Avian Bornavirus RNA Research Articles

Avian Bornaviruses in North American Gulls

Avian bornaviruses, recently described members of the family Bornaviridae, have been isolated from captive parrots and passerines as well as wild waterfowl in which they may cause lethal neurologic disease. We report detection of avian bornavirus RNA in the brains of apparently healthy gulls. We tested 439 gull brain samples from 18 states, primarily in the northeastern US, using a reverse-transcriptase PCR assay with primers designed to detect a conserved region of the bornavirus M gene. Nine birds yielded a PCR product of appropriate size. Sequencing of PCR products indicated that the virus was closely related to aquatic bird bornavirus 1 (ABBV-1). Viral RNA was detected in Herring Gulls (Larus argentatus), Ring-billed Gulls (Larus delawarensis), and Laughing Gulls (Leucophaeus atricilla). Eight of the nine positive birds came from the New York/New Jersey area. One positive Herring Gull came from New Hampshire. Histopathologic examination of one well-preserved brain from a Herring Gull from Union County New Jersey, showed a lymphocytic encephalitis similar to that observed in bornavirus-infected parrots and geese. Bornavirus N protein was confirmed in two Herring Gull brains by immunohistochemistry. Thus ABBV-1 can infect gulls and cause encephalitic brain lesions similar to those observed in other birds.

Investigation into the possibility of vertical transmission of avian bornavirus in free-ranging Canada geese (Branta canadensis)

To investigate the possibility of in ovo infection with avian bornavirus (ABV) in wild Canada geese (Branta canadensis), 53 eggs were opportunistically collected at various stages of embryonic development from 16 free-ranging goose nests at a large urban zoo site where ABV infection is known to be present in this species. ABV RNA was detected in the yolk of one of three unembryonated eggs using real-time reverse transcription polymerase chain reaction. ABV RNA was not identified in the brains from 23 newly hatched goslings or 19 embryos, nor from three early whole embryos. Antibodies against ABV were not detected in the plasma of any of the hatched goslings using an enzyme-linked immunosorbent assay. Possible reasons for the failure to detect ABV RNA in hatchlings or embryos include low sample size, eggs deriving from parents not actively infected with ABV, the testing of only brain tissue, and failure of the virus to replicate in Canada goose embryos. In conclusion, this preliminary investigation demonstrating the presence of ABV RNA in the yolk of a Canada goose egg provides the first evidence for the potential for vertical transmission of ABV in waterfowl.

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.

Vertical Transmission of Avian Bornavirus in Psittaciformes: Avian Bornavirus RNA and Anti-Avian Bornavirus Antibodies in Eggs, Embryos, and Hatchlings Obtained from Infected Sun Conures (Aratinga solstitialis)

Fertilized eggs were obtained from four pairs of sun conures (Aratinga solstitialis) infected with avian bornavirus (ABV) genotype 2, as determined by the sequence of the P24 gene. ABV RNA could be detected in early embryos of all four pairs. ABV RNA also was detected in brain, liver, and eyes of late-stage embryos of one of the pairs (Pair 4) and in blood of a 2-wk-old hatchling of this pair, demonstrating that vertical transmission can occur. ABV RNA could be detected in the liver but not in the brain or eyes of the late-stage embryos of another pair (Pair 3). Although it could be detected in the undeveloped eggs of the female parent and 8-day-old embryos, bornaviral RNA could not be found in the brain and liver of the late-stage embryos or in feathers and blood of young (5-9-wk-old) hatchlings of a third pair (Pair 2). At 11 wk, ABV RNA could be detected again in feathers and blood of these hatchlings and in the brain of one of the hatchlings of Pair 2 that suddenly died. ABV RNA could however be detected in throat swabs of the 5- and 9-wk-old hatchlings and their parents (Pair 2). Although the continued presence of ABV RNA in feathers and blood below the detection level of the reverse transcription-PCR used cannot be excluded, this result also may be attributable to feeding by the infected parents. Analysis by enzyme-linked immunosorbent assay showed that egg yolks and serum of late-stage embryos contain variable amounts of non-neutralizing anti-ABV-P40, -P10, -P24, and -P16 antibodies, the ratio of which reflected the antibody ratio in the serum of the female parent. Antibodies against the viral glycoprotein, which are considered neutralizing in mammals, and against ABV RNA polymerase were not detected. Whereas 5-wk-old hatchlings of the pair (Pair 2) that produced ABV RNA-free late-stage embryos were free of anti-ABV antibodies, such antibodies could be detected again in the serum of these hatchlings at 9 wk of age, before the age that bornaviral RNA could again be detected in feathers and blood. At 16 wk, these antibodies became abundant. The finding that late-stage embryos, presumably free of ABV RNA, can be obtained from eggs from infected parents suggests that hand- or foster-raising of such birds may be a method to obtain birnavirus-free offspring from some infected birds.

