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To the Editor: Proventricular dilatation disease (PDD) is a fatal disease in psittacines that jeopardizes critical species conservation projects, such as that involving the Spix’s macaw (Cyanopsitta spixii), the world’s most endangered bird species (1). The disease is characterized by lymphoplasmacytic infiltrations in the enteric and central nervous systems (2). Consequently, gastrointestinal and neurologic disorders are the major clinical manifestations. Only recently has the cause of the disease been identified by characterization of a newly discovered member of the family Bornaviridae, the avian bornavirus (ABV), which has been detected in affected psittacines (3,4). The relationship of an infection with ABV and the occurrence of PDD has been described in natural cases (5,6) and in experimental trials (7,8). However, birds that are infected with ABV but that are clinically healthy have also been recognized (6). Infected birds can shed viral RNA intermittently (9), and not all infected birds seroconvert (5). For psittacine flock management, control of an AΒV infection is critical, e.g., by repeated testing of breeding stock and removal of ABV-positive birds (2,5). However, in breeding projects of rare species, every individual is genetically important and cannot be lost. Therefore, pairing infected, but clinically healthy, birds separately from birds that test negative for the virus might represent an option. For this possibility to be viable, whether vertical transmission of ABV can take place must be further clarified. A study investigating the distribution of ABV in tissues of PDD-positive birds has demonstrated ABV antigen in follicular cells, which may point toward vertical transmission (9). To investigate vertical transmission of ABV, we examined 30 dead-in-shell embryos of various psittacine species that originated from ABV-infected flocks with a history of PDD. First, the eggshell was disinfected and opened at the blunt end by using sterile equipment. The brain and proventriculus of each embryo were analyzed for the presence of ABV RNA by using 2 different real-time reverse transcription PCRs, as described by Honkavuori (4), with the primer pair 1034–1322 and, in case of a negative result, the additional primer set 1367. Sampling, RNA extraction, and PCR were repeated by using the same methods to exclude possible cross-contamination of samples. Afterwards, the complete embryo was placed in 10% buffered formalin, and histopathologic examination and immunohistochemical (IHC) testing were carried out (10) with antibodies directed against the viral phosphoprotein and X protein. If ABV RNA or ABV antigen was demonstrated, crop and cloacal swab specimens and serum of the parents of the positive embryo were immediately taken and used either for ABV RNA detection with the above described PCR or for the detection of specific ABV antibodies by indirect immunofluorescence assay (10). This procedure was chosen because earlier sampling of the parents might have caused breeding interruption, and which eggs of which parents would be available for investigation was not clear. In 2 of the 30 embryos investigated, ABV RNA was detected by using the 1034 PCR. The repeated procedure provided the same results in the same embryos. One embryo was a Major Mitchell cockatoo (Cacatua leadbeateri) (cycle threshold 31.41) and the other a red-crowned Amazon (Amazona viridigenalis) (cycle threshold 35.1). None of the investigated embryos demonstrated histopathologic lesions typical of an ABVinfection. IHC testing did not show any positive results. However, in the ABV-positive Amazon embryo, an equivocal result was obtained. The swab specimens of both parents of the Major Mitchell cockatoo tested positive for ABV RNA, but serum did not demonstrate specific ABV antibodies. The crop swab specimen of the female red crowned Amazon was positive for ABV RNA, but serum was negative for ABV antibodies; the male bird tested negative by PCR but demonstrated an ABV-specific antibody titer of 80. These results highlight the potential risk for vertical transmission of ABV and the conclusion that ABV-infected parents can most likely produce infected offspring. However, test results were positive for only 2 of 30 eggs. Because potentially dead-in-shell embryos are usually further incubated by the breeder to ensure embryonic death, the possibility cannot be excluded that ABV RNA was already degraded in some cases, thus causing false-negative results. On the other hand, these eggs might have originated from ABV-negative parents. The quality of the samples might also have caused the questionable IHC results. In PDD-affected, ABV-positive flocks >30% of the birds could be infected (5,6) and the virus is shed intermittently (9). Therefore, pairing ABV-positive birds, incubating their eggs artificially, and raising the chicks separately until they show negative test results, might be an option for breeding projects. However, when vertical transmission occurs (and, if so, its incidence) is unknown. Whether ABV infection of the embryos was the cause of death remains unclear. Even if typical lesions were not detected, the poor quality of the material might have hidden such lesions. However, embryonic infection that does not result in embryonic death is the basic requirement for successful vertical transmission. These preliminary results warrant further studies investigating the possibility of vertical transmission by ABV-infected pairs, especially to minimize the risk for such transmission to endangered species with restricted breeding opportunities.

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 (ACH) 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. NeuroReport 12:3767-3772 (C) 2001 Lippincott Williams & Wilkins.

