Fig. 1.

AiV-A1 A846/88 does not infect prototypic human immortalised cell lines but moderately replicates at the apical side of HIE. (A) AiV-A1 A846/88 was pre-absorbed (MOI=0.1) to the indicated cell lines for 1 h at 4 °C. The cells were washed, medium was added, and the contents of the wells were collected separately at 0 hpi (T0) and 72 hpi (T72). Note that no titre was detected for HCEC-1CT cells at 72 hpi. N = 3. (B) Each cell line was transfected with 1 μg of AiV-A1 A846/88 RNA and different amounts of lipofectamine 2000 (1, 2 and 4 μl); after three days the well contents were collected. Each bar represents the average from the three different lipofectamine amounts. (C–H) HIE-containing wells were simultaneously infected apically and basolaterally with 10⁷ PFU of AiV-A1 A846/88 or CVB3 Nancy for 1 h at 37 °C. After viral challenge, the epithelia were washed once with PBS before continuing the infection at 37 °C. In 24-hour intervals (T0–T96) the basolateral and apical media of the HIE were collected and replaced with new maintenance medium. At the indicated times, the respective membrane was cut out for total RNA extraction. (C and F) Plots of the viral titres in the apical and basolateral SN collected from infected HIE in 24-hour intervals. Note that no progeny virus was detected at some time points in the basolateral SN. Each symbol represents an individual HIE insert. (D and G) Graphs showing the average of the inferred cumulative amount of virus produced per HIE insert at each time point pi. (E and H) vRNA determined by RT-qPCR at the given times. In (F) N = 1 and in (I) N = 3. (I) Top and transverse views of HIE at 24 hpi labelled with mAb J2 antibody (dsRNA - red), Hoechst (nuclei – cyan) and phalloidin (actin filaments - green). Scale bars, 10 μm. (J) Quantification of AiV-A1 A846/88 VP1-positive cells in HIE per 10 fields of view for each time point. N = 3. (K) Quantification of CVB3 dsRNA-positive cells in HIE per 13 fields of view at 24 hpi. N = 1. (L) Immunofluorescence images stained for dsRNA (red) and viral capsid protein VP1 (green) 24 h after AiV-A1 challenge. The box represents the zoomed area shown at the right. Scale bars, 10 μm. Shown is the mean ± SEM.