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.Borna disease virus (BDV) is the causative agent of severe meningoencephalitis in horses, sheep, and other animal species in central Europe (7,13,21,22), and it is suspected to contribute to human psychiatric disorders worldwide (1, 2, 6, 8-12, 14, 15, 20). The pathogenesis of Borna disease is mediated by a T-cell-dependent immune mechanism. All natural isolates of BDV that have been available to date, independent of species (humans, horses, sheep, cats, dogs, etc.), area (Europe, the United States, Japan), and year of isolation (1929 to 1998), show a remarkably high sequence conservation of the 8.9-kb RNA genome (5,11,16,17). Human BDV isolates are 95 to 100% identical to animal-derived BDV at the nucleotide level and are 97 to 100% identical at the amino acid level (5,11,16). BDV is therefore considered a potential zoonotic agent. Because serum antibody titers are frequently low in naturally infected individuals and BDV serology has several other limitations, reverse transcriptase PCR (RT-PCR) technology using primers that match sequences of the viral N or P genes (1,2,5,6,8,9,12,15,16,17,19) is now widely used for the diagnosis of BDV infection. In this report, we describe a novel subtype of BDV which escapes detection by currently used diagnostic RT-PCR protocols. MATERIALS AND METHODSImmunohistochemistry. Paraffin-embedded brain sections were stained with the monoclonal antibody BO18, directed against the N protein, and a polyclonal mouse antiserum, directed against the X protein of BDV (3), as described previously (21,22). Staining of cultured cells grown on glass coverslips was carried out according to standard procedures, by using polyclonal antisera raised against purified recombinant X and P proteins of BDV He/80.Sample preparation and RNA extraction. Samples from hippocampus and rhinencephalon were homogenized by using liquid nitrogen and were resuspended in diethyl pyrocarbon...
The RNA genome of Borna disease virus (BDV) shows extraordinary stability in persistently infected cell cultures. We performed bottleneck experiments in which virus populations from single infected cells were allowed to spread through cultures of uninfected cells and in which RNase protection assays were used to identify virus variants with mutations in a 535-nucleotide fragment of the M-G open reading frames. In one of the cell cultures, the major virus species (designated 2/1) was a variant with two point mutations in the G open reading frame. When fresh cells were infected with a low dose of a virus stock prepared from 2/1-containing cells, only a minority of the resulting persistently infected cultures contained detectable levels of the variant, whereas the others all seemed to contain wild-type virus. The BDV variant 2/1 remained stable in the various persistently infected cell cultures, indicating that the cells were resistant to superinfection by wild-type virus. Indeed, cells persistently infected with prototype BDV He/80 were also found to resist superinfection with strain V and vice versa. Our screen for mutations in the viral M and G genes of different rat-derived BDV virus stocks revealed that only one of four stocks believed to contain He/80 harbored virus with the original sequence. Two stocks mainly contained a novel virus variant with about 3% sequence divergence, whereas the fourth stock contained a mixture of both viruses. When the mixture was inoculated into the brains of newborn mice, the novel variant was preferentially amplified. These results provide evidence that the BDV genome is mutating more frequently than estimated from its invariant appearance in persistently infected cell cultures and that resistance to superinfection might strongly select against novel variants.Genome replication of RNA viruses is error prone, because RNA-dependent RNA polymerases lack proofreading activity (10, 18). Nevertheless, field isolates of certain RNA viruses exhibit a high degree of genetic stability over many decades (10). The selective forces which restrict virus variability are presumably complex and nonuniform. In most cases, the mechanisms of restriction are largely unknown.Borna disease virus (BDV) is a newly classified nonsegmented negative-strand RNA virus that can persistently infect the central nervous systems of a broad range of warm-blooded animals and possibly humans without destruction of its host cells (11,13,21,29). Natural and experimental infections with BDV usually result in immune-system-mediated neurological disease and behavioral abnormalities (5,13,14,34). Sequence comparisons between old and recent BDV isolates of diseased animals from regions of endemicity in Central Europe revealed viral genome conservation of greater than 95%, in spite of the fact that some of these viruses were passaged many times in experimental animals or in cell culture (3, 28). Recent evidence indicates that BDV strains from outside the classical regions of endemicity (e.g., Japan, Sweden, and the United S...
Borna disease virus (BDV) replicates and transcribes its negative-sense RNA genome in the nucleus. The BDV phosphoprotein (P) is localized in the nucleus of infected cells and cells transfected with P expression constructs. To identify the nuclear localization signal (NLS) of P, COS-7 cells were transfected with wild-type or mutant forms of P fused with green fluorescent protein (GFP). Whereas GFP alone was exclusively cytoplasmic, P or P-GFP were nuclear. Analysis of carboxy-and aminoterminal truncation mutants of P indicated that amino acids (aa) 20-37 are sufficient to promote efficient nuclear accumulation of the fusion protein.Residual nuclear import of GFP was observed with portions of P including aa 33-134 or aa 134-201, suggesting the presence of additional NLS motifs. The major NLS of P appears to be bipartite. It consists of two basic aa domains, R22RER25 and R30PRKIPR36, separated by four non-basic aa, S26GSP29.Borna Disease virus (BDV) is a non-segmented negativestrand RNA virus that causes persistent central nervous system infection and behavioural disturbances in warm-blooded animals (Ludwig et al., 1988 ; Rott & Becht, 1995). It encodes at least six proteins : the nucleoprotein (N), phosphoprotein (P) (Thiedemann et al., 1992 ; Thierer et al., 1992), atypical glycoprotein (gp18) (Kliche et al., 1994 ; Stoyloff et al., 1994), type I membrane glycoprotein (p57) (Gonzalez-Dunia et al., 1997 ; Schneider et al., 1997), polymerase (pol) and X-protein (Briese et al., 1994 ; Cubitt & de la Torre, 1994 ; Wehner et al., 1997). BDV replicates in the nucleus (Briese et al., 1992 ; Cubitt et al., 1994) and employs the cellular splicing machinery for the maturation of some of its viral transcripts (Schneemann et al., e-mail ilipkin!uci.edu 1995). Thus, transport of viral RNAs and proteins between nucleus and cytoplasm is an essential feature of the BDV lifecycle. A functional nuclear localization signal (NLS) was recently described for the N protein (Kobayashi et al., 1998 ; Pyper & Gartner, 1997). Three lines of evidence indicate that P may also contain an NLS and mediate nuclear import of other BDV proteins : (1) transient expression of P results in its nuclear accumulation ; (2) X interacts with P but not N ; and (3) the presence of P in transfected and infected cells shifts the distribution of X from the cytoplasm to the nucleus (Schwemmle et al., 1998).To identify regions of P that mediate nuclear localization we employed a strategy similar to that used to characterize the NLS of lymphoid enhancer factor-1 (Prieve et al., 1996). A set Fig. 1. Localization of P, GFP and P-GFP fusion proteins after transient expression in COS-7 cells. Plasmids encoding BDV-P, P-GFP and GFP, or P-GFP mutants lacking the amino-terminal 32 aa (N∆32), 93 aa (N∆93) and 133 aa (N∆133) or the carboxy-terminal 67 aa (C∆67), 107 aa (C∆107) or 166 aa (C∆166), were transiently expressed in COS-7 cells. The fusion proteins were detected by immunofluorescence after incubation with antisera to P or GFP. 0001-5790 # 1999 SGMJH
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