Borna disease virus 1 (BoDV‐1) is the causative agent of Borna disease, an often fatal neurologic condition of domestic mammals, including New World camelids, in endemic areas in Central Europe. Recently, BoDV‐1 gained further attention by the confirmation of fatal zoonotic infections in humans. Although Borna disease and BoDV‐1 have been described already over the past decades, comprehensive reports of Borna disease outbreaks in domestic animals employing state‐of‐the‐art diagnostic methods are missing. Here, we report a series of BoDV‐1 infections in a herd of 27 alpacas (Vicugna pacos) in the federal state of Brandenburg, Germany, which resulted in eleven fatalities (41%) within ten months. Clinical courses ranged from sudden death without previous clinical signs to acute or chronic neurologic disease with death occurring after up to six months. All animals that underwent necropsy exhibited a non‐suppurative encephalitis. In addition, six apparently healthy seropositive individuals were identified within the herd, suggesting subclinical BoDV‐1 infections. In infected animals, BoDV‐1 RNA and antigen were mainly restricted to the central nervous system and the eye, and sporadically detectable in large peripheral nerves and neuronal structures in other tissues. Pest control measures on the farm resulted in the collection of a BoDV‐1‐positive bicoloured white‐toothed shrew (Crocidura leucodon), while all other trapped small mammals were negative. A phylogeographic analysis of BoDV‐1 sequences from the alpacas, the shrew and BoDV‐1‐positive equine cases from the same region in Brandenburg revealed a previously unreported endemic area of BoDV‐1 cluster 4 in North‐Western Brandenburg. In conclusion, alpacas appear to be highly susceptible to BoDV‐1 infection and display a highly variable clinical picture ranging from peracute death to subclinical forms. In addition to horses and sheep, they can serve as sensitive sentinels used for the identification of endemic areas.

Shrews, insectivorous small mammals, pertain to an ancient mammalian order. We screened 693 European and African shrews for hepatitis B virus (HBV) homologs to elucidate the enigmatic genealogy of HBV. Shrews host HBVs at low prevalence (2.5%) across a broad geographic and host range. The phylogenetically divergent shrew HBVs comprise separate species termed crowned shrew HBV (CSHBV) and musk shrew HBV (MSHBV), each containing distinct genotypes. Recombination events across host orders, evolutionary reconstructions, and antigenic divergence of shrew HBVs corroborated ancient origins of mammalian HBVs dating back about 80 million years. Resurrected CSHBV replicated in human hepatoma cells, but human-and tupaiaderived primary hepatocytes were resistant to hepatitis D viruses pseudotyped with CSHBV surface proteins. Functional characterization of the shrew sodium taurocholate cotransporting polypeptide (Ntcp), CSHBV/MSHBV surface peptide binding patterns, and infection experiments revealed lack of Ntcp-mediated entry of shrew HBV. Contrastingly, HBV entry was enabled by the shrew Ntcp. Shrew HBVs universally showed mutations in their genomic preCore domains impeding hepatitis B e antigen (HBeAg) production and resembling those observed in HBeAg-negative human HBV. Deep sequencing and in situ hybridization suggest that HBeAg-negative shrew HBVs cause intense hepatotropic monoinfections and low within-host genomic heterogeneity. Geographical clustering and low MSHBV/CSHBVspecific seroprevalence suggest focal transmission and high virulence of shrew HBVs. HBeAg negativity is thus an ancient HBV infection pattern, whereas Ntcp usage for entry is not evolutionarily conserved. Shrew infection models relying on CSHBV/MSHBV revertants and human HBV will allow comparative assessments of HBeAg-mediated HBV pathogenesis, entry, and species barriers.hepatitis B virus | viral evolution | zoonosis | shrew | E antigen T he hepatitis B virus (HBV, genus Orthohepadnavirus) is a ubiquitous pathogen that causes 887,000 deaths annually, predominantly due to cirrhosis and hepatocellular carcinoma after chronic hepatitis B (CHB) (1). Distantly related hepadnaviruses were identified recently in animals other than humans and apes (1). The newly discovered animal viruses revealed that prototypic properties of HBV such as envelopment (2) and presence of an X gene (3) emerged de novo during orthohepadnavirus evolution.Hepadnaviruses are ancient pathogens, likely infecting vertebrates for over 200 million years (3). Placental mammals evolved ∼99 million years ago (mya) and form 2 major clades termed Laurasiatheria and Euarchontoglires (4). The known laurasiatherian HBV hosts belong to several species within the orders Chiroptera (bats) and to one species each within the orders Carnivora (cat) and Artiodactyla (duiker). HBV hosts within the Euarchontoglires include Significance Hepatitis B viruses (HBVs) have existed for millions of years. We describe divergent HBV species in shrews, which are ancient insectivorous mammals. The shrew viruses co...

The variegated squirrel bornavirus 1 (VSBV-1) is a recently discovered emerging viral pathogen which causes severe and eventually fatal encephalitis in humans after contact to exotic squirrels in private holdings and zoological gardens. Understanding the VSBV-1 epidemiology is crucial to develop, implement and maintain surveillance strategies for detection and control of animal and human infections. Based on a newly detected human encephalitis case in a zoological garden, epidemiological squirrel trade investigations and molecular phylogeny analyses of VSBV-1 with temporal and spatial resolution were conducted. Phylogenetic analyses indicated a recent emergence of VSBV-1 in European squirrel holdings and several animal-animal and animal-human spill-over infections. Virus phylogeny linked to squirrel trade analysis showed the introduction of a common ancestor of the known current VSBV-1 isolates into captive exotic squirrels in Germany, most likely by Prevost's squirrels (Callosciurus prevostii). The links of the animal trade between private breeders and zoos, the likely introduction pathway of VSBV-1 into Germany, and the role of a primary animal distributor were elucidated. In addition, a seroprevalence study was performed among zoo animal caretakers from VSBV-1 affected zoos. No seropositive healthy zoo animal caretakers were found, underlining a probable high-case fatality rate of human VSBV-1 infections. This study illustrates the network and health consequences of uncontrolled wild pet trading as well as the benefits of molecular epidemiology for elucidation and future prevention of infection chains by zoonotic viruses. To respond to emerging zoonotic diseases rapidly, improved regulation and control strategies are urgently needed.

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 transcriptionquantitative 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.

Island populations may have a higher extinction risk due to reduced genetic diversity and need to be managed effectively in order to reduce the risk of biodiversity loss. The Eurasian red squirrels (Sciurus vulgaris) in the south of England only survive on three islands (the Isle of Wight, Brownsea and Furzey islands), with the Isle of Wight harbouring the largest population in the region. Fourteen microsatellites were used to determine the genetic structure of red squirrel populations on the Isle of Wight, as well as their relatedness to other populations of the species. Our results demonstrated that squirrels on these islands were less genetically diverse than those in Continental mainland populations, as would be expected. It also confirmed previous results from mitochondrial DNA which indicated that the squirrels on the Isle of Wight were relatively closely related to Brownsea island squirrels in the south of England. Importantly, our findings showed that genetic mixing between squirrels in the east and west of the Isle of Wight was very limited. Given the potential deleterious effects of small population size on genetic health, landscape management to encourage dispersal of squirrels between these populations should be a priority.

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