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.

Recombinant Modified Vaccinia Virus Ankara (MVA) Vaccines Efficiently Protect Cockatiels Against Parrot Bornavirus Infection and Proventricular Dilatation Disease.

Parrot bornaviruses (PaBVs) are the causative agents of proventricular dilatation disease (PDD), a chronic and often fatal neurologic disorder in Psittaciformes. The disease is widely distributed in private parrot collections and threatens breeding populations of endangered species. Thus, immunoprophylaxis strategies are urgently needed. In previous studies we demonstrated a prime-boost vaccination regime using modified vaccinia virus Ankara (MVA) and Newcastle disease virus (NDV) constructs expressing the nucleoprotein and phosphoprotein of PaBV-4 (MVA/PaBV-4 and NDV/PaBV-4, respectively) to protect cockatiels (Nymphicus hollandicus) against experimental challenge infection. Here we investigated the protective effect provided by repeated immunization with either MVA/PaBV-4, NDV/PaBV-4 or Orf virus constructs (ORFV/PaBV-4) individually. While MVA/PaBV-4-vaccinated cockatiels were completely protected against subsequent PaBV-2 challenge infection and PDD-associated lesions, the course of the challenge infection in NDV/PaBV-4- or ORFV/PaBV-4-vaccinated birds did not differ from the unvaccinated control group. We further investigated the effect of vaccination on persistently PaBV-4-infected cockatiels. Remarkably, subsequent immunization with MVA/PaBV-4 and NDV/PaBV-4 neither induced obvious immunopathogenesis exacerbating the disease nor reduced viral loads in the infected birds. In summary, we demonstrated that vaccination with MVA/PaBV-4 alone is sufficient to efficiently prevent PaBV-2 challenge infection in cockatiels, providing a suitable vaccine candidate against avian bornavirus infection and bornavirus-induced PDD.

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.

Immunopathology of Fatal Human Variegated Squirrel Bornavirus 1 Encephalitis, Germany, 2011-2013.

Variegated squirrel bornavirus 1 (VSBV-1) is a zoonotic virus that causes fatal encephalitis in humans who are infected after contact with exotic squirrels. We analyzed the brain lesions and the immune responses in all 4 known human cases that showed panencephalitis. Inflammatory infiltrates in areas positive for VSBV-1 RNA and antigen consisted of CD4+ and CD8+ T cells, with perivascular B-cell accumulation. Strong microglial response and bizarre astroglial expansion were present. Areas of malacia contained neutrophils and foamy microglia and macrophages. Immunopathologic examination during infection showed cleavage of caspase 3 in brain cells adjacent to CD8+ cells and widespread p53 expression, hallmarks of apoptosis. Cerebrospinal fluid analyses over time demonstrated increasing protein concentrations and cell counts, paralleled by pathologic lactate elevations in all patients. The most severe cerebrospinal fluid and histologic changes occurred in the patient with the highest viral load, shortest duration of disease, and most medical preconditions.

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.

Viral vector vaccines protect cockatiels from inflammatory lesions after heterologous parrot bornavirus 2 challenge infection.

Avian bornaviruses are causative agents of proventricular dilatation disease (PDD), a chronic neurologic and often fatal disorder of psittacines including endangered species. To date no causative therapy or immunoprophylaxis is available. Our previous work has shown that viral vector vaccines can delay the course of homologous bornavirus challenge infections but failed to protect against PDD when persistent infection was not prevented. The goal of this study was to refine our avian bornavirus vaccination and infection model to better represent natural bornavirus infections in order to achieve full protection against a heterologous challenge infection. We observed that parrot bornavirus 2 (PaBV-2) readily infected cockatiels (Nymphicus hollandicus) by combined intramuscular and subcutaneous injection with as little as 102.7foci-forming units (ffu) per bird, whereas a 500-fold higher dose of the same virus administered via peroral and oculonasal route did not result in persistent infection. These results indicated that experimental bornavirus challenge infections with this virus should be performed via the parenteral route. Prime-boost vaccination of cockatiels with Newcastle disease virus (NDV) and modified vaccinia virus Ankara (MVA) vectors expressing the nucleoprotein and phosphoprotein genes of PaBV-4 substantially blocked bornavirus replication following parenteral challenge infection with 103.5ffu of heterologous PaBV-2. Only two out of six vaccinated birds had very low viral levels detectable in a few organs. As a consequence, only one vaccinated bird developed mild PDD-associated microscopic lesions, while mock-vaccinated controls were not protected against PaBV-2 infection and inflammation. Our results demonstrate that NDV and MVA vector vaccines can protect against invasive heterologous bornavirus challenge infections and subsequent PDD. These vector vaccines represent a promising tool to combat avian bornaviruses in psittacine populations.

