Orthopoxvirus vaccinia strain:Copenhagen VVdelEdelK+RhTRS1 Genome sequencing. Orthopoxvirus vaccinia strain:Copenhagen VVdelEdelK+RhTRS1

To establish a productive infection in new host species, viruses must overcome barriers mediated by diverse and rapidly evolving host restriction factors such as protein kinase R (PKR). To model the process of host switching, we employed a recombinant vaccinia virus (VVΔEΔK+RhTRS1) expressing the rhesus cytomegalovirus PKR antagonist RhTRS1, which inhibits PKR in some African green monkey cells, but not in human or rhesus cells. Serial passaging VVΔEΔK+RhTRS1 in minimally permissive AGM fibroblasts resulted in rapid amplification of the rhtrs1 locus, which improved VVΔEΔK+RhTRS1 replication in AGM fibroblasts and expanded the virus host range to human and rhesus cells. Notably, serial passage of VVΔEΔK+RhTRS1 in human fibroblasts did not improve virus replication. These results support the hypothesis that amplification of a weak viral antagonist of a host defense in one species may be a critical intermediate step enabling cross-species transmission into new hosts that are nonpermissive to the initial virus.