Genus Bornavirus

Type Species	Borna disease virus	(BDV)

Virion Properties

Morphology

Electron microscopy studies of negatively stained Borna disease virus (BDV) infectious particles have shown that virions have a spherical morphology with a diameter of 90   ± 10  nm containing an internal electron-dense core (50 to 60  nm) and a limiting outer membrane envelope that does not appear to be covered with projections (Fig. 1).

Physicochemical and Physical Properties

Virion Mr and the S_20,w are not known. Partially purified BDV infectious particles have a buoyant density in CsCl of 1.16-1.22  g/cm³, in sucrose of 1.22  g/cm³, in renografin of 1.13  g/cm³. Virus infectivity is rapidly lost by heat treatment at 56°C. Virions are relatively stable at 37°C, and only minimal infectivity loss is observed after 24 hours incubation in the presence of serum. Virions are inactivated below pH 5.0, as well as by treatment with organic solvents, detergents, and exposure to UV radiation. Infectivity is completely and rapidly destroyed by chlorine-containing disinfectants or formaldehyde treatment.

Nucleic Acids

The genome consists of a single molecule of a linear, non-segmented negative-stranded (NNS) RNA (about 8.9  kb in size and Mr of approximately 3 10⁶). The RNA genome is not polyadenylated. Extracistronic sequences are found at the 3 (leader) and 5 (trailer) ends of the BDV genome. BDV 3-terminal genomic sequences have a high A+U content with a U/A ratio of ~ 2:1. The ends of the BDV genome RNA exhibit partial inverted complementarity. Full-length plus-strand (antigenomic) RNAs are present in infected cells and in viral ribonucleoproteins. Defective RNAs have not been identified in BDV-infected cells and tissues. As yet only two complete BDV genome sequences have been reported. These two sequences have approximately 95% homology at the nucleotide level, predict the same BDV genomic organization and differ by only two nucleotides in absolute genome size.

Proteins

Six major ORFs are found in the BDV genome sequence (Fig. 2). These ORFs code for polypeptides with predicted Mr of 40 10³ (p40), 24 10³ (p24), 10 10³ (p10), 16 10³ (p16), 56 10³ (p56) and 180 10³ (p180), respectively. Based on their positions in the viral genome and abundance in infected cells and virion particles, together with their biochemical and sequence features, p40, p24 and p16 BDV polypeptides correspond to the viral nucleoprotein (NP), the phosphoprotein (P) transcriptional activator, and matrix (M) proteins, respectively, found in other NNS RNA viruses. Two isoforms of the BDV NP (p40 and p38) are found in BDV-infected cells. These two forms of the viral NP appear to be encoded by two different mRNA species. Differential usage of two in-frame initiation codons present in the BDV p40 gene may also contribute to the production of BDV p40/38. BDV p24 is an acidic polypeptide (predicted pHi of 4.8), that has a high Ser-Thr content (16%), with phosphorylation at serine residues which is mediated by both PKC and casein kinase II. These features are consistent with those of the phosphoprotein (P) transcriptional activator found in other NNS RNA viruses. An additional ORF, p10, encodes for a polypeptide of Mr 10 10³ present in BDV-infected cells. BDV p10 starts within the same mRNA transcription unit, 46  nts upstream from p24 and overlaps, in a different frame, with the 71 N-terminal amino acids of p24.

