Introduction and spread of variegated squirrel bornavirus 1 (VSBV-1) between exotic squirrels and spill-over infections to humans in Germany

Abstract

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 the 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.

Keywords: Bornavirus; animal trade; seroprevalence study; squirrel; surveillance; time-resolved phylogeny; variegated squirrel bornavirus 1 (VSBV-1); zoonotic infection.

Figure 1.

Bayesian maximum clade credibility (MCC) tree of VSBV-1 based on complete coding sequences showing the spread of the virus between different private holdings and zoos. Branches are coloured by the host and represent the transitions between different host species for the virus and the host of the common ancestor of all VSBV-1 strains, and the circle at the nodes the most probable host species of their descendant nodes (see colour codes). Spill-over infections to humans are shown in red. Human cases B and C are not included, as no complete virus genomes were available. Virus infections in zoos are shown in shaded areas. Statistical support of grouping from Bayesian posterior probabilities (clade credibility ≥90%) is indicated with an asterisk. Taxon information includes GenBank accession number, sample designation, host, year of detection and location of origin. Time in years is reported in the axis below the tree.

Figure 2.

Maximum-likelihood (ML) phylogenetic tree of VSBV-1 based on complete coding sequences showing the phylogenetic placement of the human-derived strains compared with squirrel-derived strains. Sequence information is coloured to illustrate different hosts. Spill-over infections to humans are shown in red. Human cases B and C are not included, as no complete virus genomes were available. Virus infections in zoos are shown in shaded areas. ML bootstrap replicates scores (>70%) are shown next to the nodes. Taxon information includes GenBank accession number, sample designation, host, year of detection and location. Scale bar indicates nucleotide substitutions per site.

Figure 3.

Spatio-temporal reconstruction of trade between zoos and private holdings with confirmed VSBV-1 positive squirrels. Timeline of squirrel presence and trading routes, including human cases and detection of VSBV-1-positive squirrels. Each horizontal line, differently coloured to illustrate the host species, represents the presence of at least one squirrel of that species at the named location over time. The vertical coloured lines indicate the trading route of one or more squirrels from one location to another at the distinct point in time. Squirrel holdings in zoos are shown in shaded areas. Filled red boxes on the coloured lines represent the quarter of the year in which one or more VSBV-1 positive squirrels were detected in the respective institution. Red frames mark the time period of reported neurological disease of proven, probable and possible human VSBV-1 encephalitis cases. Red arrows mark the assumed transfer of one or more infected squirrels. Streamlining was performed by omitting unaffected squirrels and holdings with no ties to affected animals and institutions, respectively. Affected holdings without reliable data were also not included.

Figure 4.

Presence of individual Prevost’s squirrels in zoo D and contact zoos aligned with the occurrence of the two occupation-related human cases. Each light green line represents the presence (horizontal) or the transfer (vertical) of one individual Prevost’s squirrel in or between involved zoos, which are depicted by black (zoo D) and grey frames. Filled red or dark green ends of the pathways mark positive and negative testing results for VSBV-1 RNA, respectively. Red frames indicate the period of symptom onset in human cases D.1 and D.2. Continuous red lines mark squirrels diagnosed with a VSBV-1-infection, dotted red lines represent squirrels assumed to be infected.