Andrews, T.; Isaac, A.; Duffy, S.

RNA viruses frequently shift between infecting different hosts 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 genetic architecture of host specialization on a virus\'s future evolutionary potential are understudied. In this study, we explored the ability of extreme specialists to re-expand their host ranges, particularly the ease and mutational mechanisms by which they might accomplish this such as reversion of host-range-narrowing mutations or mutations at other sites. Using previously evolved specialized strains of model dsRNA bacteriophage phi6 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 phi6, 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 phi6 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.