PARROT BORNAVIRUSES IN PSITTACINES KEPT IN CAPTIVITY IN THE STATE OF SANTA CATARINA, BRAZIL.

Parrot bornaviruses are responsible for proventricular dilatation disease (PDD) in psittacines. This study aimed to determine the occurrence and factors associated with Parrot bornaviruses infection in psittacines kept in captivity in a state in the southern region of Brazil. A cross-sectional study was carried out with 192 birds from two facilities (A and B) in 2019, using choanal, esophageal, and cloacal swabs and feathers, totaling 768 samples subjected to reverse-transcription polymerase chain reaction (RT-PCR), for the matrix (M) protein gene with a final product of 350 base pairs (bp). Genetic sequencing of three positive samples was performed by the Sanger method. In the study, the overall virus occurrence was 35.9% (69/192), with 40.4% (42/104) in Facility A and 30.7% (27/88) in Facility B. Sequencing analysis of the samples revealed the presence of Parrot bornavirus 2 (PaBV-2) in both facilities. Swab samples from the choanal (40/69), esophageal (30/69), cloacal (35/69), and feather (15/69) tested positive, facilitating the molecular diagnosis of Parrot bornaviruses. The results indicated that there is no single ideal sample type for antemortem molecular diagnosis of this virus. Simultaneously testing all four samples at the same time point yielded more diagnoses than testing any single sample among the four. Most of the 29 sampled psittacine species were native, and 46.9% of the birds (90/192) consisted of endangered species. Among the psittacines that tested positive, 88.4% (61/69) were clinically healthy, and 8.7% (6/69) exhibited clinical or behavioral signs, including behavioral changes, alterations in feathering, and changes in body score at the time of collection. This study showcases the application of minimally invasive sampling for diagnosing Parrot bornaviruses, enabling sample collection when the birds are restrained for clinical evaluation. This approach facilitates a prompt and effective antemortem diagnosis, thereby serving as an efficient screening method for parrots kept in captivity.

Surveillance of Parrot Bornavirus in Taiwan Captive Psittaciformes.

Parrot bornavirus (PaBV) is an infectious disease linked with proventricular dilatation disease (PDD) with severe digestive and neurological symptoms affecting psittacine birds. Despite its detection in 2008, PaBV prevalence in Taiwan remains unexplored. Taiwan is one of the leading psittacine bird breeders; hence, understanding the distribution of PaBV aids preventive measures in controlling spread, early disease recognition, epidemiology, and transmission dynamics. Here, we aimed to detect the prevalence rate of PaBV and assess its genetic variation in Taiwan. Among 124 psittacine birds tested, fifty-seven were PaBV-positive, a prevalence rate of 45.97%. Most of the PaBV infections were adult psittacine birds, with five birds surviving the infection, resulting in a low survival rate (8.77%). A year of parrot bornavirus surveillance presented a seasonal pattern, with peak PaBV infection rates occurring in the spring season (68%) and the least in the summer season (25%), indicating the occurrence of PaBV infections linked to seasonal factors. Histopathology reveals severe meningoencephalitis in the cerebellum and dilated cardiomyopathy of the heart in psittacine birds who suffered from PDD. Three brain samples underwent X/P gene sequencing, revealing PaBV-2 and PaBV-4 viral genotypes through phylogenetic analyses. This underscores the necessity for ongoing PaBV surveillance and further investigation into its pathophysiology and transmission routes.

Betanodavirus meningoencephalitis in an Atlantic blue marlin.

Viral nervous necrosis (viral encephalopathy and retinopathy) is caused by piscine nodavirus (Nodaviridae, Betanodavirus). Since 1986, this highly infectious virus has caused mass mortalities of up to 100% in farmed saltwater and freshwater fish around the world (with the exception of South America and Antarctica), affecting >60 species across 10 orders. The Atlantic blue marlin (Makaira nigricans Lacépède, 1802) is a top-level predator found throughout the tropical waters of the Atlantic and Indo-Pacific oceans. Despite their popularity as a sportfish, relatively little is known about the Atlantic blue marlin and other billfish. We describe here chronic betanodavirus infection in a juvenile Atlantic blue marlin, which is, to our knowledge, the first report of disease in M. nigricans.

