The variation in the TM domain of E protein played a potential role in PEDV-host interaction. (A) Alignment of the amino acid sequences of the V4 region in E protein from DR13, ca-DR13, 85-7 parent, C30, C40, and E40 strains. C30, C40, and E40 shared the same variation, with a deletion of 16LWLFV20 and the L25P mutation. (B) Analysis of the putative TM domain of the PEDV parent and variant E protein with TMHMM Server v.2.0 . The TM domain was predicted as I15 to L37 and I10 to L32, respectively. (C) Comparative analysis of the mRNA levels of HSPA5 (encoding GRP78) and the protein production of GRP78 among the parent-, C30-, and C40-infected Vero cells. (D, E) Comparative analysis of the mRNA levels of the genes encoding IL-6 and IL-8 among the parent-, C30-, and C40-infected Vero cells by qPCR assay. The β-Actin gene served as an endogenous control. Error bars indicated the means of three independent experiments. (F and G) and (H) Comparative analysis of the mRNA levels of the genes encoding GRP78, IL-6, and IL-8 among the recombinant plasmids-transfected Vero cells by qPCR assay. The corresponding amount of empty plasmid (pIRES2-EGFP) was used as the mock control. (I) Comparison of the cell apoptosis level by flow cytometry with dual Annexin V-PI cell labeling. The infected cells were collected at 36 hpi, and the mock-infected cells were used as the control. Fluorescence-activated cell sorting was used to detect the fluorescent signals of Annexin V and PI, using channels FL-1 and FL-2, respectively. The figure was representative of two independent experiments. The graph representing the percentage of fluorescent signals of Annexin V and PI in each quadrant was shown on the left. (J) Comparative analysis of the levels of early apoptosis. C30, C40, and E40 showed higher early apoptosis levels than the 85-7 parent, A40, B40, and D40 strains.