Phosphorylation of one or more viral proteins is probably an essential step in the life cycle of every member of the nonsegmented negative-strand RNA virus (mononegavirales [MNV]) group. Since no virally encoded protein kinases have been discovered in this group, phosphorylation is effected entirely by host cell kinases. The virally encoded P proteins of the MNV are the only ones consistently phosphorylated with a stoichiometry > or =1. The P protein of vesicular stomatitis virus (VSV), and perhaps also of respiratory syncytial virus, are the only ones for which a function of phosphorylation has been established. Phosphorylation by casein kinase 2 at one or more identified sites in the VSV P protein activates transcriptional activity by promoting formation of a homotrimer, which is then capable of binding the RNA polymerase and attaching it to the N protein-RNA template. A second phosphorylation of VSV P protein by a different kinase also occurs, dependent upon prior modification by casein kinase 2, but its function is not definitely known. Phosphorylation of the other MNV P proteins may serve a different purpose. No evidence has been obtained yet for any function for phosphorylation of any other MNV protein.

Background In a stable of eight horses in Northern Iceland, six horses presented with clinical signs, such as ataxia and reduced appetite, leading to euthanasia of one severely affected horse. Serological investigations revealed no evidence of active equine herpes virus type 1 infection, a common source of central nervous system disease in horses, nor equine arteritis virus and West Nile virus. Another neurotropic virus, Borna disease virus, was therefore included in the differential diagnosis list. Findings Serological investigations revealed antibodies against Borna disease virus in four of five horses with neurological signs in the affected stable. One horse without clinical signs was seronegative. Four clinically healthy horses in the stable that arrived and were sampled one year after the outbreak were found seronegative, whereas one of four investigated healthy horses in an unaffected stable was seropositive. Conclusions This report contains the first evidence of antibodies to Borna disease virus in Iceland. Whether Borna disease virus was the cause of the neurological signs could however not be confirmed by pathology or molecular detection of the virus. As Iceland has very restricted legislation regarding animal imports, the questions of how this virus has entered the country and to what extent markers of Bornavirus infection can be found in humans and animals in Iceland remain to be answered.

Bornaviruses are the only animal RNA viruses that establish a persistent infection in their host cell nucleus. Studies of bornaviruses have provided unique information about viral replication strategies and virus–host interactions. Although bornaviruses do not integrate into the host genome during their replication cycle, we and others have recently reported that there are DNA sequences derived from the mRNAs of ancient bornaviruses in the genomes of vertebrates, including humans, and these have been designated endogenous borna-like (EBL) elements. Therefore, bornaviruses have been interacting with their hosts as driving forces in the evolution of host genomes in a previously unexpected way. Studies of EBL elements have provided new models for virology, evolutionary biology and general cell biology. In this review, we summarize the data on EBL elements including what we have newly identified in eukaryotes genomes, and discuss the biological significance of EBL elements, with a focus on EBL nucleoprotein elements in mammalian genomes. Surprisingly, EBL elements were detected in the genomes of invertebrates, suggesting that the host range of bornaviruses may be much wider than previously thought. We also review our new data on non-retroviral integration of Borna disease virus.

AVIAN bornavirus (ABV), a newly discovered agent, has been identified as the causal agent of proventricular dilation disease (PDD) in psittacine birds (Honkavuori and others 2008, Kistler and others 2008). Subsequent research, including bird inoculation studies (Gancz and others 2009, Gray and others 2010) and outbreak investigations (Kistler and others 2010) have provided strong supporting evidence. PDD is a significant pathological syndrome, with high mortality affecting primarily psittacine birds, that has been reported worldwide since the late 1970s. Characteristic pathological findings of PDD …

Background Avian bornaviruses (ABV) are a recently described group of intranuclear negative-stranded RNA viruses (Order Mononegavirales, Family Bornaviridae). At least 13 different ABV genotypes have been described. One genotype, the Canada goose genotype (ABV-CG), has been isolated from geese and swans and is widely distributed across North America. Results We have isolated and characterized a previously undescribed genotype of avian bornavirus from the brains of wild ducks. This new genotype, provisionally designated ABV genotype MALL, was detected in 12 of 83 mallards, and 1 of 8 wood ducks collected at a single location in central Oklahoma. The virus was cultured on primary duck embryo fibroblasts, fragments were cloned, and its genome sequence of 8904 nucleotides determined. This new genotype has 72% nucleotide identity and 83% amino acid identity with the ABV-CG genotype previously shown to be present in geese and swans. Histologic and immunohistochemical examination of the brains and eyes of four positive ducks indicated the presence of virus-infected neurons and glia in their cerebrums and retinas in the absence of inflammation. Conclusions More than one genotype of ABV is circulating in North American waterfowl. While the infected ducks were not observed to be suffering from overt disease, based on the immunohistochemistry, we speculate that they may have suffered some visual impairment. Electronic supplementary material The online version of this article (doi:10.1186/s12985-014-0197-9) contains supplementary material, which is available to authorized users.