The absence of superinfection exclusion of Borna disease virus 2 maintains genomic polymorphisms in persistently infected cells

Abstract Viruses belonging to the genus Orthobornavirus within the family Bornaviridae are known to infect various vertebrate species, including mammals and birds. Within the genus, the species Orthobornavirus bornaense includes two mammalian viruses: Borna disease virus 1 (BoDV-1), the prototype of the family, and its closely related virus, BoDV-2. BoDV-1 was identified as the causative agent of Borna disease (BD) in horses, sheep, humans, and other mammals. BoDV-2 was first detected in a pony in eastern Austria in 1999 that exhibited severe and incurable neurological symptoms. Although BoDV-2 shares approximately 80% nucleotide identity with BoDV-1, its virological properties, including host range, replication ability, and pathogenicity, remain unclear. In this study, we aimed to investigate the virological properties of BoDV-2 by re-evaluating its whole-genome sequence using RNA sequencing. Compared to the published reference sequence, we identified two nonsynonymous nucleotide substitutions in the large (L) gene. One of these substitutions was found to be critical for the restoration of polymerase activity, enabling the successful recovery of recombinant BoDV-2 (rBoDV-2) through reverse genetics. We also 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. In addition, our studies showed that BoDV-2 does not induce superinfection exclusion in cells, allowing 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. Importance Mammalian bornaviruses are a general term for viruses belonging to the genus Orthobornavirus that infect mammalian species, e.g., Borna disease virus 1 and 2 (BoDV-1 and -2) and variegated squirrel bornavirus 1 (VSBV-1). Although BoDV-1 and VSBV-1 are reported to be associated with fatal encephalitis in humans, the infectivity and pathogenicity of BoDV-2 in humans remain unclear. To determine the virological properties of BoDV-2, we developed a reverse genetics system for BoDV-2. By using recombinant BoDV-2s, we identified several nucleotides that affect the growth ability of BoDV-2 and revealed the molecular mechanisms through which BoDV-2 maintains genetic heterogeneity in persistently infected cells. This reverse genetics system will accelerate the biological studies of BoDV-2 and contribute to the development of countermeasures against mammalian bornaviruses.