Sequence variability of Borna disease virus: Resistance to superinfection may contribute to high genome stability in persistently infected cells

The RNA genome of Borna disease virus (BDV) shows extraordinary stability i n persistently infected fell cultures, We performed bottleneck experiments in which virus populations from single infected cells were allowed to sprea d through cultures of uninfected cells and in which RNase protection assays were used to identify virus variants with mutations in a 535-nucleotide fr agment of the M-G open reading frames, In one of the cell cultures, the maj or 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 o f a virus stock prepared front 2/1-containing cells, only a minority of the resulting persistently infected cultures contained detectable levels of th e variant, whereas the others all seemed to contain wild-type virus. The BD V variant 2/1 remained stable in the various persistently infected cell cul tures, indicating that the cells were resistant to superinfection by wild-t ype virus. Indeed, cells persistently infected with prototype BDV He/80 wer e also found to resist superinfection with strain V and vice versa, Our scr een for mutations in the viral M and G genes of different rat-derived BDV v irus stocks revealed that only one of four stocks believed to contain He/80 harbored virus with the original sequence. Two stocks mainly contained a n ovel virus variant with about 3% sequence divergence, whereas the fourth st ock contained a mixture of both viruses, When the mixture was inoculated in to the brains of newborn mice, the novel variant was preferentially amplifi ed, These results provide evidence that the BDV genome is mutating more fre quently than estimated from its invariant appearance in persistently infect ed cell cultures and that resistance to superinfection might strongly selec t against novel variants.