RNA splicing in Borna disease virus, a nonsegmented, negative-strand RNA virus.

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus related to rhabdoviruses and paramyxoviruses. Unlike animal viruses of these two families, BDV transcribes RNAs in the nuclei of infected cells and produces high levels of transcripts containing multiple open reading frames. Previous Northern blot analysis of RNA from BDV-infected rat brain tissue has shown that two viral transcripts, a 6.1-kb RNA and a 1.5-kb RNA, lack regions that are internal to two otherwise identical transcripts, the 7.1-kb RNA and the 2.8-kb RNA, respectively (T. Briese, A. Schneemann, A. Lewis, Y. Park, S. Kim, H. Ludwig, and W. I. Lipkin, Proc. Natl. Acad. Sci. USA 91:4362-4366, 1994). To determine the precise location of this deletion, we performed reverse transcription PCR analysis using total RNA from BDV-infected rat brain tissue. This investigation resulted in the identification of two introns in the 7.1- and 2.8-kb RNAs, which can be alternatively spliced to yield additional RNA species, including the 6.1- and 1.5-kb RNAs. Transient transfection of COS-7 cells with a cDNA clone of the 2.8-kb RNA resulted in the production of both the 2.8-kb RNA and the 1.5-kb RNA, confirming the theory that the 2.8-kb RNA is a sufficient substrate for splicing in mammalian cells. Splicing has not previously been observed in nonsegmented, negative-strand RNA viruses and presumably serves as a mechanism by which expression of BDV proteins is regulated in infected cells.

Detection of Borna disease virus RNA in naturally infected animals by a nested polymerase chain reaction.

Borna disease virus in naturally infected horses, a donkey and sheep was detected for the first time by amplification of viral RNA using PCR. In contrast to a control group of healthy horses, brain tissue was positive by this assay in all animals with neurological symptoms. The use of a second round of PCR with nested primers following Southern hybridization confirmed the specificity and increased the sensitivity of the test. Comparison with conventional methods recommends this technique for monitoring of BDV infections at a molecular level.

Molecular characterization of Borna virus RNAs.

Borna disease virus is cell-associated in infected animals. Antibodies in animals are directed against BDV proteins of 38/39, 24, and 14.5 kD. cDNA clones that encode these proteins hybridize to five mRNAs of 10.5, 3.6, 2.1, 1.4, and 0.85 kb. The 10.5, 3.6, 2.1, and 0.85 kb RNAs are 3' co-terminal; the 1.4 kb RNA is contained within the 10.5, 3.6, and 2.1 kb species but is not 3' co-terminal. A negative strand 10 kb RNA is also present in infected cells. To determine which of the large 10 kb species represents the genomic RNA, strand-specific probes were used for Northern analyses of RNA from infectious particles isolated by Freon extraction of BDV-infected rat brain. RNA purified from these particles contained both positive and negative sense 10 kb species. Treatment of particles with RNaseA before isolation of RNA resulted in detection of only negative strand species, suggesting that BDV is a negative strand RNA virus. However, the genomic organization of BDV is unlike any known negative strand RNA virus.

Sequence conservation in field and experimental isolates of Borna disease virus.

Coding and noncoding sequences were analyzed from field and experimental isolates of Borna disease virus. For a 24-kDa protein, maximum divergence was 1.5% at the predicted amino acid level and 3.1% at the nucleotide level. For a 40-kDa protein, maximum divergence was 1.1% at the predicted amino acid level and 4.1% at the nucleotide level. The highest variability in sequence (10%) was found in a 40-nucleotide stretch of genomic RNA between coding sequences for the 40- and 24-kDa proteins. The degree of sequence conservation in these isolates, passaged in various host species in vivo and in vitro over a period of 64 years, is unusual for negative-strand RNA viruses.

In borna disease virus infected rabbit neurons 100 nm particle structures accumulate at areas of Joest-Degen inclusion bodies.

Borna disease virus infected rabbits were chosen to search for electronmicroscopic structures. Intensively investigated hippocampal neurons showed intranuclear inclusions; 100 nm particle-like structures surrounded by 20 nm granular forms were prominent. In connection with elsewhere reported in situ hybridization studies of virus-specific RNA to areas of the Joest-Degen inclusions we suggest that these particle structures may represent Borna virus. Jost-Degen (8) found intranuclear inclusion bodies in neurons to be pathognomonic for Borna disease (BD) in the horse. Half a century later these structures were suggested to represent BD virus (BDV)-specific antigen aggregates (15). A century later we characterized the virus to contain a single and negative stranded RNA of 8.5 kb, which transcribes in the nucleus (5) and could show that virus complementary RNA seems to hybridize spot-like to nuclear areas, probably representing the Jost-Degen inclusions (7). Electron microscopic (EM) findings on structures in BDV infected brain cells and about particle-like structures obtained from infected tissue culture cells have been reported by different groups (1, 2, 3, 4, 5, 6, 9, 11, 12, 13). The demonstration of crystalline aggregates and filament bundles in the cytoplasma and karyospheridia (nuclear bodies) were prominent. Such structures were seen in infected rabbits, hamsters, rats mice and naturally infected horses. The phenotypic description of filamentous structures, crystalline aggregates in the cytoplasma and large karyospheridia were prominent. Based on our experience with BDV infections in rabbits we selected this species for ultrastructural studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Natural Borna disease in domestic animals others than horses and sheep.