In contrast to other NNS RNA viruses, BDV p16, the putative BDV M protein, is glycosylated, appears to be present in the virus envelope and it may participate in viral attachment to the cell. BDV ORF4 (p56) overlaps, in a different frame, with the C-terminus of ORF p16, and is capable of encoding a 503 amino acids polypeptide with a predicted Mr of 56 10³. Based on its sequence features, BDV p56 is the counterpart of the virus surface glycoproteins (G) found in other NNS RNA viruses. The p56 gene directs the synthesis of two glycosylated polypeptides of about Mr 84 or 94 10³ (GP-84/94) and 43 10³ (GP-43). GP-84/94 corresponds to the full length of the p56 gene, whereas GP-43 represents the C-terminus of ORF p56. Both GP-84/94 and GP-43 are associated with BDV infectious particles. Antibodies to p56 have neutralizing activity, suggesting that BDV p56 gene products play an important role in the early steps of BDV infection. BDV ORF5 (p180) is capable of encoding a polypetide with a predicted Mr of 180 10³, whose deduced amino acid sequence displays strong homology to other NNS-RNA virus polymerases, members of the L protein family. An additional ORF predicted in mRNA species generated via RNA splicing would encode a variant BDV L with a predicted Mr of 190 10³ (BVp190). BDVp190 corresponds to BDVp180 with 153 amino acids added to its N-terminus.

Lipids

Not known.

Carbohydrates

Not known.

Genome Organization and Replication.

The negative-sense BDV RNA genome codes for at least six ORFs in the order 3-NP-P/p10-M-G-L-5. The genomic polarity has a very limited coding capability, and none of its predicted ORFs has a favorable translational start signal; further they are not flanked by putative transcription start and termination/polyadenylation signals. Therefore, it seems unlikely that BDV uses an ambisense coding strategy. BDV has the property, unique among known NNS RNA animal viruses, of a nuclear site for genome transcription and replication. Full-length genome complementary RNA molecules (antigenomes) act as templates for new viral genome RNA synthesis. Genome and antigenome RNA molecules are neither capped nor polyadenylated. These RNAs exist as nucleocapsids in the nucleus of infected cells. It is unknown whether RNA species corresponding to the leader RNA are transcribed in BDV-infected cells.

BDV cell entry occurs by receptor-mediated endocytosis. Both the virus G and M proteins have been implicated in entry. The identity of the BDV cellular receptor is unknown. In endosomes, low pH-dependent fusion occurs between viral and cell membranes. This fusion event releases the BDV ribonucleoprotein (RNP) which are then transported to the cell nucleus where viral transcription and replication occur. Sequential and polar transcription results in decreasing molar quantity of BDV transcripts from the 3- to the 5-encoded cistrons. The viral mRNAs are polyadenylated and their 5-ends contain a blocking group, presumably a cap structure. Virus specific mRNA synthesis is not inhibited by -amanitin and sequences at the 5 of the BDV mRNAs are homogeneous and genome encoded. Thus, it is unlikely that transcription initiation of BDV mRNAs involves a cap-snatching mechanism similar to the one used by influenza viruses. Monocistronic viral mRNAs in BDV-infected cells are detected only for the NP and P/p10 genes (Fig. 2). The BDV G and L polymerase gene products are synthesized from downstream ORFs within polycistronic mRNAs. Mapping of subgenomic BDV mRNAs present in infected cells to the viral genome revealed that the BDV genome contains three transcription initiation sites (S signals), and four transcription termination/polyadenylation sites (E signals) (Fig. 2). The S signals contain a semi-conserved U-rich motif that is partially copied into the respective transcripts. A similar motif is not found within the S signals of previously described NNS RNA viruses. BDV E signals consist of six or seven U residues preceded by a single A residue, resembling the E signal motif found in other NNS RNA viruses. The BDV genome lacks the characteristic configuration of E signal / intergenic (IG) region / S signal, found at the gene boundaries of other NNS RNA viruses. Instead, BDV transcription units and transcriptive signals frequently overlap (Fig. 2). Two of the BDV primary transcripts are post-transcriptionally processed by the cellular RNA splicing machinery. Two introns (I and II) have been identified in the BDV genome. BDV introns I and II span nt 1932-2025 and 2410 to 3703, respectively, in the BDV antigenomic sequence (Fig. 2). Splicing of intron I places the amino acid in position 13 of M next to a stop codon, whereas splicing of intron II, and I+II, results in a mRNA containing a predicted ORF that corresponds to the first 58 amino acids of G fused to a new C-terminus of 20 amino acids. RNA species resulting from splicing of intron II, and I+II, predicts also an additional ORF that would encode a variant BDV L protein with 153 amino acids added to the N-terminus. Whether these new predicted BDV polypeptides are synthesized in infected cells is unknown. RNA splicing can also modulate the efficiency of termination-reinitiation of translation and leaky scanning mechanisms, thus contributing to the regulation of the expression of BDV M, G and L gene products.

