In Vitro and In Ovo Host Restriction of Aquatic Bird Bornavirus 1 in Different Avian Hosts.

Aquatic bird bornavirus 1 (ABBV-1) is associated with chronic meningoencephalitis and ganglioneuritis. Although waterfowl species act as the natural host of ABBV-1, the virus has been sporadically isolated from other avian species, showing the potential for a broad host range. To evaluate the host restriction of ABBV-1, and its potential to infect commercial poultry species, we assessed the ability of ABBV-1 to replicate in cells and embryos of different avian species. ABBV-1 replication was measured using multi- and single-step growth curves in primary embryo fibroblasts of chicken, duck, and goose. Embryonated chicken and duck eggs were infected through either the yolk sac or chorioallantoic cavity, and virus replication was assessed by immunohistochemistry and RT-qPCR in embryonic tissues harvested at two time points after infection. Multi-step growth curves showed that ABBV-1 replicated and spread in goose and duck embryo fibroblasts, establishing a population of persistently infected cells, while it was unable to do so in chicken fibroblasts. Single-step growth curves showed that cells from all three species could be infected; however, persistence was only established in goose and duck fibroblasts. In ovo inoculation yielded no detectable viral replication or lesion in tissues. Data indicate that although chicken, duck, and goose embryo fibroblasts can be infected with ABBV-1, a persistent infection is more easily established in duck and goose cells. Therefore, ABBV-1 may be able to infect chickens in vivo, albeit inefficiently. Additionally, our data indicate that an in ovo model is inadequate to investigating ABBV-1 host restriction and pathogenesis.

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.

Viral Equine Encephalitis, a Growing Threat to the Horse Population in Europe?

Neurological disorders represent an important sanitary and economic threat for the equine industry worldwide. Among nervous diseases, viral encephalitis is of growing concern, due to the emergence of arboviruses and to the high contagiosity of herpesvirus-infected horses. The nature, severity and duration of the clinical signs could be different depending on the etiological agent and its virulence. However, definite diagnosis generally requires the implementation of combinations of direct and/or indirect screening assays in specialized laboratories. The equine practitioner, involved in a mission of prevention and surveillance, plays an important role in the clinical diagnosis of viral encephalitis. The general management of the horse is essentially supportive, focused on controlling pain and inflammation within the central nervous system, preventing injuries and providing supportive care. Despite its high medical relevance and economic impact in the equine industry, vaccines are not always available and there is no specific antiviral therapy. In this review, the major virological, clinical and epidemiological features of the main neuropathogenic viruses inducing encephalitis in equids in Europe, including rabies virus (Rhabdoviridae), Equid herpesviruses (Herpesviridae), Borna disease virus (Bornaviridae) and West Nile virus (Flaviviridae), as well as exotic viruses, will be presented.

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.

Studies on immunity and immunopathogenesis of parrot bornaviral disease in cockatiels.

We have demonstrated that vaccination of cockatiels (Nymphicus hollandicus) with killed parrot bornavirus (PaBV) plus recombinant PaBV-4 nucleoprotein (N) in alum was protective against disease in birds challenged with a virulent bornavirus isolate (PaBV-2). Unvaccinated birds, as well as birds vaccinated after challenge, developed gross and histologic lesions typical of proventricular dilatation disease (PDD). There was no evidence that vaccination either before or after challenge made the infection more severe. Birds vaccinated prior to challenge largely remained free of disease, despite the persistence of the virus in many organs. Similar results were obtained when recombinant N, in alum, was used for vaccination. In some rodent models, Borna disease is immune mediated thus we did an additional study whereby cyclosporine A was administered to unvaccinated birds starting 1day prior to challenge. This treatment also conferred complete protection from disease, but not infection.

Parrot Bornavirus (PaBV)-2 isolate causes different disease patterns in cockatiels than PaBV-4.

