogen was presented, and leaflets were distributed including a QR-coded link to the online questionnaire and contact addresses. The questionnaire (Supplements) was designed as a basic version for registration, from which an extended version with more detailed questions on the husbandry condition and management could be reached after completion. With the basic version, information was gathered on (a) species and number of squirrels kept, (b) the purpose of holding (hobby or breeding), (c) documentation of movements of bred animals and (d) personal data (name and address). Informed consent was obtained from all participants. Finally, the participants could register for a mailing list with information on VSBV-1 and for the involvement in the subsequent diagnostic study.
Concomitantly, online platforms for animal trade, squirrel-specific groups, adverts in special magazines and Web-based exotic animal markets were screened for current offers of squirrels of any species. Sellers were registered passively with the data available on the website and the offered species. They were informed about the purpose of the investigation and motivated to participate in the survey, as were all people who had sent in samples for VSBV-1 diagnostics to the Friedrich-Loeffler-Institut. These diagnostic services had been offered since 2015.
For the zoo sector, a publicly available online database on German zoological inventories (https://www.zooti erlis te. de, 2018-04-01) and the inventories listed in the Zoo Information Management System (ZIMS) for German zoos with one or more records on sciurid species were pre-screened and the entries confirmed or corrected by phone calls, emails or personal contact. Meetings of national and international zoological societies and small mammal specialist groups were visited to spread information and acquire participating institutions.
In general, individual squirrel species were kept separately, whereas the exchange between enclosures within one holding for breeding and other reasons was frequent for animals of the same species. Therefore, all individuals of one species were presumed to form one epidemiologic unit, further on referred to as a subpopulation, if one owner/breeder/institution kept more than one species.
Registered private and zoological populations were compared with the only known and presumably selection-biased data set Schlottau, Jenckel, et al., 2017), further referred to as the pilot cohort. This pilot cohort included the squirrels of 33 private breeders or owners and 19 zoological gardens

| Sampling
For the subsequent cross-sectional studies on subpopulation level within both populations, sample sizes were calculated by the formula.
with Z representing the value from standard normal distribution that corresponds to the desired confidence level (Z = 1.96 for 95% CI), p as the estimated true prevalence and e as the desired precision (Cochran, 1977).
The sample size for estimating the prevalence in the private and zoo population was calculated for a confidence interval of 95% and different precision values. For the private population, a precision of 10% was chosen to take the expected lower compliance into account; for the zoo population, the common precision of 5% was maintained.
The prevalence estimate p was taken from the pilot cohort. To generate less biased estimators from these pilot data, the proportions p i of positive subpopulations within each species (i.e. stratum) were adjusted to the assumed true frequency distribution of all species as measured throughout the registration process by applying the formula.
For the zoo population, the following correction for a finite population size was applied to the sample size n resulting from the equation above (Thrusfield & Christley, 2018): with N as the sum of all registered subpopulations (pilot plus new register).

| Detection of VSBV-1 RNA
All registered owners and zoos were contacted and motivated to sample all their squirrels by dry oral swabs and/or faecal samples if possible. Previous studies had shown equal suitability of both sample types . If no individual swabs or faecal samples could be taken, pooled faecal samples were collected from the enclosure of the respective subpopulation. Informed consent was obtained from all owners and institutions participating in this study.
Samples were analysed by real-time quantitative reverse transcription-PCR (RT-qPCR) using the system established in former studies (Hoffmann et al., 2015;Schlottau, Jenckel, et al., 2017). Briefly, in an extraction step with TRIzol reagent

| Statistical analyses
To estimate the apparent prevalence, the samples were stratified by subpopulation. Data processing and statistical analyses were performed using r Version 3.5.1.; the packages utils, propCI, dplyr, stats, treemap and ggplot2 were used for descriptive statistics, calculations and graphical visualization. To determine whether the observed frequencies of the study and the pilot data followed the same distribution, Pearson's chi-squared goodness-of-fit test was performed. Confidence intervals were calculated using the Clopper-Pearson exact method (Clopper et al., 1934).

