Cell culture co- and superinfection experiments suggest that transmission during captivity contributes to the presence of reptarenavirus S and L segment swarms in boid inclusion body disease-positive snakes.

Boid inclusion body disease (BIBD) caused by reptarenaviruses affects captive constrictor snake collections worldwide. The disease manifests by the formation of cytoplasmic inclusion bodies in various tissues. Curiously, a snake with BIBD nearly always carries a swarm of reptarenavirus small and large segments rather than a single pair, and the composition of the swarm can vary between tissues. The role of reptarenavirus coinfections in BIBD pathogenesis remains unknown, and it is unclear whether reptarenavirus infection affects the susceptibility to superinfection or to secondary infections. For mammarenaviruses, co- and/or superinfection can occur if the infecting viruses are genetically divergent enough, and we hypothesized reptarenaviruses to behave similarly. To study this hypothesis, we employed boa constrictor kidney- and brain-derived cell cultures to perform a set of co- and superinfection experiments with one hartmanivirus and five reptarenavirus isolates. While all tested viruses replicated well in the boid kidney cells, experiments on the brain-derived cells showed differences in the replication efficacy between the viruses, suggesting that reptarenaviruses could differ in their target cell spectra. The quantification of viral RNA released from infected cells as a proxy for virus replication did not reveal overt differences between mono- and coinfections. Passaging of coinfected cell cultures revealed that one of the reptarenavirus isolates requires a coinfecting reptarena- or hartmanivirus to establish a persistent infection. Superinfection experiments on persistently reptarenavirus-infected cell lines suggested some interference between genetically similar viruses. We hypothesized that such interference would be mediated by the viral Z protein (ZP) specifically locking the genetically similar viral polymerase in a catalytically inactive state. Curiously, experiments on ZP-expressing cell lines indicated ZP overexpression not to significantly affect the amount of released viral RNA. Our experiments showed very little co- or superinfection interference between genetically dissimilar reptarenaviruses, reflecting the naturally occurring reptarenavirus coinfections in snakes with BIBD.