BDV-infected cells exhibit a heterogeneous pattern of viral antigen expression. BDV NP, P, and p10 polypeptides are expressed both in the nucleus and cytoplasm. NP and P are the viral antigens expressed at higher levels, and they are expressed by the majority of the cells within an infected population. In contrast, only 1 to 10% of the infected cells express detectable levels of BDV G. Expression of full length BDV G (GP-84/94) is restricted to the ER and nuclear envelope. The subcellular distribution of the BDV M has not been characterized. Both M and G are post-translationally modified by glycosylation. M has been characterized as a biantennary complex-type glycoprotein sensitive to endoglycosidase F (endo F), but not to endoglycosidase H or O-glycosidase. BDV G undergoes post-translational cleavage by the cellular protease furin, with the resulting C-terminus (GP-43) reaching the cell surface. Cleavage of GP-84/94 likely occurs in the trans-Golgi compartment. GP-84/94 and GP-43 are sensitive to both Endo F and H. The newly exposed N-terminus of GP-43 is highly hydrophobic, and BDV-infected cells form extensive syncytia upon low-pH treatment. These findings suggest that GP-43 is involved in pH-dependent fusion after internalization of BDV by receptor-mediated endocytosis.

The assembly process and site of virus maturation have not been identified and budding of BDV particles from infected cells has been documented only from the surface of BDV-infected MDCK cells after treatment with n-butyrate. BDV RNP accumulate in the nucleus and, as with other NNS RNA viruses, they are also infectious on the basis of an ability to direct synthesis of BDV macromolecules, as well as the production of BDV cell-associated infectivity upon transfection of BDV-susceptible cells. Thin sections of BDV-infected cells revealed the presence of intracytoplasmic virus-like particles with morphological characteristics similar to those described for partially purified cell-free BDV infectious particles. These particles showed no association with cisternae of the endoplasmic reticulum, the Golgi complex, or other intracytoplasmic membranes.

Antigenic Properties

BDV possess a number of distinct antigenic determinants. The so-called soluble antigen (s-antigen) obtained from the supernatant after ultracentrifugation of ultrasonicated BDV-infected brain tissue, contains the viral NP, P and M proteins. Serum antibodies from BDV-infected animals frequently recognize all the components of the s-antigen, but rarely recognize the viral G products. BDV field isolates from the same or different animal species, as well as viruses recovered from experimental infections with different histories of passages exhibit strong serological cross-reactivity. There is only one recognized serotype of BDV, but monoclonal antibodies have revealed minor antigenic differences among BDV isolates. Complement independent IgG-specific neutralizing antibodies have been documented in experimentally infected animals. Titers of neutralizing antibodies are usually very low and dependent on the infected host species. Both BDV M and G proteins have been implicated in virus neutralization.

Biological Properties

Horses and sheep have been regarded as the main natural hosts of BDV. In these species BDV can cause a fatal neurologic disease, Borna disease (BD). Evidence indicates that the natural host range of BDV is wider than originally thought. Naturally occurring BDV infections have been documented in cattle, rabbits, cats, and ostriches. In addition, sporadic cases of natural infection with BDV have been reported in several other species including donkeys, mules, and llamas. Moreover, experimental infections have revealed a remarkable wide host range for BDV, from birds to rodents and non-human primates. BDV-induced neurobehavioral abnormalities in animals are reminiscent of some human neuropsychiatric disorders. Serological data and recent molecular epidemiological studies indicate that BDV can infect humans, and is possibly associated with certain neuropsychiatric disorders.

