Abstract
Mammalian orthobornaviruses, such as Borna disease virus 1 (BoDV-1) and variegated squirrel bornavirus 1, are zoonotic pathogens that cause fatal encephalitis in humans. BoDV-2, another mammalian orthobornavirus with high genetic homology to BoDV-1, is believed to share the same geographical distribution as BoDV-1, indicating its potential risk to human health. However, due to the limited number of isolations, the virological characteristics of BoDV-2, such as pathogenicity and infectivity, remain largely unexplored. Here, we re-evaluated the whole-genome sequence of BoDV-2 and established a reverse genetics system to investigate its virological properties. Compared to the published reference sequence, we identified two nonsynonymous nucleotide substitutions in the large (L) gene, one of which was critical for restoring polymerase activity, enabling the successful recovery of recombinant BoDV-2 (rBoDV-2). Additionally, we identified two nonsynonymous single-nucleotide polymorphisms (SNPs) in the L gene and one in the phosphoprotein (P) gene. Substitution of these SNPs significantly enhanced the growth ability of rBoDV-2. Furthermore, our studies demonstrated that BoDV-2 does not induce superinfection exclusion in cells, allowing the persistence of low-fitness genome variants for an extended period of time. These findings help to characterize the virological properties of BoDV-2 and shed light on how bornaviruses maintain genetic diversity in infected cells.