Psittaciform 1 bornavirus (PaBV) has already been shown to be the aetiologic agent of proventricular dilatation disease, a significant disease of birds. However, the pathogenesis of PaBV infection has not yet been resolved and valid data regarding the pathogenicity of different PaBV species are lacking. Thus, the present study was aimed to characterize the influence of two different PaBV species on the course of disease. Eighteen cockatiels were inoculated intracerebrally (i.c.) or intravenously (i.v.) with a PaBV-2 isolate under the same conditions as in a previous study using PaBV-4. Birds were surveyed and sampled for 33 weeks to analyse the course of infection and disease in comparison to that of PaBV-4. Similar to PaBV-4, PaBV-2 induced a persistent infection with seroconversion (from day 6 p.i. onwards) and shedding of viral RNA (from day 27 p.i. onwards). However, in contrast to PaBV-4, more birds displayed clinical signs and disease progression was more severe. After PaBV-2 infection, 12 birds exhibited clinical signs and 10 birds revealed a dilated proventriculus in necropsy. After PaBV-4 infection only four birds revealed clinical signs and seven birds showed a dilatation of the proventriculus. Clinically, different courses of disease were observed after PaBV-2 infection, mainly affecting the gastrointestinal tract. This had not been detected after PaBV-4 infection where more neurological signs were noted. The results provide evidence for different disease patterns according to different PaBV species, allowing the comparison between the infection with two PaBV species, and thus underlining the role of viral and individual host factors for disease outcome.

Aquatic Bird Bornavirus 1 in Wild Geese, Denmark.

To investigate aquatic bird bornavirus 1 in Europe, we examined 333 brains from hunter-killed geese in Denmark in 2014. Seven samples were positive by reverse transcription PCR and were 98.2%-99.8% identical; they were also 97.4%-98.1% identical to reference strains of aquatic bird bornavirus 1 from geese in North America.

No contact transmission of avian bornavirus in experimentally infected cockatiels (Nymphicus hollandicus) and domestic canaries (Serinus canaria forma domestica).

Avian bornaviruses (ABV) are the causative agents of proventricular dilatation disease (PDD), a widely distributed disease of parrots. Distinct ABV lineages were also found in various non-psittacine avian species, such as canaries, but the pathogenic role of ABV in these species is less clear. Despite the wide distribution of ABV in captive parrots and canaries, its mode of transmission is poorly understood: both horizontal transmission via the urofaecal-oral route and vertical transmission are discussed to play a role. In this study we investigated pathology and horizontal transmission of ABV in domestic canaries (Serinus canaria forma domestica) and cockatiels (Nymphicus hollandicus), two natural host species commonly used for experimental ABV infections. ABV inoculation resulted in persistent infection of all inoculated animals from both species. ABV-infected cockatiels exhibited PDD-like symptoms, such as neurologic signs or shedding of undigested seeds. In contrast, infected domestic canaries did not develop clinical disease. Interestingly, we did not detect viral RNA in cloacal swabs and organ samples or ABV-specific antibodies in serum samples of contact-exposed sentinel birds from either species at any time during a four months observation period. Our results strongly indicate that horizontal transmission of ABV by direct contact is inefficient in immunocompetent fully fledged domestic canaries and cockatiels.

Avian bornavirus and proventricular dilatation disease: diagnostics, pathology, prevalence, and control.

Avian bornavirus (ABV) has been shown the cause of proventricular dilatation disease (PDD) in psittacines. Many healthy birds are infected with ABV, and the development of PDD in such cases is unpredictable. As a result, the detection of ABV in a sick bird is not confirmation that it is suffering from PDD. Treatment studies are in their infancy. ABV is not restricted to psittacines. It has been found to cause PDD-like disease in canaries. It is also present at a high prevalence in North American geese, swans, and ducks. It is not believed that these waterfowl genotypes can cause disease in psittacines.

The X proteins of bornaviruses interfere with type I interferon signalling.