Vertical Transmission of Avian Bornavirus in Psittaciformes: Avian Bornavirus RNA and Anti-Avian Bornavirus Antibodies in Eggs, Embryos, and Hatchlings Obtained from Infected Sun Conures (Aratinga solstitialis)

Vertical Transmission of Avian Bornavirus in Psittaciformes: Avian Bornavirus RNA and Anti-Avian Bornavirus Antibodies in Eggs, Embryos, and Hatchlings Obtained from Infected Sun Conures (Aratinga solstitialis)

The Detection of Avian Bornavirus Within Psittacine Eggs

Avian bornavirus (ABV) is a known cause of proventricular dilatation disease in parrots and encephalitis in waterfowl and is a significant cause of both morbidity and mortality in captive birds. Transmission is thought to occur primarily by the fecal-oral route. In an aviary setting, controlling the disease involves a thorough understanding of the complete transmission cycle, including determining whether vertical transmission occurs. In this study, vertical transmission of ABV was evaluated by using 61 eggs obtained from birds in 2 aviaries where proventricular dilatation disease was prevalent, and the presence of ABV had been confirmed by fecal reverse transcription-polymerase chain reaction by using a primer set designed to detect ABV M protein. The contents of these eggs were then tested for the presence of ABV RNA. Of the eggs tested, 10 were determined to contain ABV RNA. These eggs ranged from apparently nonviable to those that contained developing embryos. ABV was detected in the brain tissue of 2 embryos. It remains to be proven that infected chicks can hatch from these eggs to complete the vertical transmission cycle; however, these findings suggest that vertical transmission of ABV may occur.

Follow-Up Investigations on Different Courses of Natural Avian Bornavirus Infections in Psittacines

To study the course of natural avian bornavirus (ABV) infection, 63 psittacines of three bird collections where ABV had been demonstrated were investigated over a period of 1 yr. The psittacines were clinically observed and swabs of crop and cloaca as well as serum samples were collected three separate times at intervals of 2-6 mo. According to the results of detection of ABV RNA by reverse transcriptase polymerase chain reaction (RT-PCR) and of anti-ABV antibodies by indirect immunofluorescence assay (IIFA), 43 of the birds were found to be infected with ABV. Based on variations in virus shedding and antibody production in combination with the occurrence of proventricular dilatation disease (PDD) -related clinical signs, pathological findings, and lethal outcome, four different groups of infected psittacines and a fifth group of noninfected psittacines were identified. Group 1 comprised six birds with various courses of ABV infection and forms of clinical PDD. Groups 2-4 included all birds with subclinical ABV infections: Group 2 contained 13 birds that were consistently (subgroup A, 6 birds) or inconsistently (subgroup B, 7 birds) ABV positive by PCR and serology; group 3 was composed of 13 psittacines exhibiting only anti-ABV antibodies; and 8 birds that had positive ABV RNA detection in crop and cloaca, but did not develop anti-ABV specific antibodies, were classified in group 4. Twenty-three out of the 63 psittacines remained free of detectable ABV RNA or anti-ABV antibodies over the whole observation period (group 5). Based on the results, it seems that birds with high ABV RNA load in crop and cloaca combined with high anti-ABV antibodies have a high risk of the development of PDD, indicating that the humoral antibodies do not protect against the disease. The meaning of the detection of ABV RNA and antibodies at a low and inconsistent level for the single bird as well as for the epidemiology of the ABV infection remained unclear in this field study and needs to be further investigated.

