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.

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.

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.

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.

Age-dependent development and clinical characteristics of an experimental parrot bornavirus-4 (PaBV-4) infection in cockatiels (Nymphicus hollandicus).

Parrot bornavirus (PaBV) is a pathogen often found in psittacine populations. Infected, clinically healthy carrier birds are of major importance for epidemiology, but the underlying pathomechanism of this carrier status is poorly understood. The age, implying the maturation status of the immune system, at the time of infection might be significant for the clinical outcome. Therefore, two groups of 11 cockatiels of different ages (adult and newly hatched) were inoculated with a PaBV-4 isolate intravenously. The trial lasted for 233 days and all birds were observed for clinical signs, PaBV-RNA shedding and anti-PaBV antibody production. At the end of the trial, histopathology, immunohistochemistry, PCR and virus re-isolation were performed. All 22 birds seroconverted and shed PaBV-RNA during the investigation period; the juvenile group earlier and more homogeneously. Nine of 11 birds of the adult group developed clinical signs; five birds died or had to be euthanized before the end of the study. In the juvenile group none of the birds developed clinical signs and only one bird died due to bacterial septicaemia. Eight birds of the adult group, but none of the juvenile group, showed a dilatation of the proventriculus. PaBV-RNA detection and virus re-isolation were successful in all birds. Immunohistochemically, PaBV antigen was found in all birds. Histopathology revealed mononuclear infiltrations in organs in birds of both groups, but the juveniles were less severely affected in the brain.Thus, PaBV infection at an age with a more naïve immune system makes the production of carrier birds more likely.RESEARCH HIGHLIGHTS PaBV infection at a young age might favour the development of carrier birds.Cockatiels infected at a very young age showed inflammation but no clinical signs.The juvenile group started seroconversion and PaBV-RNA shedding earlier.Seroconversion and PaBV-RNA shedding occurred more homogeneously in the juveniles.

First isolation, in-vivo and genomic characterization of zoonotic variegated squirrel Bornavirus 1 (VSBV-1) isolates.

The variegated squirrel bornavirus 1 (VSBV-1), a member of the family Bornaviridae, was discovered in 2015 in a series of lethal human infections. Screening approaches revealed kept exotic squirrels as the putative source of infection. Infectious virus was successfully isolated by co-cultivation of infected primary squirrel cells with permanent cell lines. For in vivo characterization, neonatal and adult Lewis rats were inoculated either intracranially, intranasally or subcutaneously. After 4.5 months, three out of fifteen neonatal intracranially inoculated rats were VSBV-1 genome positive in the central nervous system without showing clinical signs. Pathohistological examination revealed a non-purulent encephalitis. While infection of immune incompetent rats (neonatal) using the type species of mammalian bornaviruses, the Borna disease virus 1, proceed to an immune tolerant status, VSBV-1 infection could result in inflammation of neuronal tissue. Sequencing showed minor adaptations within the VSBV-1 genome comparing to the viral genomes from infected squirrels, cell cultures or rat tissues. In conclusion, we were able to generate the first VSBV-1 isolates and provide in vivo animal model data in Lewis rats revealing substantial differences between VSBV-1 and BoDV-1. Furthermore, the presented data are a precondition for insights into the transmission and pathogenesis of this novel zoonotic pathogen.

Wounds as the Portal of Entrance for Parrot Bornavirus 4 (PaBV-4) and Retrograde Axonal Transport in Experimentally Infected Cockatiels (Nymphicus hollandicus).

In this study, we investigated the natural route of infection of psittacine bornavirus (PaBV), which is the causative agent of proventricular dilatation disease (PDD) in psittacines. We inoculated two infection groups through wounds with a PaBV-4 isolate. In nine cockatiels (Nymphicus hollandicus) we applied a virus suspension with a titer of 103 50% tissue culture infection dose (TCID50) via palatal lesions (Group P, P1-9). In a second group of three cockatiels, we applied a virus suspension with a titer of 104 TCID50 to footpad lesions (Group F, F1-3). In two cockatiels, the control (or "mock") group, we applied a virus-free cell suspension (Group M, M1-2) via palatal lesions. The observation period was 6 mo (Groups P and M) or 7 mo (Group F). We monitored PaBV-4 RNA shedding and seroconversion. At the end of the study, we examined the birds for the presence of inflammatory lesions, PaBV-4 RNA, and antigen in tissues, as well as virus reisolation of brain and crop material. We did not observe any clinical signs typical of PDD during this study. We also did not see seroconversion or PaBV RNA shedding in any bird during the entire investigation period, and virus reisolation was not successful. We only found PaBV-4 RNA in sciatic nerves, footpad tissue, skin, and in one sample from the intestine of Group F. In this group, the histopathology revealed mononuclear infiltrations mainly in skin and footpad tissue; immunohistochemistry showed positive reactions in spinal ganglia and in the spinal cord, and slightly in skin, footpad tissues, and sciatic nerves. In Groups P and M we found no viral antigen or specific inflammations. In summary, only the virus application on the footpad lesion led to detectable PaBV RNA, mononuclear infiltrations, and positive immunohistochemical reactions in tissues of the experimental birds. This could suggest that PaBV spreads via nervous tissue, with skin wounds as the primary entry route.