Genetic trends in parrot Bornavirus: a clinical analysis.

Parrot Bornavirus (PaBV) has been reported to cause indigestion and other wasting symptoms such as weight loss and lethargy. The pathogenesis of PaBV has yet to be fully elucidated. This study reports PaBV infections in South Korea and suggests a trend in the genetic information gathered from clinical cases. A total of 487 birds with or without clinical symptoms were tested for bornavirus. Twelve of 361 asymptomatic birds tested positive for bornavirus, while 15 of 126 birds with various symptoms tested positive. A segment of approximately 1,540 bps including the N, X, P and M proteins were obtained from 23 of the positive strains and analyzed with other strains found on GenBank that had clinical information. PaBV was type 2 and 4 in South Korea, and certain amino acid sequences showed a difference between symptom presenting animals and asymptomatic animals in the X protein and P protein. When considering that some asymptomatic cases may have been latent infections at the time of examination, it is plausible these trends may grow stronger with time. Majority of PaBV was type 4 in South Korea. If these trends are confirmed, diagnosis of potentially pathogenic PaBVs in a clinical manner will be possible during the early stages of infection.

Surveillance for Avian Bornavirus in Colorado and Wyoming, USA, Raptor Populations.

Avian bornavirus (ABV) is known to infect at least 80 avian species and is associated with avian bornaviral ganglioneuritis (ABG). Avian bornaviral ganglioneuritis is characterized by a lymphoplasmacytic infiltration of the nervous tissue, mainly affecting the nerves that supply the gastrointestinal tract of birds. This disease is diagnosed commonly in psittacines under human care and has been demonstrated in wild bird species; however, its occurrence in raptors is largely unknown. Because of the commonality of ABV in the pet bird population, there is concern about the spread of this virus to other companion avian species, such as falconry birds, as well as wildlife. This prospective study used reverse-transcription quantitative PCR (RT-qPCR) to survey free-ranging Colorado and Wyoming, US, raptor populations for ABV. Quantitative PCR was performed on mixed conjunctival-choanal-cloacal swabs collected from live birds (n=139). In dead birds, a combination of mixed swabs (n=265) and tissue samples of the brain (n=258), heart (n=162), adrenal glands (n=162), liver (n=162), kidney (n=139), spinal cord (n=139), and brachial plexus (n=139) were evaluated. All 1,565 swab and tissue samples RT-qPCR results from the 404 birds evaluated were negative. Based on these results and a lack of clinical signs suggestive of ABG, ABV is likely not a prevalent pathogen in Colorado and Wyoming raptor populations at this time.

Molecular detection of bornavirus in parrots imported to China in 2022.

