Narrowed host ranges do not constrain future host range expansion in RNA phage Φ6.

RNA viruses frequently shift between different host species and emerge on novel hosts. Part of this evolutionary process can involve specialization, when viruses adapt to increase their fitness on a particular host, often at the expense of their ability to infect other hosts. This trajectory has the potential to lead to extreme host narrowing, which excludes all other previously accessible hosts. The consequences of the evolutionary history of host specialization on a virus's future evolutionary potential are understudied. Using previously evolved specialized strains of model dsRNA bacteriophage Φ6, which had lost their ability to infect some hosts, we challenged specialists to adapt to their ancestral and other challenge hosts and identified resulting mutations. We found that these specialists readily re-gained their broader host ranges, at rates comparable to previously observed emergence events in Φ6, indicating a lack of potential constraint from their mutational backgrounds due to epistasis. While some viral strains achieved host range re-expansion by reversing the original host-narrowing mutations gained during specialization, others used secondary mutations, which were found to be parallel mutations previously associated with Φ6 entry into those host species. This study contributes to our understanding of the evolutionary dynamics of host shifting in RNA viruses and their strategies to re-expand following specialization, which is relevant to spillback events and recurring host shifts that are observed in nature.IMPORTANCEThe consequences of RNA virus specialization on a virus's future evolutionary potential are not well understood. This study addresses the gap in the literature by investigating the mechanisms of viral host range re-expansion using a model RNA virus. We found that RNA viruses readily re-expand their host ranges to infect both novel and previously accessible hosts, both by mutational reversion and secondary mutations. Investigating these evolutionary mechanisms improves understanding of recurring host shifts and spillback events, which occur often in nature.