Viral vector vaccines expressing nucleoprotein and phosphoprotein genes of avian bornaviruses ameliorate homologous challenge infections in cockatiels and common canaries.

Avian bornaviruses are causative agents of proventricular dilatation disease (PDD), an often fatal disease of parrots and related species (order Psittaciformes) which is widely distributed in captive psittacine populations and may affect endangered species. Here, we established a vaccination strategy employing two different well described viral vectors, namely recombinant Newcastle disease virus (NDV) and modified vaccinia virus Ankara (MVA) that were engineered to express the phosphoprotein and nucleoprotein genes of two avian bornaviruses, parrot bornavirus 4 (PaBV-4) and canary bornavirus 2 (CnBV-2). When combined in a heterologous prime/boost vaccination regime, NDV and MVA vaccine viruses established self-limiting infections and induced a bornavirus-specific humoral immune response in cockatiels (Nymphicus hollandicus) and common canaries (Serinus canaria forma domestica). After challenge infection with a homologous bornavirus, shedding of bornavirus RNA and viral loads in tissue samples were significantly reduced in immunized birds, indicating that vaccination markedly delayed the course of infection. However, cockatiels still developed signs of PDD if the vaccine failed to prevent viral persistence. Our work demonstrates that avian bornavirus infections can be repressed by vaccine-induced immunity. It represents a first crucial step towards a protective vaccination strategy to combat PDD in psittacine birds.

Impact of antigenic diversity on laboratory diagnosis of Avian bornavirus infections in birds.

Avian bornaviruses (ABVs) are a group of genetically diverse viruses within the Bornaviridae family that can infect numerous avian species and represent the causative agents of proventricular dilatation disease, an often fatal disease that is widely distributed in captive populations of parrots and related species. The current study was designed to assess the antigenic variability of the family Bornaviridae and to determine its impact on ABV diagnosis by employing fluorescent antibody assays. It was shown that polyclonal rabbit sera directed against recombinant bornavirus nucleoprotein, X protein, phosphoprotein, and matrix protein provided sufficient cross-reactivity for the detection of viral antigen from a broad range of bornavirus genotypes grown in cell culture. In contrast, a rabbit anti-glycoprotein serum and 2 monoclonal antibodies directed against nucleoprotein and phosphoprotein proteins reacted more specifically. Antibodies were readily detected in sera from avian patients infected with known ABV genotypes if cells persistently infected with a variety of different bornavirus genotypes were used for analysis. For all sera, calculated antibody titers were highest when the homologous or a closely related target virus was used for the assay. Cross-reactivity with more distantly related genotypes of other phylogenetic groups was usually reduced, resulting in titer reduction of up to 3 log units. The presented results contribute to a better understanding of the antigenic diversity of family Bornaviridae and further emphasize the importance of choosing appropriate diagnostic tools for sensitive detection of ABV infections.

Avian bornavirus in free-ranging waterfowl: prevalence of antibodies and cloacal shedding of viral RNA.