BACKGROUND: Avian bornavirus (ABV) is a neurotropic virus, it has been established as the primary causative agent of proventricular dilatation disease (PDD). However, substantial international trade and transnational trafficking of wild birds occur, potentially enabling these birds to harbor and transmit pathogens to domestic poultry, adversely affecting their well-being. Real-time RT-PCR was employed to detect the presence of PaBV-4 in parrots imported to China in 2022. RESULTS: In 2022, a total of 47 cloacal swabs from 9 distinct species of parrots were collected at the Wildlife Rescue Monitoring Center in Guangdong, China. The purpose of this collection was to detect the presence of PaBV-4. Using real-time PCR techniques, it was determined that the positive rate of PaBV-4 was 2.12% (1 out of 47) in parrots. The PaBV-4 virus was detected in a Amazona aestiva that had been adopted for one month. Conversely, all other species tested negative for the virus. Subsequently, the whole genome of the PaBV-4 GD2207 strains was sequenced, and the homology and genetic evolution between these strains and previously published PaBV-4 strains on GenBank were analyzed using DNAStar and MEGA7.0 software. The findings revealed that the full-length genome of PaBV-4 consisted of 8915 nucleotides and encoded six proteins. Additionally, it exhibited the highest nucleotide similarity (99.9%) to the GZ2019 strain, which causes death and severe clinical symptoms in Aratinga solstitialis. Furthermore, when compared to other strains of PaBV-4, the GD2207 strain demonstrated the highest amino acid homology with GZ2019. The phylogenetic analysis demonstrated that the GD2207 strain clustered with various strains found in Japanese, American, and German parrots, indicating a close genetic relationship with PaBV-4, but it revealed a distant relationship with PaBV-5 Cockg5 from America. Notably, the GD2207 was closely associated with the GZ2019 strain from Aratinga solstitialis in China. CONCLUSION: This study presents the preliminary identification of PaBV-4 in Amazona aestiva parrots, emphasizing its importance as the predominant viral genotype linked to parrot infections resulting from trade into China. Through genetic evolution analysis, it was determined that the GD2207 strain of PaBV-4 exhibits the closest genetic relationship with GZ 2019 (Aratinga solstitialis, China), M14 (Ara macao, USA), AG5 (Psittacus erithacus, USA) and 6758 (Ara ararauna, Germany) suggesting a shared ancestry.

Reverse genetics of parrot bornavirus 4 reveals a unique splicing of the glycoprotein gene that affects viral propagation.

Viruses can utilize host splicing machinery to enable the expression of multiple genes from a limited-sized genome. Orthobornaviruses use alternative splicing to regulate the expression level of viral proteins and achieve efficient viral replication in the nucleus. Although more than 20 orthobornaviruses have been identified belonging to eight different viral species, virus-specific splicing has not been demonstrated. Here, we demonstrate that the glycoprotein (G) transcript of parrot bornavirus 4 (PaBV-4; species Orthobornavirus alphapsittaciforme), a highly virulent virus in psittacines, undergoes mRNA splicing and expresses a soluble isoform termed sGP. Interestingly, the splicing donor for sGP is not conserved in other orthobornaviruses, including those belonging to the same orthobornavirus species, suggesting that this splicing has evolved as a PaBV-4-specific event. We have also shown that exogenous expression of sGP does not affect PaBV-4 replication or de novo virion infectivity. In this study, to investigate the role of sGP in viral replication, we established a reverse genetics system for PaBV-4 by using avian cell lines and generated a recombinant virus lacking the spliced mRNA for sGP. Using the recombinant viruses, we show that the replication of the sGP-deficient virus is significantly slower than that of the wild-type virus and that the exogenous expression of sGP cannot restore its propagation efficiency. These results suggest that autologous or controlled expression of sGP by splicing may be important for PaBV-4 propagation. The reverse genetics system for avian bornaviruses developed here will be a powerful tool for understanding the replication strategies and pathogenesis of avian orthobornaviruses. IMPORTANCE Parrot bornavirus 4 (PaBV-4) is the dominant cause of proventricular dilatation disease, a severe gastrointestinal and central nervous system disease among avian bornaviruses. In this study, we discovered that PaBV-4 expresses a soluble isoform of glycoprotein (G), called sGP, through alternative splicing of the G mRNA, which is unique to this virus. To understand the role of sGP in viral replication, we generated recombinant PaBV-4 lacking the newly identified splicing donor site for sGP using a reverse genetics system and found that its propagation was significantly slower than that of the wild-type virus, suggesting that sGP plays an essential role in PaBV-4 infection. Our results provide important insights not only into the replication strategy but also into the pathogenesis of PaBV-4, which is the most prevalent bornavirus in captive psittacines worldwide.

Factors Influencing Vertical Transmission of Psittacine Bornavirus in Cockatiels (Nymphicus hollandicus).