Borna disease virus (BDV) is a neurotropic, negative-stranded RNA virus causing persistent infection and progressive neurological disorders in a wide range of warm-blooded animals. The role of the small non-structural X protein in viral pathogenesis is not completely understood. Here we investigated whether the X protein of BDV and avian bornavirus (ABV) interferes with the type I interferon (IFN) system, similar to other non-structural proteins of negative-stranded RNA viruses. In luciferase reporter assays, we found that the X protein of various bornaviruses interfered with the type I IFN system at all checkpoints investigated, in contrast to previously reported findings, resulting in reduced type I IFN secretion.

[Bornaviruses].

Bornaviridae is an enveloped animal virus carrying an 8.9 kb non-segmented, negative-strand RNA genome. The genus bornavirus contains two members infecting vertebrates, Borna disease virus (BDV) and avian bornavirus (ABV), which could preferably infect the nervous systems. BDV causes classical Borna disease, a progressive meningoencephalomyelitis, in horses and sheep, and ABV is known to induce proventricular dilatation disease, a fatal disease characterized by a lymphocytic, plasmacytic inflammation of central and peripheral nervous tissues, in multiple avian species. Recent evidences have demonstrated that bornavirus is unique among RNA viruses as they not only establish a long-lasting, persistent infection in the nucleus, but also integrate their own DNA genome copy into the host chromosome. In this review, I outline the recent knowledge about the unique virological characteristics of bornaviruses, as well as the diseases caused by the infection of BDV and ABV.

Unusual and severe lesions of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) acting as healthy carriers of avian bornavirus (ABV) and subsequently infected with a virulent strain of ABV.

A flock of 14 apparently healthy cockatiels, purchased from a single aviary, was tested for the presence of avian bornavirus (ABV). Twelve birds were found to be intermittently shedding ABV, predominantly genotype 4. Four of the cockatiels known to be shedding ABV4 were subsequently challenged with the tissue culture derived, virulent M24 strain of ABV4. The challenged birds remained in apparent good health until day 92 when one was found dead. The remaining three birds began to exhibit severe neurologic signs, ataxia and convulsions on day 110 and were euthanized. On necropsy, all four birds showed mild proventricular enlargement. In contrast, histopathological examination showed unusually severe and widespread tissue lesions. These included massive lymphocytic infiltration and lymphoid nodule formation within and around the ganglia throughout the gastrointestinal tract. There were similar lesions in the medullary cords of the adrenal gland, heart, spleen, liver, kidney, lungs, pancreas, testes and ovary. Immunohistochemistry demonstrated ABV P antigen not only in the cells of the central and autonomic nervous systems, but also within the mononuclear cells infiltrating the various organs. Two healthy cockatiels, one of which was a known ABV carrier, were inoculated with uninfected tissue culture cells and euthanized on day 150. These birds showed no gross lesions of proventricular dilatation disease but had a mild lymphocytic infiltration in their liver, spleen, and kidneys. Prior infection with ABV did not therefore confer significant immunity on these birds, and may have resulted in increased disease severity following challenge.

Achalasia and viral infection: new insights from veterinary medicine.

Achalasia is a serious disorder in which the movement of food and liquids through the esophagus is impaired. It is currently thought to be caused by an inflammatory process that destroys neurons in myenteric ganglia, which affect peristalsis in the esophagus. The factor(s) that precipitate this inflammatory process are unknown; possibilities include environmental agents (such as microbes or toxins) and/or cell-mediated autoimmune reactivity. Recently, infection with a newly described bornavirus has been strongly linked to a disease of exotic birds that displays many striking similarities to achalasia. These findings demonstrate that viruses can induce achalasia-like pathophysiology and have renewed interest in the search for infectious agents in this enigmatic human disease.

Use of avian bornavirus isolates to induce proventricular dilatation disease in conures.

Avian bornavirus (ABV) is a newly discovered member of the family Bornaviridae that has been associated with the development of a lethal neurologic syndrome in birds, termed proventricular dilatation disease (PDD). We successfully isolated and characterized ABV from the brains of 8 birds with confirmed PDD. One isolate was passed 6 times in duck embryo fibroblasts, and the infected cells were then injected intramuscularly into 2 healthy Patagonian conures (Cyanoliseus patagonis). Clinical PDD developed in both birds by 66 days postinfection. PDD was confirmed by necropsy and histopathologic examination. Reverse transcription-PCR showed that the inoculated ABV was in the brains of the 2 infected birds. A control bird that received uninfected tissue culture cells remained healthy until it was euthanized at 77 days. Necropsy and histopathologic examinations showed no abnormalities; PCR did not indicate ABV in its brain tissues.

