Reptarenavirus Co- and Superinfection in Cell Culture Sheds Light on the S and L Segment Accumulation in Captive Snakes

Boid inclusion body disease (BIBD) caused by reptarenaviruses affects collections of captive constrictor snakes worldwide. The disease manifests by formation of cytoplasmic inclusion bodies (IBs) in various tissues. Curiously, a snake with BIBD most often carries more than a single pair of genetically distinct reptarenavirus S and L segments, and the tissues of an infected individual can show variation in the variety of different S and L segment species. The role of reptarenavirus coinfection in development of BIBD remains unknown, and it is unclear how the infection affects the susceptibility to reptarenavirus superinfection or to secondary infection by other agents. Because cell culture studies with mammarenaviruses have demonstrated persistently infected cultures to resist superinfection by genetically similar viruses, we hypothesized that coinfection would only occur if the infecting viruses were of two different species. To study the hypothesis, we employed boa constrictor kidney- and brain-derived cell cultures to perform a set of co- and superinfection experiments with five reptarenavirus and one hartmanivirus isolates. Analysis of viral RNA released from coinfected cells using qRT-PCR did not demonstrate evident competition between reptarenaviruses of the same or different species in the boa constrictor kidney-derived cell line. The experiments on the brain-derived cell line revealed considerable differences in the replication ability of the reptarenaviruses tested, suggesting varying tissue specificity or target cell spectra for reptarenavirus species. Finally, experiments on persistently reptarenavirus infected cell lines showed reduced replication of closely related reptarenaviruses while the replication of reptarenaviruses from different species appeared unaffected. IMPORTANCESnakes with boid inclusion body disease (BIBD) often display reptarenavirus coinfections, or presence of unbalanced S and L segment ratios. Studies on mammarenaviruses suggest replication interference between closely but not between more distantly related viruses. In the study, we provide evidence that similar interference or competition between segments occurs also in the case of reptarenaviruses. Conversely, the results show that there is very little or no competition between more distantly related L or S segments, the cells release similar amounts of viral RNA segments in the case of mono and coinfection. Successful superinfections of persistently infected cell cultures suggest that the unbalanced S and L segment pools often seen in the infected animals could be a result of segment accumulation through sequential reptarenavirus co- and superinfections during breeding in captivity.