Borna disease virus is a neurotropic negativestrand RNA virus that infects a wide range of vertebrate hosts, causing disturbances in movement and behavior. We have cloned and sequenced the 8910-nucleotide viral genome by using RNA from Borna disease virus particles. The viral genome has complementary 3' and 5' termini and contains antisense information for five open reading frames. Homology to Filoviridae, Paramyxoviridae, and Rhabdoviridae is found in both cistronic and extracistronic regions. Northern analysis indicates that the virus transcribes mono-and polycistronic RNAs and uses terminatlon/polyadenylylation signals reminiscent ofthose observed in other negative-strand RNA viruses. Borna disease virus is likely to represent a previously unrecognized genus, bornaviruses, or family, Bornaviridae, within the order Mononegavirales.

Borna disease virus, an uncas ied Infectious agent, causes immune-mediated neurologic disease in a wide variety of animal hosts and may be involved in pathogenesis of selected neuropsychiatric diseases in man. 1nitialreports suggested that Borna disease virus is a singlesranded RNA virus. We describe here a method for isolatin of viral particles that has allowed definitive identification ofthe genome as containg a negative-polarity RNA 8.5 (8, 9) to 10 kilobases (kb) (6). The polarity of the viral genome has been controversial (6,(8)(9)(10)(11)). Here we report that infectious BDV particles contain a negative-polarity 8.5-kb RNA and demonstrate that viral mRNAs are synthesized in cell nuclei. MATERIALS AND METHODSVirus Particle Preparation. Oligo/TL, an human oligodendrocyte cell line (12), was infected at an estimated multiplicity of infection of 0.5 focus-forming unit per cell using BD rat brain homogenate (strain V, sixth rat passage) (13-15). Cells were passed every 2-3 days and used for virus particle preparation in passages 10 through 25. Infectious virus was titered in a cell-ELISA system (16). The confluent cell layer (1-2 x 108 cells) was washed once with 20 mM Tris-HCl (pH 7.4), overlaid with 20 mM Tris HCl, pH 7.4/250 mM MgCl2 (Tris-Mg250 buffer), and incubated for 1.5 hr at 370C to release the cell-bound virus. Supernatant was collected and spun twice at 2500 x g for 5 min. Clarified supernatant was brought to 0.002% Zwittergent 3-14 (Calbiochem) and incubated for 1 hr at room temperature. Virus particles were pelleted at 100,000 x g for 1 hr at 200C, resuspended

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