FIGURE 3.

Integrity of HEV particle and ORF2 protein, but not HEV gRNA, potently activates inflammasome response. THP‐1 macrophages were transfected with GT3 HEV for 24 hours, and IL‐1β mRNA (A) was quantified by qRT‐PCR (n = 4). IL‐1β protein level (B) in supernatant was quantified by ELISA (n = 4), and (C) mature IL‐1β and cleaved Casp‐1 in supernatant were determined by western blotting. THP‐1 macrophages were transfected with GT1 and ‐7 HEV for 24 hours. IL‐1β mRNA (D) was quantified by qRT‐PCR (n = 6), and IL‐1β protein in supernatant (E) was quantified by ELISA (n = 6). (F) Mature IL‐1β in supernatant or pro‐IL‐1β in lysates were determined by western blotting. (G) Phylogenetic tree of HEV complete genome sequences. Three representative HEV strains (GenBank Accession Numbers: AF444002, JQ679013, and KJ496144) used in this study are indicated. THP‐1 macrophages were inoculated with infectious HEV and UV‐ and heat‐inactivated HEV. (H,J) IL‐1β protein in supernatant was quantified by ELISA (n = 6). (I,K) Mature IL‐1β in supernatant and pro‐IL‐1β in lysates were determined by western blotting. (L) THP‐1 macrophages were incubated with 50 µg/mL of purified recombinant ORF2 protein, and IL‐1β was measured by ELISA after 24 hours (n = 6). Data were normalized to the control (CTR; set as 1). Data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. Abbreviations: NS, not significant (Mann‐Whitney U test); Super, supernatant