Increased or decreased numbers of CpG dinucleotide motifs in the genome of influenza A virus do not affect

Cytosine-phosphate-guanine (CpG) dinucleotide motifs are the most underrepresented motifs in RNA virus genomes, including those of the influenza A viruses (IAV). Previous studies have indicated that increasing the number of CpG motifs in IAV genomes negatively impacts viral replication. However, outcomes of these studies have been inconsistent, most likely due to variations in experimental designs. Here, the impact of altered numbers of CpG motifs in the IAV genome was investigated by carefully designing mutants with either increased or decreased frequencies of CpG motifs. Importantly, CpG mutations were excluded from regions essential for viral replication, such as predicted RNA secondary structures and alternative open reading frames, and only mutations naturally occurring in viral isolates were introduced. The resulting mutant viruses showed no significant differences in replication efficiency in human respiratory epithelial cells, nor in their ability to activate the innate immune response. Furthermore, after repeated passage in both human respiratory epithelial (A549) and MDCK cells, the introduced mutations remained stable, indicating no selection pressure on CpG motifs