Parrot bornavirus in naturally infected Brazilian captive parrots: Challenges in viral spread control.

Psittaciform orthobornaviruses are currently considered to be a major threat to the psittacine bird population worldwide. Parrot bornavirus (PaBV) was identified recently in Brazil and, since then, few studies have been conducted to understand the epidemiology of PaBV in captive psittacine birds. In the present study, natural infections by PaBV in South American parrots were investigated in two breeding facilities: commercial (A) and conservationist (B). Thirty-eight psittacine of 21 different species were presented for postmortem examination. Tissue samples were collected and investigated for the presence of PaBV-RNA using RT-PCR. In addition, clinical information about these birds was used when available. PaBV infection was detected in 73.7% of all birds investigated, indicating a wide dissemination of this virus in both facilities. From birds investigated in aviary A, 66.7% showed clinical signs, 100% had typical lesions of proventricular dilatation disease (PDD), 100% had mild to severe proventricular dilatation and 88.9% were PaBV-positive. In birds from aviary B, 27.6% showed clinical signs, 65.5% had typical lesions of PDD, 62% had mild to severe proventricular dilatation and 69% were PaBV-positive. Neurological disease was observed more frequently than gastrointestinal disease. Sequencing analysis of the matrix gene fragment revealed the occurrence of genotype 4 (PaBV-4) in both places. About 15.8% of birds in this study are threatened species. We discussed the difficulties and challenges for controlling viral spread in these aviaries and implications for South American psittacine conservation. These results emphasize the urgent need to develop a national regulatory and health standard for breeding psittacine birds in the country.

Monitoring of free-ranging and captive Psittacula populations in Western Europe for avian bornaviruses, circoviruses and polyomaviruses.

Avian pathogens such as bornaviruses, circoviruses and polyomaviruses are widely distributed in captive collections of psittacine birds worldwide and can cause fatal diseases. In contrast, only little is known about their presence in free-ranging psittacines and their impact on these populations. Rose-ringed parakeets (Psittacula krameri) and Alexandrine parakeets (Psittacula eupatria) are non-native to Europe, but have established stable populations in parts of Western Europe. From 2012-2017, we surveyed free-ranging populations in Germany and France as well as captive Psittacula individuals from Germany and Spain for avian bornavirus, circovirus and polyomavirus infections. Samples from two out of 469 tested free-ranging birds (0.4%; 95% confidence interval [CI-95]: 0.1-1.5%) were positive for beak and feather disease virus (BeFDV), whereas avian bornaviruses and polyomaviruses were not detected in the free-ranging populations. In contrast, avian bornaviruses and polyomaviruses, but not circoviruses were detected in captive populations. Parrot bornavirus 4 (PaBV-4) infection was detected by RT-PCR in four out of 210 captive parakeets (1.9%; CI-95: 0.7-4.8%) from four different holdings in Germany and Spain and confirmed by detection of bornavirus-reactive antibodies in two of these birds. Three out of 160 tested birds (1.9%; CI-95: 0.5-5.4%) possessed serum antibodies directed against budgerigar fledgling disease virus (BuFDV). PaBV-4 and BuFDV were also detected in several psittacines of a mixed holding in Germany, which had been in contact with free-ranging parakeets. Our results demonstrate that Psittacula parakeets are susceptible to common psittacine pathogens and their populations in Western Europe are exposed to these viruses. Nevertheless, the prevalence of avian bornaviruses, circoviruses and polyomaviruses in those populations is very low.RESEARCH HIGHLIGHTS Psittacula parakeets are susceptible to bornavirus, circovirus and polyomavirus infection.Introduced Psittacula populations in Europe have been exposed to these viruses.Nevertheless, they may be absent or present at only low levels in free-ranging Psittacula populations.Free-ranging populations in Europe pose a minor threat of transmitting these viruses to captive Psittaciformes.

Investigation of astrovirus and bornavirus in the cerebrospinal fluid of dogs clinically diagnosed with meningoencephalitis of unknown etiology.

