Isolation and characterization of a new subtype of Borna disease virus

Abstract

Borna disease virus (BDV), the causative agent of severe meningoencephalitis in a wide variety of animal species, has been considered to be genetically invariable and to form a single type within the genus Bornavirus of the family Bornaviridae. BDV infections are of particular interest, because for the first time a virus infection appears to be linked to human psychiatric disorders. We now describe a new subtype of BDV isolated from a horse which was euthanatized due to severe, incurable neurological disease. The nucleotide sequence of this new strain, named No/98, differs from the reference strains by more than 15%, and the subtype is difficult to detect by standard reverse transcriptase PCR protocols. The nucleotide exchanges of the novel BDV isolate have surprisingly little effect on the primary structures of most viral proteins, with the notable exception of the X protein (p10), which is only 81% identical to its counterpart in reference strains. Our data indicate that the genome of BDV is far more variable than previously assumed and that naturally occurring subtypes may escape detection by currently used diagnostic assays.

FIG. 1

Immunohistochemical staining of paraffin-embedded brain tissue by using the avidin-biotin complex technique and the monoclonal antibody BO18. (A) Specifically labeled neuron within the hippocampus region (magnification, ×335). (B) Positive immunostaining of hippocampal neurons. Note the numerous intranuclear Joest-Degen inclusion bodies (arrows), which are considered to be characteristic of BDV infection (magnification, ×335).

FIG. 2

Comparison of nucleotide and amino acid sequences of BDV No/98 and common laboratory strains. (A) Gene order and coding strategy of BDV. Overlapping RT-PCR products of No/98 corresponding to nucleotide positions 25 to 4234 of reference strain V (3) were sequenced. This fragment includes the complete coding sequences for the viral proteins N, X, P, M, and G and part of the L gene, as indicated. (B) Comparison of nucleotide (nt) and amino acid (aa) sequences of No/98 and laboratory strains V (3) and He/80 (4). Percentage of overall sequence identity (all) or sequence identities of the particular gene products at nucleotide and amino acid levels are indicated. Numbers in parentheses show percentage of sequence similarity when conserved amino acid exchanges are taken into account. Amino acid exchanges were rated conservative when the affected residues had similar biochemical properties. Any exchanges within the following groups of amino acids were considered conservative (single letter code): AILVM, STC, FY, NQ, WF, DE, and KR. Numbers boxed with gray highlight the striking dissimilarity of the X gene product. (C) Strongly biased conservation of the P protein sequence in the X-P gene overlap region. Positions of nonconservative amino acid exchanges in the X (n = 14) and P (n = 3) proteins are indicated by vertical bars. The comparison of strains V and No/98 is shown.

FIG. 3

Phylogenetic trees of BDV strains. Nucleotide sequence comparisons of fragments from the N gene coding region (corresponding to nucleotides 262 to 829 of reference strain V) and from the P gene coding region (corresponding to nucleotides 1482 to 1814 of reference strain V) were performed. Distances between strains indicate percentage of sequence divergence. Sequence information used to construct these trees was taken from the following sources: No/98, this report and GenBank no. AF136236; V, reference and GenBank no. U04608; He/80, reference and GenBank no. L27077; WT-1 N gene, reference and GenBank no. S67502; WT-1 P gene, reference and GenBank no. S67507; Hu1 N gene, reference and GenBank no. U58594; Hu1 P gene, reference and GenBank no. L76234; Hu3 N gene, reference and GenBank no. U58596; Hu3 P gene, reference and GenBank no. L76236. Sequences designated MDCK, D1-3, D2-1, and H2-1 were taken from the work of Iwata et al. (9).

FIG. 4

No/98 is poorly recognized by conventional diagnostic RT-PCR assays. To examine at which sensitivity levels a published RT-PCR assay is able to detect the novel BDV subtype compared to the classical subtype, we prepared cell mixtures, each containing 5 × 10⁵ uninfected Vero cells and the indicated numbers of Vero cells infected with either BDV No/98 or He/80 and subjected them to PCR analysis. (A) Conventional RT-PCR (19); (B) conventional nested PCR (19); (C) modified RT-PCR using primer pairs (described above) that amplify both No/98 and He/80 genomes.