Viral Genome Sequencing from Cell Supernatant

Viral RNA extraction from cell supernatant was performed as described above. A set of specific primer pairs (Table S3) was used to produce overlapping amplicons covering the entire viral genome with the SuperScript III One-Step RT-PCR System (Thermo Scientific). Complete genome sequencing was performed using the Ion PGM Sequencer (Thermo Scientific). Read sequences were analyzed as previously described [7 (link)]. To assess the genetic diversity of viral populations, mutation frequency for each position was calculated as the number of reads with a mutation compared to the reference, divided by the total number of reads at that site (minimum coverage of 500). For the analysis, only substitutions with a frequency of at least 1% were taken into account (Table S4).