We surveyed free-ranging Canada Geese (Branta canadensis), Trumpeter Swans (Cygnus buccinator), Mute Swans (Cygnus olor), and Mallards (Anas platyrhynchos) to estimate the prevalence of antibodies to avian bornavirus (ABV) and of cloacal shedding of ABV RNA in southern Ontario, Canada. Blood samples and cloacal swabs were collected from 206 free-ranging Canada Geese, 135 Trumpeter Swans, 75 Mute Swans, and 208 Mallards at 10 main capture sites between October 2010 and May 2012. Sera were assessed for antibodies against ABV by enzyme-linked immunosorbent assay and swabs were evaluated for ABV RNA using real-time reverse-transcription PCR. Serum antibodies were detected in birds from all four species and at each sampling site. Thirteen percent of the geese caught on the Toronto Zoo site shed ABV RNA in feces compared with 0% in geese sampled at three other locations. The proportions of shedders among Mute Swans, Trumpeter Swans, and Mallards were 9%, 0%, and 0%, respectively. Birds that were shedding viral RNA were more likely to have antibodies against ABV and to have higher antibody levels than those that were not, although many birds with antibodies were not shedding. We confirmed that exposure to, or infection with, ABV is widespread in asymptomatic free-ranging waterfowl in Canada; however, the correlation between cloacal shedding, presence of antibodies, and presence of disease is not fully understood.

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.

Diagnosis of Avian bornavirus infection in psittaciformes by serum antibody detection and reverse transcription polymerase chain reaction assay using feather calami.

Avian bornavirus (ABV) is the causative agent of proventricular dilatation disease (PDD), a highly devastating and contagious disease of psittacines (parrots and parakeets), which has resulted in the death of many captive birds. Accurate diagnosis of bornavirus infection is therefore important for the identification and isolation of infected birds. The current study showed that nonvascular contour (chest) feather calami provide a ready and minimally invasive source of RNA for the detection of ABV by reverse transcription polymerase chain reaction (RT-PCR). Storage of the feathers at room temperature for at least a month did not affect the results. Serological analysis by enzyme-linked immunosorbent assay (ELISA) showed that identification of anti-bornaviral nucleoprotein P40 antibodies can identify many birds with a past or present infection. The presence of anti-avian bornaviral P24 phosphoprotein and P16 matrix protein antibodies was quite variable, rendering these antibodies less useful for diagnosis of ABV infection. The significance of the present findings is that the use of nonvascular feathers as a source of RNA allows sample collection under conditions where storage of other samples would be difficult. Serum detection by ELISA of anti-P40 antibodies allows the identification of infected birds when RT-PCR fails.

Histopathology and the detection of avian bornavirus in the nervous system of birds diagnosed with proventricular dilatation disease.

Avian bornavirus (ABV) is currently considered a probable etiologic agent of proventricular dilatation disease (PDD) of psittacines. We tested 24 stored avian brain samples, processed for histopathology and retained following their submission for necropsy or histopathology to the Schubot Exotic Bird Center diagnostic laboratory in 1992. Thirteen of these samples were from birds diagnosed at that time as suffering from PDD. The remaining 11 samples were diagnosed as suffering from diseases other than PDD. Immunohistochemistry was performed using an antiserum directed against the ABV nucleoprotein (N-protein). Stained slides were read by an investigator unaware of their prior histopathology results. Cells containing ABV N-protein were present in the nervous tissues of all 13 PDD cases. One bird not previously diagnosed with PDD also had ABV N-protein in its brain. A review of this bird's necropsy report indicated that it was, most probably, also suffering from PDD. The remaining 10 non-PDD birds had no detectable N-protein in their brains. The N-protein was present in the cerebrum, cerebellum and spinal cord. These findings support other studies that indicate that ABV is an etiological agent of PDD.

Presence of avian bornavirus RNA and anti-avian bornavirus antibodies in apparently healthy macaws.

Recently a novel avian bornavirus has been described that has been suggested to be the possible etiological agent for proventricular dilatation disease or macaw wasting disease. This article describes two macaws that shed avian bornaviral RNA sequences and demonstrated anti-avian bornavirus antibodies as revealed by reverse transcriptase polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot, yet are free of outward clinical signs of the disease.