Temperature affects reptarenavirus growth in a permissive host-derived in vitro model

Reptarenaviruses cause Boid inclusion body disease (BIBD), a lethal disease primarily affecting captive boa constrictors. The presence of cytoplasmic inclusion bodies (IBs), mainly composed of viral nucleoprotein (NP), in various cell types is characteristic to and a diagnostic criterion of BIBD. We have previously reported that reptarenavirus replication and IB formation are efficient in cell cultures that are maintained at 27-30 °C but not in cells that are kept at 37 °C, the temperature commonly used for mammalian cell cultures. Here, considering the poikilothermic nature of snakes, we studied the ideal temperature(s) for reptarenavirus propagation and the expression of reptarenavirus NP. We incubated Boa constrictor kidney-derived I/1Ki cells at different temperatures (24-36 °C), inoculated them with University of Giessen virus 1 (UGV-1) and monitored both cell growth and virus proliferation. Cell growth was optimal at 30-34 °C and was not significantly affected by UGV-1 infection. Viral RNA release per cell was highest at ambient temperatures between 28 and 32 °C, as determined by qRT-PCR. However, the cells passaged at day 15 post-inoculation released viral RNA at comparable levels even when kept at slightly lower temperatures (24-26 °C). Morphometric analyses undertaken on sections of cell pellets immunostained for reptarenavirus NP found the expression to be most intense at 32 and 34 °C in freshly inoculated cells, and at 28-32 °C in passaged cells. The NP expression positively correlated with the amount of viral RNA released per cell. Our results indicate that the optimal temperature ranges for boid cell growth and reptarenavirus replication (as judged based on antigen expression and RNA release) overlap at about 32 °C. They also suggest that environmental temperature modulation could represent a strategy to impair reptarenavirus replication and, potentially, the spread of reptarenaviruses within and between snake collections.