Sequence Variability of Borna Disease Virus: Resistance to Superinfection May Contribute to High Genome Stability in Persistently Infected Cells

Abstract

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...

Epidemiology and host spectrum of Borna disease virus infections

“…Its incidence, even in the best-known hosts, horse and sheep in the classical endemic region, has (Rott et al, 1985;Staeheli et al, 2000;Sauder et al, 2002). In addition to IFA, BDV antibodies have also been detected using other serological methods, but as the results are quite incomparable with each other ( declined from the large outbreaks, which affected thousands of animal patients in the 19th century, to 21-69 cases year 21 (reviewed by Staeheli et al, 2000;Friedrich Loeffler Institut, 2012). Whether this results from improved hygiene (including rodent control) of stables, decline of a particular vector species and/or other factors remains to be elucidated.…”

“…Evidence also exists of infections in ostrich (Malkinson et al, 1995), fox (Dauphin et al, 2001), mallard and jackdaw (Berg et al, 2001), wild voles (Kinnunen et al, 2007), macaque (Hagiwara et al, 2008) and raccoon (Hagiwara et al, 2009). In addition, this neurotropic virus experimentally infects tree shrew, rhesus monkey, chicken, rat, mouse, hamster, Mongolian gerbil, guinea pig (reviewed by Staeheli et al, 2000;Pletnikov et al, 2002) and bank vole . Despite the wide possible host range, the incidence of BD in species other than horses and sheep appears low (reviewed by Dürrwald et al, 2006a;Weissenböck et al, 1998).…”

“…1). The highest clinical incidence in animals and the verified classical BD cases, however, are restricted to central Europe (reviewed by Dürrwald et al, 2006a;Staeheli et al, 2000). As several PCR amplicons retrieved from animals and humans outside the classical endemic central European area resemble those of control strains, many PCR results have been suspected to be caused by contamination (Dürrwald et al, 2006b;.…”

“…Although BDV has an RNA genome and lacks the proofreading activity of its polymerase, it has also been demonstrated to be surprisingly stable in persistently infected cell cultures (Formella et al, 2000). A strict selection pressure, probably related to a complicated transmission chain including several species, such as that of rabies virus (Holmes et al, 2002), may provide constraints to genomic variation, as mutations may easily weaken the fitness of BDV in one or more hosts crucial for its survival (reviewed by Dürrwald et al, 2006a).…”

“…It is known as a veterinary pathogen that, in addition to ungulates and carnivores, also infects humans at least occasionally and has its suspected host among small wild mammals (reviewed by Staeheli et al, 2000;Kinnunen et al, 2007;Puorger et al, 2010). Animal Borna disease (BD) has classically been described as a chronic, progressive meningoencephalomyelitis, causing both neurological and behavioural symptoms in horses and sheep (reviewed by Dürrwald & Ludwig, 1997).…”