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LUNG Publication-derived

Enterovirus 71 infection of human airway organoids

Source van der Sanden et al., 2018 · Academic Medical Center, Amsterdam · 10.1038/s41426-018-0077-2

👤 Sabine M. G. van der Sanden, Norman Sachs, Sylvie M. Koekkoek, Gerrit Koen, Dasja Pajkrt, Hans Clevers, Katja C. Wolthers ⏱ 28 days 📋 9 phases 🧫 Human airway basal stem cells

Abstract

This protocol describes the infection of human airway organoids with enterovirus 71 (EV71) clinical isolates and mutant strains to identify viral infectivity determinants. The protocol enables study of EV71–host interactions in a human physiological airway epithelial model, demonstrating strain-dependent replication kinetics and identifying VP1-145 as a key capsid protein determinant of infectivity.

Cell source
Human airway basal stem cells
Application
Viral infectivity determinant identification and disease modeling

Protocol overview

59 steps across 9 phases

Primary human airway organoid culture establishment and maintenance Days −28 to 0
  1. 1 Obtain patient tissue and establish organoid lines
  2. 2 Embed organoid cells in Basement Membrane Extract
  3. 3 Overlay BME droplets with airway organoid culture medium
  4. 4 Culture organoids in humidified incubator
  5. 5 Change medium every 4 days
  6. 6 Passage organoids every 2 weeks
Cell culture of viral amplification strains Days −14 to −1
  1. 1 Culture RD and HT-29 cells for viral amplification
  2. 2 Amplify EV71 clinical isolates
Airway organoid infection with EV71 clinical isolates Days 1–3 post-infection
  1. 1 Prepare organoids for infection
  2. 2 Distribute split organoids and prepare infection medium
  3. 3 Perform 4-hour infection
  4. 4 Wash infected organoids
  5. 5 Resuspend organoids in gel and distribute to wells
  6. 6 Overlay gel with airway organoid culture medium
  7. 7 Collect medium and organoid samples at time points
  8. 8 Store samples for downstream analysis
Viral RNA detection and quantification by real-time PCR Days 4–5
  1. 1 Extract total RNA from lysed organoids
  2. 2 Elute viral RNA in elution buffer
  3. 3 Prepare cDNA synthesis
  4. 4 Perform EV-specific real-time PCR
  5. 5 Normalize and calculate fold-increase in viral RNA
Quantification of infectious viral particles by CCID50 titration Days 4–9
  1. 1 Prepare cell cultures for CCID50 assay
  2. 2 Prepare serial dilutions of medium samples
  3. 3 Inoculate cells with diluted virus
  4. 4 Score cytopathic effect (CPE) in all wells
  5. 5 Calculate CCID50 using Spearman–Kärber formula
Capsid amino acid sequence analysis Days 6–10
  1. 1 Extract viral RNA from clinical stocks and organoid medium
  2. 2 Elute viral RNA
  3. 3 PCR amplify capsid-encoding regions
  4. 4 Purify PCR products
  5. 5 Sequence PCR products by cycle sequencing
  6. 6 Assemble capsid consensus sequences
  7. 7 Identify amino acid changes at key residues
Site-directed mutagenesis to generate VP1-145 mutant viruses Days 11–18
  1. 1 Prepare full-length EV71 cDNA clone templates
  2. 2 Design and synthesize mutagenic primers
  3. 3 Perform site-directed mutagenesis PCR
  4. 4 Digest methylated template DNA
  5. 5 Transform mutated plasmids into competent cells
  6. 6 Select and verify mutant clones by sequencing
  7. 7 Linearize verified cDNA clones
  8. 8 Synthesize viral RNA in vitro
  9. 9 Transfect viral RNA into RD cells
  10. 10 Observe CPE and harvest virus
  11. 11 Amplify mutant virus in fresh RD cells
  12. 12 Quantify mutant viral stocks
Characterization of mutant virus replication kinetics in RD cells Days 19–21
  1. 1 Prepare RD cell monolayers in 96-well plates
  2. 2 Infect RD cells with mutant viruses
  3. 3 Wash infected cells and add growth medium
  4. 4 Harvest infected cells at multiple time points
  5. 5 Determine viral titers at each time point
  6. 6 Plot and analyze replication kinetics
Infection of airway organoids with mutant viruses and replication kinetics analysis Days 22–24
  1. 1 Prepare 14-day-old airway organoids for infection
  2. 2 Distribute split organoids and infect with mutant viruses
  3. 3 Wash and embed infected organoids
  4. 4 Overlay gel-embedded organoids with culture medium
  5. 5 Collect medium and organoid samples
  6. 6 Perform viral RNA detection and quantification
  7. 7 Perform CCID50 titration on organoid culture medium
  8. 8 Plot replication kinetics in organoids

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in van der Sanden et al., 2018. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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