BACKGROUND: Non-suppurative encephalitides in a variety of species, including humans and dogs, have been linked to infection by astroviruses and bornaviruses. HYPOTHESIS/OBJECTIVES: To determine whether or not ribonucleic acid of astroviruses or bornaviruses was present in the cerebrospinal fluid (CSF) of dogs with clinically diagnosed meningoencephalomyelitis of unknown etiology (MUE). ANIMALS: Twenty-five client-owned dogs evaluated by CSF analysis at a single university referral hospital. METHODS: Prospective case-control study. Cerebrospinal fluid was collected from clinically diagnosed MUE and control cases and evaluated by reverse-transcriptase polymerase chain reaction for the presence of astrovirus and bornavirus. RESULTS: Neither astrovirus nor bornavirus nucleic acids were identified in CSF collected from 20 clinically diagnosed MUE and 5 control cases. CONCLUSIONS AND CLINICAL IMPORTANCE: The negative results of this investigation suggest that astrovirus and bornavirus are not commonly detectable in CSF of dogs with MUE.

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.

Detection of Avian Bornavirus in Wild and Captive Passeriformes in Brazil.

Avian bornaviruses (ABVs) are the causative agents of proventricular dilatation disease (PDD), a fatal neurologic disease considered to be a major threat to psittacine bird populations. We performed a reverse transcription PCR survey to detect the presence of canary avian bornavirus (CnBV) in birds of order Passeriformes related to different clinical manifestations, such as sudden death, neurologic signs, apathy, anorexia, excessive beak growth, and PDD. A total of 227 samples from captive and wild canaries were included, of which 80 samples were captive birds, comprising saffron finches (n = 71) and common canary (n = 9), and 147 samples were wild birds distributed among a variety of several species. Two samples from captive birds (2/80) were positive for ABV, and in wild birds, only one sample was positive for ABV. The positive samples were subjected to DNA sequencing, and only the CnBV-1 serotype was found, which was the first time it was detected outside of Germany (Austria/Hungary), where it was first detected in 2009. Phylogenetic analysis confirmed that avian bornavirus serotype CnBV-1 is present in order Passeriformes in Brazil.

Detección de bornavirus aviar en aves paseriformes silvestres y en cautiverio en Brasil. Los bornavirus aviares (ABV, por sus siglas en inglés) son los agentes causantes de la enfermedad de la dilatación proventricular (PDD), una enfermedad neurológica mortal considerada como una de las principales amenazas para las poblaciones de aves psitácidas. Se realizó un muestreo por transcrpción reversa y PCR para detectar la presencia de bornavirus de los canarios (CnBV) en aves de orden Passeriformes relacionadas con diferentes manifestaciones clínicas, como muerte súbita, signos neurológicos, apatía, anorexia, crecimiento excesivo del pico y enfermedad de dilatación proventricular. Se incluyeron un total de 227 muestras de canarios en cautividad y silvestres, de las cuales 80 muestras fueron de aves en cautiverio, incluyendo jilgueros dorados (n =71) y canarios comunes (n = 9) y 147 muestras fueron aves silvestres distribuidas entre una variedad de especies. Dos muestras de aves cautivas (2/80) fueron positivas para bornavirus aviar; en aves silvestres, solo una muestra fue positiva para bornavirus aviar. Las muestras positivas se sometieron a secuenciación de ADN y solo se encontró el bornavirus de canarios serotipo 1, que es la primera vez que se detecta fuera de Alemania (Austria/Hungría), donde se detectó por primera vez en el año 2009. El análisis filogenético confirmó que el bornavirus de canarios serotipo 1 está presente en el orden Passeriformes en Brasil.

Analysis of exotic squirrel trade and detection of human infections with variegated squirrel bornavirus 1, Germany, 2005 to 2018.

Following the discovery in 2015 of the variegated squirrel bornavirus 1 (VSBV-1) in fatal encephalitis cases among exotic squirrel breeders and a zoo animal caretaker in Germany, a case definition was developed. It was employed during trace-back animal trade investigations and sero-epidemiological studies among breeders and zoo animal caretakers of holdings with VSBV-1 infected squirrels. During the investigation, two possible human cases who had died of encephalitis were identified retrospectively among the squirrel breeders. Moreover, one probable human case was detected among the breeders who had a positive memory T-cell response to VSBV-1 antigen and antibodies against VSBV-1. The low rate of seropositivity found among living persons in risk groups that handle exotic squirrels privately or at zoos may reflect rareness of exposure to VSBV-1 during animal contact, a high lethality of infection or a combination of these factors. As a precaution against human exposure, testing of exotic squirrels for VSBV-1 infection and/or avoiding direct contact with exotic squirrels in zoos and private holdings is strongly advised.