Analysis of naturally occurring avian bornavirus infection and transmission during an outbreak of proventricular dilatation disease among captive psittacine birds.

A proventricular dilatation disease (PDD) outbreak provided the opportunity to investigate the transmissibility of avian Bornavirus (ABV) and its linkage to PDD under natural conditions. Upon exposure to a bird with a fatal case of PDD, 10 birds became symptomatic and died. ABV2 RNA was recovered from available tissues. Further screening revealed that 12/46 exposed birds were ABV2(+). Three chicks boarded at this aviary developed PDD. They harbored the same ABV2 isolate and transmitted it to five of eight chicks in their home aviary. These findings demonstrate that ABV infection precedes the development of PDD. ABV-specific Western blotting and reverse transcription-PCR indicate that ABV2 is not strictly neurotropic.

Avian bornaviruses in psittacine birds from Europe and Australia with proventricular dilatation disease.

To determine whether avian bornaviruses (ABVs) were a factor in proventricular dilatation disease (PDD), we used immunohistochemistry, reverse transcription-PCR, and nucleotide sequence analysis to examine paraffin wax-embedded or frozen tissue samples of 31 psittacine birds with this disease. PDD is a fatal disease of psittacine birds associated with nonsuppurative encephalitis and ganglioneuritis of the upper intestinal tract. Tissue samples had been collected from 1999 through 2008 in Austria, Switzerland, Hungary, and Australia. Immunohistochemical demonstration of viral antigen within the brain and vegetative nerve system of the gastrointestinal tract provides strong evidence for a causative role of ABVs in this condition. Partial sequences of nucleoprotein (p40) and matrix protein (gp18) genes showed that virus in most of our cases belonged to the ABV-2 and ABV-4 groups among the 5 genogroups described so far. Viral sequences of 2 birds did not match any of the described sequences and clustered together in a new branch termed ABV-6.

Novel avian bornavirus in a nonpsittacine species (Canary; Serinus canaria) with enteric ganglioneuritis and encephalitis.

A canary bird (Serinus canaria) died with nonsuppurative ganglioneuritis of the proventriculus and gizzard and encephalitis, lesions comparable to proventricular dilatation disease (PDD) of psittacine birds. Recently, several genotypes of a novel avian bornavirus have been linked to PDD. In the canary, bornaviral antigen was detected by immunohistochemistry in both neural and extraneural tissues. The widespread viral dissemination was confirmed by reverse transcription-PCR. Sequence analysis revealed a unique genotype of avian bornavirus. This observation suggests that bornaviruses are natural pathogens of several avian species and that the family Bornaviridae comprises more viral genotypes (or viral species) than previously assumed.

Experimental induction of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) inoculated with brain homogenates containing avian bornavirus 4.