| Study population
In the private sector, the online questionnaire was completed by 133 participants, of which 117 stated that they kept one or more squirrels at the time, when the study was conducted. Ninety-three German participants answered the question on the species of squirrels kept, of which eleven kept more than one species. With this, data on 117 subpopulations were collected. To this self-registration data, passively compiled data on owners and breeders, for example by incoming diagnostic requests (n = 39) and by offline and online presentation of squirrel holdings (n = 84), were merged. Taken together, information on 240 subpopulations from 147 different private holdings was collected (for further details, refer Figure 1).
Twenty-three different species were kept by private owners (Figure 2 and Table 1). The most common species were Swinhoe's striped squirrels (23.9% of all registered subpopulations) and Siberian chipmunks (21.0%), followed by the indigenous Eurasian squirrel (16.8%). Together, these three species accounted for more than 60% of all registered subpopulations. The species of highest interest concerning previous VSBV-1 occurrence, the variegated squirrel and the Prevost's squirrel, were far less common, each constituting about 5% of the squirrel subpopulations.
In 35 of the holdings (23.8%), two or more different species were kept.

| Sampling
In brief, the sample size calculation revealed that sampling of at least 24 subpopulations was required. The data were stratified by the frequency of different species in the registered subpopulations.
Between March 2018 and January 2020, samples of overall 353 individual animals from 58 different subpopulations were received and analysed. This sample exceeded the minimum sample size and reflected approximately the expected stratification (Table 1).

| Analysis
During the cross-sectional study, VSBV-1 RNA was not detected in any of the tested samples (Table 1)    In the zoological institutions, 26 different sciurid species were kept ( Figure 3 and Table 2). In 39 institutions (57% of all registered), two or more different species were kept. Thirty of these institutions (77%) housed subpopulations of two or more subfamilies. The most common species (25% of all subpopulations) were the prairie dog (Cynomys ludovicianus Ord) of the subfamily of ground squirrels (Xerinae), followed by the Eurasian red squirrel (15%) and the Swinhoe's striped squirrel (13%). Prevost's squirrels are currently kept by 9% of the registered zoos; variegated squirrels are among the rarely kept species with only five records (3%).

| Sampling
As described above, sample size calculation was adjusted to a lim-  (Table 2).

| Analysis
VSBV-1 RNA was detected in samples from one individual Prevost's squirrel, which leads to an apparent prevalence of 1.9% (95% CI 0%-9.9%) VSBV-1-positive squirrel subpopulations in zoos (Table 2)  Note: Frequency distribution of subpopulations in the squirrel population in private holdings registered and sampled during this study (2018)(2019)(2020) including results on the presence of VSBV-1 in comparison with the pilot data on private squirrel holdings retrieved from the VSBV-1 screening studies (2015-2017) Schlottau, Jenckel, et al., 2017).
TA B L E 1 Squirrel subpopulations in private holdings-register and crosssectional study on the presence of VSBV-1 conducted in 2016 (Schlottau, Jenckel, et al., 2017).  Note: Frequency distribution of subpopulations in the squirrel population kept in zoos registered and sampled during this study (2018)(2019)(2020) including results on the presence of VSBV-1 in comparison with the pilot data on private squirrel holdings retrieved from the VSBV-1 screening studies (2015-2017) Schlottau, Jenckel, et al., 2017).  Zoos answered unsupervised and anonymously, without verification of the input. False inputs were discouraged by informing on the strictly anonymized data policy. In contrast to the private sector, the registered zoos can be assumed almost complete, with very few missing data of institutions that could not be contacted or failed to reply.