The transmission of parrot bornavirus is still not fully understood. Although horizontal transmission through wounds can be one route, vertical transmission is still discussed. PaBV RNA and PaBV antigen were detected in psittacine embryos, but isolation of the virus failed, raising doubts about this route. In this study, cockatiels were infected either as adults (adult group) or during the first 6 days after hatching (juvenile group) and raised until sexual maturity to breed and lay eggs. A total of 92 eggs (adult group: 49, juvenile group: 43) were laid and incubated until day 17. The embryos and yolk samples were examined by RT-PCR for PaBV RNA and by infectivity assay for infectious virus. In the adult group, 14/31 embryos (45.2%) and 20/39 (51%) of the yolk samples demonstrated PaBV RNA in the PCR. Isolation of PaBV was not possible in any embryo of this group, but it was achieved in six yolk samples from one female. Anti-PaBV antibodies were detected in the yolk samples after seroconversion of all female parents. In the juvenile group, 22/29 embryos (74.9%) were positive for PaBV RNA. In 9/21 embryos (42.9%), PaBV isolation was possible. PaBV RNA was detected in 100% and infectious virus in 41% of the yolk samples. Anti-PaBV antibodies were detected in all yolk samples. For the first time, successful vertical transmission of PaBV was proven, but it seems to depend on the age when the parent birds are infected. Therefore, the age of the bird at time of infection may be an important factor in the occurrence of vertical transmission.

Tissue Distribution of Parrot Bornavirus 4 (PaBV-4) in Experimentally Infected Young and Adult Cockatiels (Nymphicus hollandicus).

Proventricular dilatation disease (PDD) caused by parrot bornavirus (PaBV) infection is an often-fatal disease known to infect Psittaciformes. The impact of age at the time of PaBV infection on organ lesions and tissue distribution of virus antigen and RNA remains largely unclear. For this purpose, tissue sections of 11 cockatiels intravenously infected with PaBV-4 as adults or juveniles, respectively, were examined via histology, immunohistochemistry applying a phosphoprotein (P) antibody directed against the bornaviral phosphoprotein and in situ hybridisation to detect viral RNA in tissues. In both groups of adult- and juvenile-infected cockatiels, widespread tissue distribution of bornaviral antigen and RNA as well as histologic inflammatory lesions were demonstrated. The latter appeared more severe in the central nervous system in adults and in the proventriculus of juveniles, respectively. During the study, central nervous symptoms and signs of gastrointestinal affection were only demonstrated in adult birds. Our findings indicate a great role of the age at the time of infection in the development of histopathological lesions and clinical signs, and thus provide a better understanding of the pathogenesis, possible virus transmission routes, and the development of carrier birds posing a risk to psittacine collections.

Canary Bornavirus (Orthobornavirus serini) Infections Are Associated with Clinical Symptoms in Common Canaries (Serinus canaria dom.).

While parrot bornaviruses are accepted as the cause of proventricular dilatation disease (PDD) in psittacine birds, the pathogenic role of bornaviruses in common canaries is still unclear. To answer the question of whether canary bornaviruses (species Orthobornavirus serini) are associated with a PDD-like disease in common canaries (Serinus canaria f. dom.), the clinical data of 201 canary bird patients tested for bornaviruses using RT-PCR assays, were analyzed for the presence of PDD-like gastrointestinal or central nervous system signs and for other viruses (mainly circovirus and polyomavirus), yeasts and trichomonads. Canary bornavirus RNA was detected in the clinical samples of 40 out of 201 canaries (19.9%) coming from 28 of 140 flocks (20%). All nucleotide sequences obtained could unequivocally be determined as canary bornavirus 1, 2, or 3 supporting the current taxonomy of the species Orthobornavirus serini. PDD-like signs were found associated with canary bornavirus detection, and to a lesser extent, with circoviruses detection, but not with the detection of polyomaviruses, yeasts or trichomonads. The data indicate that canary bornaviruses contribute to a PDD-like disease in naturally infected canaries, and suggest a promoting effect of circoviruses for the development of PDD-like signs.

Experimental Infection of Embryonic Cells and Embryonated Eggs of Cockatiels (Nymphicus hollandicus) with Two Parrot Bornavirus Isolates (PaBV-4 and PaBV-2).