In 24 cats (Uppsala, Sweden) with neurological signs of "staggering disease" and typical neuropathology, 44% had Borna disease virus (BDV)-specific antibodies. In 173 cat sera (Berlin, Germany) of animals with unknown record, 7% were BDV positive. Out of 24 cats with undefined neurological disorders, 13% were BDV positive. Similarities in staggering disease of cats and Borna disease of horses and sheep suggest related etiological agents.

Borna disease virus in peripheral blood mononuclear and bone marrow cells of neonatally and chronically infected rats.

Borna disease virus (BDV) establishes a persistent infection in cells of the nervous system in rats. The response, or lack thereof, of the immune system to BDV infection of neurons is responsible for the presence or absence, respectively, of Borna disease. We recently demonstrated transmission of BDV by bone marrow cells from neonatally infected rats. Our findings suggested the possibility of a heretofore unsuspected interaction between BDV and the immune system, that of direct effects of BDV infection on the cells of the immune system. This report enlarges upon the previous findings and confirms the presence of BDV RNA in bone marrow cells of neonatally infected rats, using a reverse transcription-polymerization chain reaction-enzyme immunosorbent assay (RT-PCR-EIA). In addition, we detected BDV RNA in peripheral blood mononuclear cells of neonatally infected rats, and in rats inoculated as adults in the chronic, but not the acute, stage of infection. In addition, the RT-PCR-EIA technique identified BDV RNA in cerebrospinal fluid, nasal secretions, saliva, urine and stool. BDV-sequences were not detected in the plasma of infected animals nor in the body fluids and tissues of normal rats.

Borna disease virus: nature of the etiologic agent and significance of infection in man.

This review presents data on the characterization of Borna disease virus (BDV) and its potential as a possible causative agent in humans. The isolation of: (i) BDV-specific cDNA clones that encode various BDV-specific proteins and (ii) partially purified virus particles led to the conclusion that the viral genome consists of negative-sense, single-stranded RNA. The organization of the BDV-specific RNA species appears to be a nested set of overlapping subgenomic RNA transcripts. Furthermore, evidence is presented that BDV can infect humans and may cause certain psychiatric and neurological disorders. This concept is supported by: (i) the finding of virus-specific antibodies in sera of patients with neuropsychiatric diseases and (ii) results obtained during attempts to isolate BDV or a BDV-related agent from the cerebrospinal fluid of seropositive patients.

Biology and neurobiology of Borna disease viruses (BDV), defined by antibodies, neutralizability and their pathogenic potential.

Borna disease viruses (BDV) isolated from more than 20 naturally infected horses, 2 sheep and a possible feline isolate were included in these studies. Most of these wild-type viruses were grown in rabbit cells. Specifically rabbit-adapted viruses establish persistent infection in immortalized cell lines of various animal species. Brain-, tissue culture-, and cell-free released viruses could all be neutralized with antibodies from naturally and experimentally infected animals (horse; hamster, rat, rabbit, mouse, and chicken), with highest titres in birds. Splenectomized rabbits, which were subsequently infected with BDV, efficiently produced high titres of neutralizing antibodies. All of the neutralizing sera and cerebrospinal fluids from infected animals inhibited tissue culture spread of BDV. Experimental infection and hyperimmunization induced antibodies directed against the major components of the soluble antigen (60, 40/38, 25 and 14.5 kD proteins). Analysis of the s-antigen complex with these sera and 6 stable monoclonal antibodies revealed that it consists of 40/38 and 25 kD proteins. Although each of these antibodies detected intracellular virus-specific structures they did not recognize outer plasma membrane antigens, showed no cross-reactivity, and had no neutralizing capacity. Unifying pathogenetic concepts of this neurotropic virus and its structural elements are discussed.

Borna disease virus in mice: host-specific differences in disease expression.

We developed a mouse model of Borna disease to facilitate immunopathogenesis research by adaptation of Borna disease virus to mice through serial passage in mouse brain tissue. Borna disease virus replication, antibody production, inflammation, and Borna disease expression in several different strains of mice were examined.

Infection with Borna disease virus: molecular and immunobiological characterization of the agent.

Borna disease virus (BDV), which seems to be distinct from all other known viruses, exhibits a unique mechanism of pathogenesis. This review highlights several aspects of the biology of infection with this virus and summarizes the preliminary characterization of the agent. Studies on BDV may help to illuminate several important areas of neurobiology, including the mechanisms regulating the replication of a new type of RNA virus in the nuclei of neural cells, the neuroinvasiveness and neurotropism of such viruses, their T cell-mediated immunopathology, tolerance in newborn animals to persistent viral infection of the central nervous system, and behavioral diseases and eating disorders induced by such agents.

