Propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures - PubMed (original) (raw)

. 2018 Jun;141(6):2074-2084.

doi: 10.1016/j.jaci.2017.07.018. Epub 2017 Aug 8.

Francisco Brito 2, Sacha Benaoudia 3, Léna Royston 1, Valeria Cagno 1, Mélanie Fernandes-Rocha 4, Isabelle Piuz 1, Evgeny Zdobnov 2, Song Huang 3, Samuel Constant 3, Marc-Olivier Boldi 5, Laurent Kaiser 4, Caroline Tapparel 6

Affiliations

Propagation of respiratory viruses in human airway epithelia reveals persistent virus-specific signatures

Manel Essaidi-Laziosi et al. J Allergy Clin Immunol. 2018 Jun.

Abstract

Background: The leading cause of acute illnesses, respiratory viruses, typically cause self-limited diseases, although severe complications can occur in fragile patients. Rhinoviruses (RVs), respiratory enteroviruses (EVs), influenza virus, respiratory syncytial viruses (RSVs), and coronaviruses are highly prevalent respiratory pathogens, but because of the lack of reliable animal models, their differential pathogenesis remains poorly characterized.

Objective: We sought to compare infections by respiratory viruses isolated from clinical specimens using reconstituted human airway epithelia.

Methods: Tissues were infected with RV-A55, RV-A49, RV-B48, RV-C8, and RV-C15; respiratory EV-D68; influenza virus H3N2; RSV-B; and human coronavirus (HCoV)-OC43. Replication kinetics, cell tropism, effect on tissue integrity, and cytokine secretion were compared. Viral adaptation and tissue response were assessed through RNA sequencing.

Results: RVs, RSV-B, and HCoV-OC43 infected ciliated cells and caused no major cell death, whereas H3N2 and EV-D68 induced ciliated cell loss and tissue integrity disruption. H3N2 was also detected in rare goblet and basal cells. All viruses, except RV-B48 and HCoV-OC43, altered cilia beating and mucociliary clearance. H3N2 was the strongest cytokine inducer, and HCoV-OC43 was the weakest. Persistent infection was observed in all cases. RNA sequencing highlighted perturbation of tissue metabolism and induction of a transient but important immune response at 4 days after infection. No majority mutations emerged in the viral population.

Conclusion: Our results highlight the differential in vitro pathogenesis of respiratory viruses during the acute infection phase and their ability to persist under immune tolerance. These data help to appreciate the range of disease severity observed in vivo and the occurrence of chronic respiratory tract infections in immunocompromised hosts.

Keywords: Respiratory virus; cytokines; cytotoxicity; immune response; mucociliary clearance; pathogenesis; persistence; rhinovirus.

Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

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Figures

Fig 1

Fig 1

Virus production at the apical (A) and basal (B) sides of air-liquid interface culture of reconstituted human airway epithelia. Viral RNA loads measured by using qPCR from infected samples collected at the apical (Fig 1, A) or basal (Fig 1, B) side of tissues infected with 9 different respiratory viruses (n ≥ 4) are shown. Each tissue was inoculated apically with 106 RNA copies of the indicated virus and washed 3 times after 4 hours. Samples were then collected from the apical or basal face at the indicated time point. Input, Viral RNA load present in the inoculum for each viral stock; p.i., post-inoculation; Residual, residual bound virus after 3 washes.

Fig 2

Fig 2

Immunofluorescence of epithelia infected with the indicated respiratory virus. A, Three-dimensional view of noninfected and infected tissues, with ciliated cells stained in green, viruses in red, and cell nuclei in blue. For all viruses, immunofluorescence was performed at day 5 after infection, whereas for H3N2 and EV-D68, immunofluorescence was also done at day 2, as indicated. B, Two-dimensional view of tissues infected with H3N2 at day 5 after infection (virus stained in green, mucus cells in red [Muc5A], basal cells in red [P63], and cell nuclei in blue), with magnification insets of selected regions (*) in each panel. DAPI, 4′-6-Diamidino-2-phenylindole dihydrochloride.

Fig 3

Fig 3

Effect of infections on epithelia. Viral toxicities calculated from the amount of LDH released by damaged cells in the basal medium (n = 8; A) and TEER (n ≥ 2; B) were measured at days 2 and 5 post infection (p.i.). The 48-hour time point represents LDH accumulated during 48 hours; for later time points, the medium was replaced every day, and data correspond to LDH secretions within the preceding 24 hours. Dotted lines in Fig 3, B, correspond to the established threshold of tissue integrity. *P < .05, **P < .01, and ***P < .001. Asterisks in light gray show statistical significance relative to noninfected tissue, whereas black asterisks show statistical significance compared with all other respiratory viruses at each time point.

Fig 4

Fig 4

Effect of respiratory viruses on cytokine production. Chemokine (A) and cytokine (B) levels were measured in basal medium 96 hours after infection with the indicated respiratory virus (n = 4). Data correspond to cytokine secretions within the preceding 24 hours. *P < .05, **P < .01, and ***P < .001. Asterisks in light gray show statistical significance relative to noninfected tissue, whereas black asterisks show statistical significance compared with HCoV-OC43. Differences between other viruses are shown with bars. Additional cytokines (IL-33, TGF-β, IL-25, IFN-β, and thymic stromal lymphopoietin [TSLP]) were also tested but either were not significantly induced by viral infections or were not detected in basal medium (Fig E1).

Fig 5

Fig 5

Effect of respiratory viruses on MCC. Displacement velocity of polystyrene microbeads applied at the apical side of the tissue at 5 dpi with indicated virus is shown (n ≥ 5). ***P < .001 (vs noninfected).

Fig 6

Fig 6

Virus production at the apical site of air-liquid interface cultures of reconstituted human airway epithelia over a prolonged time period. Viral RNA loads measured by using qPCR from infected samples collected at the apical side of tissues infected with 9 different respiratory viruses are shown. Each tissue was inoculated with 106 RNA copies of the indicated virus, and tissues were washed 3 times after 4 hours; samples were then collected from the apical surface at the indicated time point (n = 4).

Fig 7

Fig 7

Comparison between the top 30 most significantly enriched biological processes in tissues infected with RV-C15 and RV-B48 at the indicated time. Circle size represents the number of differentially expressed genes found on that pathway, whereas the color represents the adjusted P value. The top 100 genes showing significant changes in each condition is available in Table E2, as well as the enrichment analysis for gene ontology biological processes (see Table E3) and the reactome pathway (see Table E4). SRP, Signal recognition particle.

Fig E1

Fig E1

Fig E2

Fig E2

Fig E3

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Fig E4

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Fig E5

Fig E5

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