Vertical Transmission of Avian Bornavirus in Psittacines

Vertical Transmission of Avian Bornavirus in Psittacines

Antigen tissue distribution of Avian bornavirus (ABV) in psittacine birds with natural spontaneous proventricular dilatation disease and ABV genotype 1 infection

Tissues of 10 psittacines from aviary 1 ("case birds") and 5 psittacines from different aviaries were investigated for the presence of Avian bornavirus (ABV) antigen by immunohistochemistry using a polyclonal serum specific for the viral nucleocapsid (N) protein. Seven of 10 case birds had clinical signs, and necropsy findings consistent with proventricular dilatation disease (PDD) while 3 case birds and the 5 birds from other aviaries did not exhibit signs and lesions of this disease. In birds with clinical signs of PDD, ABV antigen was largely limited to neuroectodermal cells including neurons, astroglia, and ependymal cells of the central nervous system, neurons of the peripheral nervous system, and adrenal cells. ABV antigen was present in the nuclei and cytoplasm of infected cells. In 2 case birds that lacked signs and lesions of PDD, viral antigen had a more widespread distribution and was present in nuclei and cytoplasm of epithelial cells of the alimentary and urogenital tract, retina, heart, skeletal muscle, and skin in addition to the mentioned neuroectodermal cells. ABV RNA was identified by reverse transcription polymerase chain reaction (RT-PCR) in tissues of all 7 case birds available for testing from aviary 1, including 4 birds with PDD lesions and the 3 birds without PDD lesions. Sequencing and phylogenetic analysis indicated the presence of ABV genotype 1 in all cases. Findings further substantiate a role of ABV in PDD of psittacine bird species.

Avian Bornavirus is Present in Many Tissues of Psittacine Birds with Histopathologic Evidence of Proventricular Dilatation Disease

Proventricular dilatation disease (PDD) is a neurologic disease of psittacine birds suspected to be caused by a recently identified Avian bornavirus (ABV). In the current report, data supporting the causal association of ABV with PDD are presented. Immunohistochemistry (IHC) with rabbit polyclonal antiserum raised against ABV nucleocapsid protein was used to identify cell and organ distribution of viral antigen. The ABV antigen was most consistently detected in brain, spinal cord, adrenal gland, pancreas, and kidney. Histopathologic evaluation was correlated with ABV-specific polymerase chain reaction (PCR) and immunohistochemical tests in multiple tissues from 16 psittacine birds with and without PDD. Using histopathologic diagnosis as the gold standard, the sensitivity and specificity of IHC for ABV antigens were found to be 100% and 100%, respectively. Many more tissues were positive for ABV RNA by reverse transcription PCR than were positive for pathologic changes or viral antigens by IHC, indicating the presence of subclinical or asymptomatic infection at many sites.