BDV is thought to be transmitted through salival, nasal, or conjuntival secretions. Infection may result from direct contact with these secretions or by exposure to contaminated water and food. Intranasal infection is the most likely route of natural infection, allowing BDV access to the CNS by intraaxonal migration through the olfactory nerve. Cases of BD are more frequent in some years than others and tend to occur in spring and early summer, suggesting arthropods as a potential vector. BDV has not been isolated from insects, but ticks have been implicated in the transmission of an infectious encephalomyeletis similar to BD affecting ruminants in the the Middle East.

Asymptomatic naturally infected animals of different species have been documented in Europe, North America, Africa and Asia, suggesting that the prevalence and geographic distribution of BDV may have been underestimated. However, a definite natural reservoir of BDV has not been identified. Phenotypic differences have been described among different BDV field isolates, and among viruses with different histories of passages in animals and cultured cells. Despite its wide host range and phenotypic variation, molecular epidemiological data has shown a remarkable sequence conservation of BDV, not within the same host species but also amongst sequences derived from from different animal species.

BDV is highly neurotropic and has a non-cytolytic strategy of multiplication. BDV causes CNS disease in several non-human vertebrate species which is characterized by neurobehavioral abnormalities that are often, but not always, associated with the presence of inflammatory cell infiltrates in the brain. BDV exhibits a variable period of incubation, from weeks to years, and diverse pathological manifestations that depend on the genetics, age and immune status of the host, as well as route of infection and viral determinants. Classic BD is caused by a T-cell dependent immune mechanism. Inflammatory cells are found forming perivascular cuffs and also within the brain parenchyma. Both CD4⁺ and CD8⁺ T-cells are present in the CNS cell infilatrates and contribute to the immune-mediated pathology associated with BD. BDV can also induce distinct deficiencies in emotional and cognitive functions that are associated with specific neurochemical disturbances in the absence of lymphoid infiltration. Heightened viral expression in limbic system structures, together with astrocytosis and neuronal structural alterations within the hippocampal formation are main histopathological hallmarks of BDV infection.

List of Species Demarcation Criteria in the Genus

Not applicable.

List of Species in the Genus

Official virus species names are in italics. Tentative virus species names, alternative names ( ), strains or serotypes are not italicized. Virus name and assigned abbreviation ( ) are:

Species in the Genus

Borna disease virus	[L27077, U04608]	(BDV)

Tentative Species in the Genus

None reported.

List of Unassigned Viruses in the Family

None reported.

Phylogenetic Relationships within the Family

Not applicable.

Similarity with Other Taxa

BDV has a genomic organization similar to that of other NNS RNA viruses. The size of the BDV genome (~ 8.9  kb) is significantly smaller than those of the other known members of the order Mononegavirales: Rhabdoviridae (~ 11-15  kb), Paramyxoviridae (~ 15  kb) and Filoviridae (~ 19  kb). BDV replication and transcription take place in the nucleus. This is a unique feature among known NNS RNA animal viruses, but shared with the plant nucleorhabdoviruses. Expression of the BDV genome is regulated by an overlap of transcription units and transcriptive signals, an overlap of ORFs, readthrough of transcription termination signals and differential use of translational initiation codons. There is precedent for use of each of these strategies by members of the order Mononegavirales. However, the concurrent use by BDV of such a diversity of strategies for the regulation of its gene expression is unique among known NNS RNA viruses. In addition, as with viruses belonging to the family Orthomyxoviridae, BDV uses RNA splicing to generate some of its mRNAs. This represents another unique feature in the order Mononegavirales. BDV has one single surface glycoprotein gene (G) which is responsible for viral attachment and fusion upon endocytosis and endosome acidification. This pH-dependent fusogenic activity of G requires its post-translational cleavage by the cellular protease furin. Thus, BDV G expression and function appears to be a new situation in NNS RNA viruses, representing a combination of the strategies adopted by rhabdoviruses and paramyxoviruses.

Derivation of Name

Borna refers to the city of Borna in Saxony, Germany, where many horses died in 1885 during an epidemic of a neurological disease, designated as Borna disease (BD), caused by the infectious agent presently known as Borna disease virus (BDV).