High frequency single nucleotide changes and copy number amplification support rapid evolution of vaccinia virus. Vaccinia virus Copenhagen

Viruses continually adapt to evade host immune defenses. To understand how poxviruses adapt to strong selective pressure imposed by innate immune detection pathways, we performed experimental evolution of vaccinia virus in primary human fibroblasts (1°HF). This cell environment places selective pressure on a ΔE3L virus to adapt to nucleic acid sensors like protein kinase R (PKR). Virus genome analyses after ten passages revealed both copy number variation (CNV) of the weak PKR inhibitor K3L and a number of high frequency point mutations in conserved poxvirus genes. Two independent mutations in the viral RNA polymerase were sufficient to provide a fitness advantage compared to the parent ΔE3L virus, suggesting an unexpected role for core genes in evading cellular immune responses. One of these polymerase mutations displayed hallmarks of a selective sweep that was enhanced by the presence of K3L CNV. The genetic interaction between CNV and a point mutation in independent genes provides new insights into the multiple evolutionary benefits of CNV in population dynamics during poxvirus evolution.