TA B L E 2
Here, limitations in terms of selection and compliance were staffing levels of the institution, which likely correlates with its size and its financial equipment. This demonstrated the success of the recruiting plan by providing detailed information and subsequent voluntary diagnostic testing of kept squirrels. Moreover, it led to a more precise prevalence estimate. Nonetheless, the assumption of the upper limit of the confidence interval, 6.2%, as the worst-case apparent prevalence is recommendable.
For the zoo population, the required sample size was also exceeded, which underscores the assumption of a high compliance among the institutions. According to the results of telephone interviews, only small zoos ran by one person or a family had no time or staff for sampling. These institutions often housed European squirrels, explaining the gap in sampling within this subpopulation.
Given the suspected lifelong persistent infection of sciurids and the lack of therapeutic agents, euthanasia of VSBV-1-infected individuals might be the only possibility to eliminate the hazard from squirrel colonies. The success of this strategy can be delineated from the decrease in VSBV-1 prevalence in both populations, when comparing the register-adjusted prevalence from the pilot study (2015-2017; 3.2%, 95% CI 0.05%-18.1%) to the apparent prevalence in the cross-sectional study (2018-2020; 1.9%, 95% CI 0%-9.9%).
Considering the relatively large time intervals of the surveys, this decrease also suggests a low reproduction ratio of VSBV-1 infections within the squirrel population in captivity, although the exact mode of transmission is not yet understood. This observation may be beneficial for the further reduction in the risk of VSBV-1 infections in humans and animals in Germany.
The only positive subpopulation of Prevost's squirrels was also found to be affected in the pilot study (Schlottau, Jenckel, et al., 2017). After removing the positive animal, the subpopulation was not monitored until December 2019, when sampling revealed the remaining contact animal of the previously positive animal to be VSBV-1-infected. This illustrates the importance of regular sampling, especially of those species known to be susceptible, to minimize the risk of transmission to humans. It should be noted that, this institution (like every other affected holding) had also been advised by the Friedrich-Loeffler-Institut to (re)test their squirrel population every 3 months.
In summary, the observed low prevalence and the presumptively low infection rate within the subpopulations may indicate a controllable and bounded outbreak of still unknown origin. But also, it proves the still existing risk of positive squirrels in German holdings.
Taking into account the high lethality of human VSBV-1 infections, the importance of a thorough surveillance-for example regular diagnostics-of at least the known susceptible species is not debatable.
If not enforceable by the authorities, especially the zoos and private breeders selling their animals should feel responsible to insure the reduction or elimination of all occupational and public health risks.

| CON CLUS ION
With this study, the insight into squirrel husbandry in Germany, certainly one of many niches in animal-human interface, is broadened. Simultaneously the occurrence of the new emerging zoonotic VSBV-1, causing a fatal disease in humans, is elucidated. Initially, the populations at risk had to be defined, before the prevalence studies could be conceptualized and conducted. Due to lacking regulation, estimating and reaching the population at risk was challenging and time-consuming. The deficiency of data regarding not just squirrel but most exotic animal ownership is inherent in the German system and in other countries of the world. The described approach may serve as a blueprint for epidemiologic assessment of emerging diseases in exotic animals. However, a profound solution would be the implementation of a general unified registration system for exotic pets. From a one health perspective, it is urgent to increase the surveillance of trade and ownership of exotic pets to prevent the potential introduction of and exposure to EID in general (Reaser et al., 2008). Traded and kept under the radar, animals carrying medium to highly virulent pathogens could easily become a hazard for human public health. Moreover, we would like to thank all participating animal owners and zoos for their support and commitment.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

AUTH O R CO NTR I B UTI O N S
VA, MB, FJC and TH designed the study. VA carried out the survey.
DR, KS and DH carried out the laboratory analysis of the samples.
VA, CF and SA conceived and performed statistical analysis. VA drafted the manuscript. MB, FJC and TH supervised the study. DR, KS, SA, CF, MB, FJC and TH provided critical feedback and helped shape the research, analysis and manuscript.

E TH I C A L A PPROVA L
Informed consent was obtained from all owners and institutions participating in this study.