Enterovirus 71 infection of human airway organoids reveals VP1-145 as a viral infectivity determinant

Fig. 1. Replication kinetics of enterovirus 71 (EV71) in human airway organoids. a, b Fold increases in viral RNA of EV71 subgenotype strains C1 (C1 91-480), C2 (2485), C4 (75-Yamagata), C5 (209-VN), B3 (SK-EV006), and B4 (C7-Osaka) in donors N39 and N41, determined by RT-PCR on lysed organoids. Data present the mean fold increase in two infection experiments + standard error of the mean (SEM). c Viral titers detected in the medium covering the gel-embedded organoids at 72 h postinfection (p.i.). Titers are expressed as the mean 50% cell culture infective dose (CCID50)/ml in two infection experiments + SEM

Fig. 2. Amino acid sequence comparison of the EV71 strain capsid regions included in the current study. Residue 145 of the VP1 capsid region is marked by a box

Fig. 3. Replication kinetics of EV71 subgenotype C1 strains with a glutamine (Q) or glutamic acid (E) at VP1 residue 145 in human airway organoids. a, b Fold increases in viral RNA of EV71 strains in donors N39 and N41, respectively. Values presented were calculated from RT-PCR assays of lysed organoids. c, d Viral titers detected in the medium covering embedded organoids of donors N39 and N41, respectively, at 0 to 72 h postinfection. Titers are expressed as the 50% cell culture infective dose (CCID50)/ml

Fig. 4. Growth characteristics of EV71 C1, C2, and B3 strains with a glutamic acid (E), glycine (G), or glutamine (Q) at VP1-145, generated by site-directed mutagenesis, in rhabdomyosarcoma (RD) cells. Data represent the mean 50% cell culture infective dose (CCID50)/ml in two infection experiments + SEM

Fig. 5. Replication kinetics of EV71 strains with a glutamic acid (E), glycine (G), or glutamine (Q) at VP1-145, generated by site-directed mutagenesis, in human airway organoids. a Fold increases in viral RNA of C1, C2, and B3 strains with VP1-145E, -G, or -Q in donor N41, determined by RT-PCR assay on lysed organoids. Data present the mean fold increase in two infection experiments + SEM. b Viral titers detected in the medium covering embedded organoids at 0–72 h postinfection. Titers are expressed as the mean 50% cell culture infective dose (CCID50)/ml in two infection experiments+SEM

Fig. 6. Comparison of EV71 VP1 amino acid sequences that were retained from the organoid culture medium 72 h postinfection