FIGURE 3.

Increasing CpG content in the N gene results in slower growth of porcine epidemic diarrhea virus (PEDV) in VeroE6 cells. (A) Analysis of generated nucleotide sequences encoding PEDV-N protein with varied CpG dinucleotides. Red lines in a sequence bar display silent mutations where the original nucleotides are replaced to increase CpG content. Synonymous codon substitutions, CpG dinucleotide compositions, and codon-paired bias (CPB) values of each sequence are listed in the Table. (B) RNA folding free energy profiles for each 100- and 20-base window of the generated CPD-N sequences compared to the native N gene sequence. The red bars indicate the high (<–30 Kcal/mol) free energy of RNA folding. (C) Construction of infectious clones using the N genes with high CpG content. PCR-amplified products of an oligonucleotide fragment from the S gene to the M gene (S-mCherry-E-M) and another fragment of various N genes (N′) were inserted into a pre-cut vector containing the rest of the viral genes (pPEDV.mCh.MluI.3UTR) by the In-Fusion ligation method. (D) VeroE6 cells were infected with each recombinant PEDV (MOI = 0.001) and virus replication was monitored at the indicated time points by the TCID₅₀ assay. Values shown are averages ± SEM of three independent experiments. (E) Representative images of virus spread in the infected cells at 96 hpi are shown.