Parrot bornavirus (PaBV) might be transmitted vertically. Cockatiel embryonic brain cells and embryonated eggs of cockatiels (ECE) were infected with PaBV-2 and PaBV-4. In embryonic brain cells, PaBV-2 and PaBV-4 showed no differences in viral spread despite the slower growth of PaBV-2 compared with PaBV-4 in CEC-32 cells. ECE were inoculated with PaBV-4 and 13-14 dpi, organs were sampled for RT-PCR, immunohistochemistry/histology, and virus isolation. In 28.1% of the embryos PaBV-4-RNA and in 81.3% PaBV-4-antigen was detected in the brain. Virus isolation failed. Division of organ samples and uneven tissue distribution of the virus limited the results. Therefore, 25 ECE were inoculated with PaBV-4 (group 1) and 15 ECE with PaBV-2 (group 3) in the yolk sac, and 25 ECE were inoculated with PaBV-4 (group 2) and 15 eggs with PaBV-2 (group 4) in the chorioallantoic membrane to use the complete organs from each embryo for each examination method. PaBV-RNA was detected in the brain of 80% of the embryos in groups 1, 2, 3 and in 100% of the embryos in group 4. In 90% of the infected embryos of group 1, and 100% of group 2, 3 and 4, PaBV antigen was detected in the brain. PaBV antigen-positive brain cells were negative for anti-neuronal nuclear protein, anti-glial fibrillary acidic protein, and anti S-100 staining. Virus was not re-isolated. These results demonstrated a specific distribution pattern and spread of PaBV-4 and PaBV-2 in the brain when inoculated in ECE. These findings support a potential for vertical transmission.

Vasculitis Associated with Parrot Bornavirus 4 Infection in a Rose-Crowned Parakeet (Pyrrhurarhodocephala).

A 3-month-old, female rose-crowned parakeet (Pyrrhura rhodocephala) was found dead after a 24-h course of lethargy and passing blood-tinged faeces. Fine white streaks were seen in the pectoral muscles on necropsy. Microscopic examination revealed typical lesions of avian ganglioneuritis and vascular necrosis in the pectoral muscles, myocardium, kidneys, air sacs, adrenal glands, pancreas and thyroid gland. These lesions were characterized by mural fibrinoid necrosis of small and medium-calibre arteries and arterioles, associated with lymphoplasmacytic inflammation, necrosis, atrophy and fibrosis of the surrounding tissues. Parrot bornavirus (PaBV) nucleoprotein was demonstrated by immunohistochemistry in smooth muscle and endothelial cells of many vessels. An avian bornavirus was isolated from kidney tissue and its identity confirmed as PaBV-4 by sequencing and phylogenetic analysis. We postulate that the vascular lesions could have been immune-mediated and that PaBV-4 may have played a role in its pathogenesis.

Avian Bornavirus Research-A Comprehensive Review.

Avian bornaviruses constitute a genetically diverse group of at least 15 viruses belonging to the genus Orthobornavirus within the family Bornaviridae. After the discovery of the first avian bornaviruses in diseased psittacines in 2008, further viruses have been detected in passerines and aquatic birds. Parrot bornaviruses (PaBVs) possess the highest veterinary relevance amongst the avian bornaviruses as the causative agents of proventricular dilatation disease (PDD). PDD is a chronic and often fatal disease that may engulf a broad range of clinical presentations, typically including neurologic signs as well as impaired gastrointestinal motility, leading to proventricular dilatation. It occurs worldwide in captive psittacine populations and threatens private bird collections, zoological gardens and rehabilitation projects of endangered species. In contrast, only little is known about the pathogenic roles of passerine and waterbird bornaviruses. This comprehensive review summarizes the current knowledge on avian bornavirus infections, including their taxonomy, pathogenesis of associated diseases, epidemiology, diagnostic strategies and recent developments on prophylactic and therapeutic countermeasures.

Immunophenotype of the inflammatory response in the central and enteric nervous systems of cockatiels (Nymphicus hollandicus) experimentally infected with parrot bornavirus 2.