Localization of avian bornavirus RNA by in situ hybridization in tissues of psittacine birds with proventricular dilatation disease

Proventricular dilatation disease (PDD) of psittacine birds is caused by a number of different genotypes of a novel viral species, avian bornavirus (ABV). Here we present an in situ hybridization (ISH) procedure using digoxigenin-labeled RNA probes for localizing viral genomic and mRNA of ABV-2 and ABV-4 in tissues of affected birds. Out of eleven immunohistochemically positive birds ISH signals were only found in seven. Partial sequencing of the viral genome had shown that four of them were infected with ABV-2, two with ABV-4 and one had a mixed infection with ABV-2 and ABV-4. ISH signals were present in the brain, in the vegetative nerve system, glandular epithelia and smooth muscle cells of the intestinal tract and in cardiomyocytes. Hybridization signals for viral genome were more abundant than signals for mRNA. As the probes were not strictly genotype-specific, four of the birds had hybridization signals with both, the ABV-2 and ABV-4 probes. The signals achieved with the homologous probes were more intense and more abundant than those resulting from heterologous probes. Taken together, the results of this study show that ISH can be used as a tool for localizing ABV sequences in tissues of birds with PDD and confirm the causative role of ABVs by showing viral replication in affected tissues.

The diagnosis of proventricular dilatation disease: Use of a Western blot assay to detect antibodies against avian Borna virus

Avian Borna virus (ABV) has recently been shown to be the causal agent of proventricular dilatation disease (PDD) a lethal neurologic disease of captive psittacines and other birds. An immunoblot assay was used to detect the presence of antibodies against avian Borna virus in the serum of affected birds. A lysate from ABV-infected duck embryo fibroblasts served as a source of antigen. The assay was used to test for the presence of antibodies to ABV in 117 birds. Thirty of these birds had biopsy or necropsy-confirmed proventricular dilatation disease (PDD), while the remaining 87 birds were apparently healthy or were suffering from diseases other than PDD. Sera from 27 of the 30 PDD cases (90%) contained antibodies to ABV. Seventy-three (84%) of the apparently “healthy” birds were seronegative. Additionally, sera from seven macaws and one parrot trapped in the Peruvian Amazon were seronegative. Positive sera recognized the bornaviral nucleoprotein (N-protein). While the presence of antibodies to ABV largely corresponded with the development of clinical PDD, 14 apparently healthy normal birds possessed detectable antibodies to ABV. The existence of a carrier state was confirmed when 13 of 15 apparently healthy cockatiels were shown by PCR to have detectable ABV RNA in their feces. Western blot assays may be of significant assistance in diagnosing proventricular dilatation disease. Many apparently healthy birds may however be seronegative while, at the same time, shedding ABV in their feces.

Presence of Avian Bornavirus RNA and Anti-avian Bornavirus Antibodies in Apparently Healthy Macaws

Presence of Avian Bornavirus RNA and Anti-avian Bornavirus Antibodies in Apparently Healthy Macaws

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.

Anatomical distribution of avian bornavirus in parrots, its occurrence in clinically healthy birds and ABV-antibody detection

Proventricular dilatation disease (PDD) is a fatal infectious disease of birds that primarily affects psittacine birds. Although a causative agent has not been formally demonstrated, the leading candidate is a novel avian bornavirus (ABV) detected in post-mortem tissue samples of psittacids with PDD from the USA, Israel and, recently, Germany. Here we describe the presence of ABV in a parrot with PDD as well as in clinically normal birds exposed to birds with PDD. In two ABV-positive post-mortem cases, the tissue distribution of ABV was investigated by quantitative real-time reverse transcription-polymerase chain reaction. Viraemia was observed in a PDD-affected bird whereas a restriction of ABV to nerve tissue was found in the non-PDD-affected bird. Healthy birds from the same aviary as the affected birds were also found to harbour the virus; 19/59 (32.2%) birds tested positive for ABV RNA in cloacal swabs, providing the first evidence of ABV in clinically healthy birds. In contrast, 39 birds from the same geographic area, but from two different aviaries without PDD cases in recent years, had negative cloacal swabs. ABV RNA-positive, clinically healthy birds demonstrated the same serological response as the animal with confirmed PDD. These results indicate that ABV infection may occur without clinical evidence of PDD and suggest that cloacal swabs can enable the non-invasive detection of ABV infection.

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

BackgroundProventricular 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).ResultsFive 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.ConclusionIn 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.