PMC

Respiratory tissues were inoculated apically with 10⁸ viral particles (normalised based on RNA quantification) of the indicated virus. Apical washes were performed 4hpi and each day afterwards and tissues were lysed 3 dpi to quantify intracellular RNA (A, B and D). mRNA levels of IFN-β (A) and IFN-λ (B) in infected versus uninfected tissue were measured by RT-qPCR while IFN-λ protein levels (C) were measured in culture medium by ELISA. D. Viral loads measured 3 dpi in tissue lysates. **P< 0.01, ***P< 0.001 ****P< 0.0001.

A. Entry assay: viruses (pre-treated or not with acid) were added to cells for 2 hours at 33°C and after extensive washing, viral RNA was extracted for quantification by real-time RT-qPCR. B. Replication assay: viruses were added to cells for 1h at 33°C and after extensive washing, cells were further incubated for 24h. Viral RNA was extracted from total cell lysate and quantified. C. Binding assay: viruses were added to cells for 1 hour at 4°C to prevent entry. After extensive washing, viral RNA was extracted and quantified. D. Binding assay as in C but with cells pretreated with sialidase. For each panel, data are expressed in percentage compared to untreated controls. **P< 0.01. ***P< 0.001. ****P< 0.0001.

EV-D94 RNA transcribed from the cloned clinical isolate E210 was transfected and passaged 6 times in HeLa cells at either 33°C (“EV-D94 adapted to 33°C”, A) or 37°C (“EV-D94 adapted to 37°C, D). A. EV-D94 adapted to 33°C contains 5 non-synonymous mutations relative to the original EV-D94 clone and presents higher titers at 33°C than at 37°C. The VP1 (B) or 2A (C) mutations were introduced independently in the original infectious clone and the two derivatives retain an optimal growth at 33°C. D) EV-D94 adapted to 37°C presents 3 non synonymous mutations relative to the original EV-D94 clone and presents higher titers at 37°C than at 33°C. The VP1 (E) or 2A (F) mutations were introduced independently in the original infectious clone and only the VP1 mutated virus retains an optimal growth at 37°C. G. Mutations in VP1 of EV-D94 adapted to 37°C (E,##) were added to the VP1 mutant adapted to 33°C (B, #) and the viral titers at both temperatures were assessed. *P< 0.05, **P< 0.01.

A. Schematic representation of the artificially engineered chimeric EV-D68/EV-D94 viruses. Construct names are indicated on the left. EV-D68 regions are represented by white boxes; EV-D94 regions are in grey. Results obtained upon transfection and passage in HeLa cells are indicated on the right, as is the percentage of nucleotide and amino acid sequence identity between the exchanged regions. V, viable construct; X, non-viable construct; V/X, viable unfit construct. B. Non-synonymous adaptation mutations observed in viral stocks after 6 passages in HeLa cells. Sequencing was performed from nt 45 to 7344. Production of viral stocks by transfection at both 33°C and 37°C was attempted for all viruses, but only EV-D94 could be recovered at 37°C. For EV-D94, the stock subsequently used for phenotypic assessment is the one prepared at 33°C. HeLa 33 and HeLa 37: viral stocks transfected and amplified at 33°C and 37°C respectively.

In vitro reconstituted human tissues were inoculated with equivalent amount of EV-D94, EV-D68 and EV-D94/D68_P1 and replication was assessed by RT-qPCR. A. 1^st Panel: Virus production at the apical tissue side: respiratory tissues were inoculated apically and washed 3 times 4 hpi. Apical samples were collected at the indicated time point for viral RNA quantification. Residual: residual bound virus after 3 washes. 2^nd Panel: Mucociliary clearance of infected respiratory tissues assessed by measuring the displacement velocity of polystyrene microbeads applied at the apical side of the tissue 5 dpi. Statistics compare infected and uninfected tissues. 3^rd Panel: Immunofluorescence of respiratory tissues 5 dpi with ciliated cells stained in green, viruses in red and cell nuclei in blue. B. Small intestine tissues were infected apically and replication was quantified as for respiratory tissues. C-D. Neurons (C) and neural tissues (D) were incubated with viral suspension and viral RNA extracted from tissue lysate after inoculation and 2 or 4 days later were compared. **P< 0.01. ***P< 0.001.