Proventricular dilatation disease is a lethal disease of psittacine birds. In this study, we characterized the local cellular immune response in the brain, proventriculus, and small intestine of 27 cockatiels (Nymphicus hollandicus) experimentally infected with parrot bornavirus 2 (PaBV-2). Perivascular cuffs in the brain were composed of CD3+ T-lymphocytes and Iba1+ macrophages/microglia in most cockatiels (n = 26). In the ganglia of the proventriculus, CD3+ T-lymphocytes (n = 17) and Iba1+ macrophages (n = 13) prevailed. The ganglia of the small intestine had a more homogeneous distribution of these leukocytes, including PAX5+ B-lymphocytes (n = 9), CD3+ T-lymphocytes (n = 8), and Iba1+ macrophages (n = 8). Our results indicate that perivascular cuffs in the brain and the inflammatory infiltrate in the proventriculus of PaBV-2-infected cockatiels is predominately composed of T-lymphocytes, while the inflammatory infiltrates in the ganglia of the small intestine are characterized by a mixed infiltrate composed of T-lymphocytes, B-lymphocytes, and macrophages.

Sendai Virus and a Unified Model of Mononegavirus RNA Synthesis.

Vesicular stomatitis virus (VSV), the founding member of the mononegavirus order (Mononegavirales), was found to be a negative strand RNA virus in the 1960s, and since then the number of such viruses has continually increased with no end in sight. Sendai virus (SeV) was noted soon afterwards due to an outbreak of newborn pneumonitis in Japan whose putative agent was passed in mice, and nowadays this mouse virus is mainly the bane of animal houses and immunologists. However, SeV was important in the study of this class of viruses because, like flu, it grows to high titers in embryonated chicken eggs, facilitating the biochemical characterization of its infection and that of its nucleocapsid, which is very close to that of measles virus (MeV). This review and opinion piece follow SeV as more is known about how various mononegaviruses express their genetic information and carry out their RNA synthesis, and proposes a unified model based on what all MNV have in common.

A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses.

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.

100-My history of bornavirus infections hidden in vertebrate genomes.

Although viruses have threatened our ancestors for millions of years, prehistoric epidemics of viruses are largely unknown. Endogenous bornavirus-like elements (EBLs) are ancient bornavirus sequences derived from the viral messenger RNAs that were reverse transcribed and inserted into animal genomes, most likely by retrotransposons. These elements can be used as molecular fossil records to trace past bornaviral infections. In this study, we systematically identified EBLs in vertebrate genomes and revealed the history of bornavirus infections over nearly 100 My. We confirmed that ancient bornaviral infections have occurred in diverse vertebrate lineages, especially in primate ancestors. Phylogenetic analyses indicated that primate ancestors were infected with various bornaviral lineages during evolution. EBLs in primate genomes formed clades according to their integration ages, suggesting that bornavirus lineages infected with primate ancestors had changed chronologically. However, some bornaviral lineages may have coexisted with primate ancestors and underwent repeated endogenizations for tens of millions of years. Moreover, a bornaviral lineage that coexisted with primate ancestors also endogenized in the genomes of some ancestral bats. The habitats of these bat ancestors have been reported to overlap with the migration route of primate ancestors. These results suggest that long-term virus-host coexistence expanded the geographic distributions of the bornaviral lineage along with primate migration and may have spread their infections to these bat ancestors. Our findings provide insight into the history of bornavirus infections over geological timescales that cannot be deduced from research using extant viruses alone, thus broadening our perspective on virus-host coevolution.

Detection of Immunoreactivity to Psittaciform Bornavirus in the Serum of a Wild Cacatuid in Victoria, Australia.

An indirect immunofluorescence serologic assay, PCR assay, and histopathology were used to screen for psittaciform orthobornaviruses (PaBV) in wild Cacatuidae in Victoria, Australia. Anti-PaBV antibodies were detected, but PCR and histopathology did not detect PaBV. This study presents the first evidence of PaBV in wild birds in Australia.

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

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.