Correlation of avian bornavirus-specific antibodies and viral ribonucleic acid shedding with neurological signs and feather-damaging behaviour in psittacine birds.

Parrot bornaviruses (PaBV) are the causative agents of proventricular dilatation disease in psittacine birds, but have also been linked to other clinical signs, including behavioural disorders and neurological signs. The aim of this study was to correlate PaBV infection in birds showing feather-damaging behaviour or neurological signs for which no other cause of disease could be identified. Psittacine birds presented to a private practice were divided into three groups: birds with neurological signs (n=28), birds showing feather-damaging behaviour (n=42) and birds presented for routine examinations (n=56). Swabs of crop and cloaca were collected and investigated for the presence of PaBV-RNA using real time RT-PCR. Additionally, serum samples were taken and examined for the presence of anti-PaBV antibodies by immunofluorescence test. PaBV infection was detected in one of the test systems in 40.5 per cent of all birds (n=126) investigated. In the clinically healthy birds (n=56), 19.6 per cent of the birds were positive in at least one of the PaBV tests, compared with 52.38 per cent of the feather-damaging (n=42) and 64.28 per cent of the neurologically diseased birds (n=28). Interestingly, the anti-PaBV antibody titres in birds with neurological signs were highest up to 1:20 480. High antibody titres (up to 1:5120) were also found in the feather-damaging group, whereas the birds of the control group, if PaBV positive, had only very low titres. Similarly, the highest viral load was found in the group of the neurologically diseased birds, followed by feather-damaging birds, whereas PaBV-positive birds in the control group demonstrated only low viral RNA shedding. A clear correlation between severity of clinical signs, amount of viral shedding and high levels of antibody titres was observed for most of the neurologically diseased birds and also for few birds with feather-damaging behaviour. For the first time, these results clearly indicate a correlation between PaBV infection and neurological signs in birds without gastrointestinal signs presented to the veterinarian in practice. It also may demonstrate a possible correlation with feather-damaging behaviour and anti-PaBV antibody presence. The antibody titre seems to represent a diagnostic tool to correlate clinical signs to PaBV as a cause.

Distribution of Viral Antigen and Inflammatory Lesions in the Central Nervous System of Cockatiels ( Nymphicus hollandicus) Experimentally Infected with Parrot Bornavirus 2.

Neurotropism is a striking characteristic of bornaviruses, including parrot bornavirus 2 (PaBV-2). Our study evaluated the distribution of inflammatory foci and viral nucleoprotein (N) antigen in the brain and spinal cord of 27 cockatiels ( Nymphicus hollandicus) following experimental infection with PaBV-2 by injection into the pectoral muscle. Tissue samples were taken at 12 timepoints between 5 and 114 days post-inoculation (dpi). Each experimental group had approximately 3 cockatiels per group and usually 1 negative control. Immunolabeling was first observed within the ventral horns of the thoracic spinal cord at 20 dpi and in the brain (thalamic nuclei and hindbrain) at 25 dpi. Both inflammation and viral antigen were restricted to the central core of the brain until 40 dpi. The virus then spread quickly at 60 dpi to both gray and white matter of all analyzed sections of the central nervous system (CNS). Encephalitis was most severe in the thalamus and hindbrain, while myelitis was most prominent in the gray matter and equally distributed in the cervical, thoracic, and lumbosacral spinal cord. Our results demonstrate a caudal to rostral spread of virus in the CNS following experimental inoculation of PABV-2 into the pectoral muscle, with the presence of viral antigen and inflammatory lesions first in the spinal cord and progressing to the brain.

Occupation-Associated Fatal Limbic Encephalitis Caused by Variegated Squirrel Bornavirus 1, Germany, 2013.

Limbic encephalitis is commonly regarded as an autoimmune-mediated disease. However, after the recent detection of zoonotic variegated squirrel bornavirus 1 in a Prevost's squirrel (Callosciurus prevostii) in a zoo in northern Germany, we retrospectively investigated a fatal case in an autoantibody-seronegative animal caretaker who had worked at that zoo. The virus had been discovered in 2015 as the cause of a cluster of cases of fatal encephalitis among breeders of variegated squirrels (Sciurus variegatoides) in eastern Germany. Molecular assays and immunohistochemistry detected a limbic distribution of the virus in brain tissue of the animal caretaker. Phylogenetic analyses demonstrated a spillover infection from the Prevost's squirrel. Antibodies against bornaviruses were detected in the patient's cerebrospinal fluid by immunofluorescence and newly developed ELISAs and immunoblot. The putative antigenic epitope was identified on the viral nucleoprotein. Other zoo workers were not infected; however, avoidance of direct contact with exotic squirrels and screening of squirrels are recommended.