BACKGROUND: Proventricular dilatation disease (PDD) is a fatal disorder of psittacine birds worldwide. The disease is characterized by lymphoplasmacytic infiltration of the central and peripheral nervous systems, leading to gastrointestinal motility and/or central nervous system dysfunction. Recently, we detected a significant association between avian bornavirus (ABV) infection and clinical signs of PDD in psittacines. However, it remains unclear whether ABV infection actually causes PDD. To address this question, we examined the impact of ABV inoculation on the cockatiel (Nymphicus hollandicus). RESULTS: Five cockatiels were inoculated via multiple routes (intramuscular, intraocular, intranasal, and oral) with a brain homogenate derived from either a PDD(+) avian bornavirus 4 (ABV4) (+) case (n = 3 inoculees) or from a PDD(-) ABV(-) control (n = 2 inoculees). The control birds remained free of clinical or pathological signs of PDD, and tested ABV(-) by RT-PCR and immunohistochemistry (IHC). In contrast, all three cockatiels inoculated with ABV4(+) brain homogenate developed gross and microscopic PDD lesions, and two exhibited overt clinical signs. In numerous tissues, ABV RT-PCR and sequence analysis demonstrated the presence of ABV4 RNA nearly identical to that in the inoculum. ABV was detected in the central nervous system of the three ABV-inoculees by IHC. Pyrosequencing to investigate the viral flora in the ABV4(+) inoculum uncovered 7 unique reads sharing 73-100% nucleotide sequence identity with previously identified ABV sequences and 24 reads sharing 40-89% amino acid sequence identity with viruses in the Retroviridae and Astroviridae families. Of these candidate viral species, only ABV RNA was recovered from tissues of the inoculated birds. CONCLUSION: In this study, the clinical and pathological manifestations of PDD were induced by inoculation of cockatiels with brain homogenates containing avian bornavirus 4. By using high throughput pyrosequencing an in-depth view of the viral content of the inoculum was achieved, revealing that of 3 candidate virus families detected, only the presence of ABV RNA correlated with the development of PDD. This study provides evidence of a causal association between ABV4 infection and PDD in cockatiels.

Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.

BACKGROUND: Proventricular dilatation disease (PDD) is a fatal disorder threatening domesticated and wild psittacine birds worldwide. It is characterized by lymphoplasmacytic infiltration of the ganglia of the central and peripheral nervous system, leading to central nervous system disorders as well as disordered enteric motility and associated wasting. For almost 40 years, a viral etiology for PDD has been suspected, but to date no candidate etiologic agent has been reproducibly linked to the disease. RESULTS: Analysis of 2 PDD case-control series collected independently on different continents using a pan-viral microarray revealed a bornavirus hybridization signature in 62.5% of the PDD cases (5/8) and none of the controls (0/8). Ultra high throughput sequencing was utilized to recover the complete viral genome sequence from one of the virus-positive PDD cases. This revealed a bornavirus-like genome organization for this agent with a high degree of sequence divergence from all prior bornavirus isolates. We propose the name avian bornavirus (ABV) for this agent. Further specific ABV PCR analysis of an additional set of independently collected PDD cases and controls yielded a significant difference in ABV detection rate among PDD cases (71%, n = 7) compared to controls (0%, n = 14) (P = 0.01; Fisher's Exact Test). Partial sequence analysis of a total of 16 ABV isolates we have now recovered from these and an additional set of cases reveals at least 5 distinct ABV genetic subgroups. CONCLUSION: These studies clearly demonstrate the existence of an avian reservoir of remarkably diverse bornaviruses and provide a compelling candidate in the search for an etiologic agent of PDD.

Cortical cholinergic decline parallels the progression of Borna virus encephalitis.

Borna disease virus (BDV)-induced meningoencephalitis is associated with the dysfunction of the cholinergic system. Temporal development of this cholinergic decline during pre-encephalitic and encephalitic stages of BDV infection remains however elusive. Changes in choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were therefore determined in the cerebral cortex, hippocampus, striatum, amygdala and cholinergic basal forebrain nuclei (ChBFN) of rats infected with BDV. Immunocytochemistry for ChAT and vesicular acetylcholine transporter (VAChT) was employed to identify morphological consequences of BDV infection on cholinergic neurons. Whereas both ChAT and AChE activities changed only slightly under pre-encephalitic conditions, the encephalitic stage was characterized by a significant decrease of ChAT activity in the cerebral cortex, horizontal diagonal band of Broca (hDBB), hippocampus and amygdala concomitant with a marked reduction of AChE activity in the cerebral cortex, hDBB and hippocampus. The striatum and medial septum remained unaffected. ChAT and VAChT immunocytochemistry revealed prominent axonal degeneration in affected cortical and limbic projection areas of ChBFN. In summary, our data indicate progressive deterioration of forebrain cholinergic systems that parallels the progression of BDV encephalitis.