Enhanced neurovirulence of borna disease virus variants associated with nucleotide changes in the glycoprotein and L polymerase genes

Abstract

Borna disease virus (BDV) infection produces a variety of clinical diseases, from behavioral illnesses to classical fatal encephalitis (i.e., Borna disease [BD]). Since the genomes of most BDV isolates differ by less than 5%, host factors are believed responsible for much of the reported variability in disease expression. The contribution of BDV genomic differences to variation in BD expression is largely unexplored. Here we compared the clinical outcomes of rats infected with one of two related BDV variants, CRP3 or CRNP5. Compared to rats inoculated with CRP3, adult and newborn Lewis rats inoculated with CRNP5 had more severe and rapidly fatal neurological disease, with increased damage to the hippocampal pyramidal neurons and rapid infection of brain stem neurons. To identify possible virus-specific contributions to the observed variability in disease outcome, the genomes of CRP3 and CRNP5 were sequenced. Compared to CRP3, there were four nucleotide changes in the CRNP5 variant, two each in the G protein and in the L polymerase, resulting in four amino acid changes. These results suggest that small numbers of genomic differences between BDV variants in the G protein and/or L polymerase can contribute to the variability in BD outcomes.

FIG. 1.

Clinical (A) and histological (B) expression of BD phenotype over time in rats inoculated as adults with CRP3 (○), CRNP5 (•), or normal rat or mouse brain (▿). (A) Severity of clinical BD (rated on a scale of 0 to 3); (B) severity of inflammation in the brain (encephalitis, rated on a scale of 0 to 4) in the BDV-infected adult rats. Crosses indicate when all of the remaining animals were morbidly ill and required euthanasia.

FIG. 2.

CA3/4 region pyramidal neurons of the hippocampus (arrows) of rats inoculated as adults (a to c) or neonates (d to f) with normal rat or mouse brain homogenate (Sham), CRP3, or CRNP5. These sections of brain were taken approximately 2 weeks p.i. and stained with H/E. Magnification, ×40.

FIG. 3.

Antibody production (A) and virus replication (B) over time in rats inoculated as adults with CRP3 (○), CRNP5 (•), or normal rat or mouse brain (▿). Asterisks indicate that anti-BDV antibody in CRNP5-infected rats was detected at significantly higher titers than in CRP3-infected rats (P < 0.001). Virus titers were not significantly different between CRP3- and CRNP5-infected rats at any time point. Crosses indicate when all of the remaining CRNP5-infected animals were morbidly ill and required euthanasia.

FIG. 4.

Virus antigen distribution in the representative sections of the brain stem at 2 weeks p.i. from rats inoculated with normal rat or mouse brain homogenate (Sham), CRP3, or CRNP5 as adults (a to f) or as neonates (g to l). The sections shown in panels a to c and g to i were stained with H/E. The sections shown in panels d to f and j to l were stained via immunohistochemistry for BDV antigens with no counterstain. Arrows indicate BDV antigen in infected neurons. Magnification, ×200.

FIG. 5.

Clinical expression of BD phenotype (A) and virus replication in the brain (B) over time for rats inoculated as neonates with CRNP5 (•), CRP3 (○), or normal rat or mouse brain homogenate (▿). The severity of BD was rated on a scale of 0 to 3. Crosses indicate when all of the remaining CRNP5-infected animals were morbidly ill and required euthanasia.

FIG. 6.

BDV gene order, coding strategy, and amino acid positions of interest. The numbers on the bottom represent nucleotides, and those on top represent amino acid positions. ORFs in the BDV genome are depicted as boxes. The broken lines show splicing between a small upstream ORF and a downstream ORF of the L-polymerase gene. The sequence of CRP3 had heterogeneity at amino acid position 1686 with both Gly and Arg. Arrows show sites of amino acid changes between CRP3 and CRNP5. Filled circles indicate sites of nucleotide changes between He/80 and CRP3.