Borna disease: association with a maturation defect in the cellular immune response.

Borna disease virus (BDV) is a negative-strand RNA virus which produces persistent infection in a variety of experimental animals. In the rat, the presence or absence of clinical signs of Borna disease, a characteristic, biphasic neurobehavioral illness, depends on host-related factors. A window of opportunity exists after birth wherein inoculation with BDV produces a persistently infected rat without signs of Borna disease or encephalitis (persistent, tolerant infection-newborn [PTI-NB] rat). Although immunopathological destruction of the nervous system does not occur in the PTI-NB rat, significant alterations in the development of the nervous system were noted, including site-specific lysis of neurons. Unlike the case with other pharmacologically produced, persistent, tolerant BDV infections, adoptive transfer of spleen cells from BDV-infected rats did not produce disease in the PTI-NB rats. PTI-NB rats developed Borna disease after being connected by parabiosis to rats with Borna disease. Bone marrow transplantation experiments revealed that bone marrow cells from PTI-NB rats produced Borna disease in lethally irradiated, BDV-infected recipient rats. Bone marrow from PTI-NB rats contained a complement of inflammatory cells capable of inducing Borna disease. Thus, the loss of BDV-specific cellular immunity appeared to occur after the release of cells from the bone marrow.

Molecular and immunopathological studies of borna disease virus infection in rats.

Borna disease virus is an agent distinct from all known viruses. Pathogenesis of its infection is also unique. This review highlights several aspects of the biology of this viral infection and the preliminary characterization of the agent. BDV can be used to answer important questions in neurobiology. These include neuroinvasiveness and neurotropism of viral agents, CD4+ T cell-mediated immunopathology and tolerance in newborn animals to a persistent viral infection in the CNS and behavioral diseases and eating disorders induced by neurotropic viruses. This review is dedicated to Prof. Dr. Rott on occasion of his 65th birthday in recognition of his immense contributions to studies on Borna disease and also for his success focusing the attention of the scientific community to this still evolving unique viral disease.

A borna virus cDNA encoding a protein recognized by antibodies in humans with behavioral diseases.

Borna disease virus (BDV) causes a rare neurological disease in horses and sheep. The virus has not been classified because neither an infectious particle nor a specific nucleic acid had been identified. To identify the genome of BDV, a subtractive complementary DNA expression library was constructed with polyadenylate-selected RNA from a BDV-infected MDCK cell line. A clone (B8) was isolated that specifically hybridized to RNA isolated from BDV-infected brain tissue and BDV-infected cell lines. This clone hybridized to four BDV-specific positive strand RNAs (10.5, 3.6, 2.1, and 0.85 kilobases) and one negative strand RNA (10.5 kilobases) in BDV-infected rat brain. Nucleotide sequence analysis of the clone suggested that it represented a full-length messenger RNA which contained several open reading frames. In vitro transcription and translation of the clone resulted in the synthesis of the 14- and 24-kilodalton BDV-specific proteins. The 24-kilodalton protein, when translated in vitro from the clone, was recognized by antibodies in the sera of patients (three of seven) with behavioral disorders. This BDV-specific clone will provide the means to isolate the other BDV-specific nucleic acids and to identify the virus responsible for Borna disease. In addition, the significance of BDV or a BDV-related virus as a human pathogen can now be more directly examined.

[The development of a purification method for Borna disease virus]. / Versuche zur Entwicklung eines Reinigungsverfahrens für das Virus der Bornaschen Erkrankung.

Borna disease virus represents an unknown neurotropic agent. It causes encephalitis in horses and sheep. The same or a similar type of virus might be responsible for psychiatric disorders in man. So far, it has been impossible to purify this agent to such an extent that it could be analyzed biochemically or electronmicroscopically. Therefore, different conventional virus purification techniques are applied in order to develop a method for obtaining purified Borna disease virus from infectious rat brain or persistently infected cell culture material.

Isolation and characterization of Borna disease agent cDNA clones.

Borna disease (BD) is a neurologic syndrome characterized by behavioral disturbances and the accumulation of specific proteins in limbic system neurons. A viral etiology has been proposed because BD can be induced in birds, rodents, and primates by inoculation with filtered brain homogenates from animals with BD. We report here the isolation and preliminary characterization of cDNA clones from a rat with BD. These clones hybridized to specific transcripts in BD rat brain and arrested in vitro translation of BD proteins. In situ hybridization experiments using RNA probes prepared from these clones showed an abundance of these transcripts in limbic system neurons. Northern (RNA) hybridizations using these RNA probes indicated that the BD agent is probably a virus with major transcripts of 8.5, 2.1, and 0.8 kilobases.