# Interaction of enterovirus A71 with the human blood-brain barrier: an in vitro study

Léa Gaume, Hélène Chabrolles, Isabelle Simon, Lucie Dehouck, Fabien

Gosselet, Audrey Mirand, Christine Archimbaud, Jean-Luc Bailly

► To cite this version:

Léa Gaume, Hélène Chabrolles, Isabelle Simon, Lucie Dehouck, Fabien Gosselet, et al. Interaction of enterovirus A71 with the human blood-brain barrier: an in vitro study. Société d'études des interfaces Sang Cerveau - SEISC, Nov 2022, Paris, France. . hal-03877075

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Submitted on 29 Nov 2022

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# Interaction of enterovirus A71 with the human blood-brain barrier: an *in vitro* study

Gaume, L.¹, Chabrolles, H.¹, Simon, I.¹, Dehouck, L.², Gosselet, F.², Mirand, A.¹, Archimbaud, C.J.¹, Bailly, J.L.¹

¹ Université Clermont Auvergne, LMGE CNRS, Clermont-Ferrand, France

² Laboratoire de la Barrière Hémato-Encéphalique, Université d'Artois, Lens, France

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## INTRODUCTION

Enterovirus A71 (EV-A71) is an RNA non-polio enterovirus that belongs to the *Picornaviridae* family. This virus is implicated in epidemic disease of hand-foot-mouth disease (HFMD) mostly in infants and children, especially in Asian countries. In Europe since 2015, the emergence of a multi-recombinant C1-like EV-A71 (C1v2015) induced outbreak of **meningitis** and **brainstem encephalitis** in Spain (2016) and France (2016, 2018). Damage to the blood-brain barrier (BBB) could explain such increase in the severity of symptoms.

The BBB is the larger interface between blood and brain, very selective, with a microvasculature of 600 km. **Endothelial cells** (ECs) constitute the wall of brain capillaries and confer to the barrier its function by expression of many adherent and tight junction proteins (ZO-1, claudin-5), specific transporters (Glut-1) and efflux pumps (P-gp, BCRP). **Pericytes**, astrocytes end feet and neurons, which compose the neurovascular unit, surrounds ECs and influences them.

This work aimed to investigate the **passage of free EV-A71 strain** through a **multi-cellular in vitro model of BBB**.

## MATERIAL & METHODS

### Evaluation of the BBB integrity after free viral infection

**Paracellular Permeability**
Basal fluorescence was measured after incubation (1h) with a hydrophilic compound : Lucifer Yellow (LY).

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**Trans-Endothelial Electrical Resistance (TEER)**
The higher the measured resistance, the stronger the tight junction.

**Immunofluorescence (IF)**

Detection of tight junction protein zonula-occludens 1 (ZO-1).

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**Functional test : presence of efflux pump P-gp**

Comparison of intracellular fluorescence of *Rhodamine 123* which naturally enter into EC, release by the efflux pumps P-gp, in the presence or not of a pump inhibitor (Elacridar).

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**CD34+-derived endothelial cells**

Cultivated on the apical side of Transwell™ and coating with Matrigel™

*In vitro* BBB models

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**Bovine pericyte**

Cultivated on the basal side of Transwell™ and coating with type I-rat collagen

Two EV-A71 strain were tested : **C1/16** and **C4/04** (MOI =1), and **echovirus 6 (E6)** was used as a positive control.

**Inflammatory environment (TNF-α)**

BBB was exposed to TNF-α (10 ng/ml)

**Luminal** and **basal** supernatant were analysed at different time post-infection (pi)

* Presence of **infectious virus** : **Titration**

* Presence of **viral RNA** :

* RT-PCR

* Immunofluorescence

**Detection of viral RNA replication (green).** Infection efficiency was evaluated by IF.

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## RESULTS

<figure><img src="image_10.png" /><figcaption>Fig. 1 Percentage of infected cells at 24h pi</figcaption></figure>

Percentage of infected cells realized from 10 confocal microscopy images.

Green = db RNA
Blue = nuclei

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Endothelial cell-CD34+ are **weakly sensitive to EV-A71: at 24h pi, less than 5% of cells** are infected; in contrast, 13% of cells are infected with the E6 positive control.

<figure><img src="image_15.png" /><figcaption>Fig. 2 Endothelial paracellular permeability after 24h and 72h post-infection</figcaption></figure>

An increase of permeability is observed 24h pi between the positive control and negative control. **No difference** is observed between the negative control and EV-A71 infections.

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**Virus yield: not quantifiable (< 10⁰,⁶)**

**Higher amounts of viral RNA** is detected in the **luminal side** than in the **basal side** (Fig 3.A). The yield of infectious virus was quantifiable only in the **luminal** side (Fig 3.B). A moderate amount was produced : less than 10,000 infectious particles at 48h pi. Of note, infectious virus is detected in small amount **in the basal side**.

<figure><img src="image_18.png" /><figcaption>Fig. 3 Supernatants RT-PCR (A) and viral titration (B) in supernatants at different time post-infection (EV-A71 C1-16)</figcaption></figure>

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The TNF-a treatment increases the permeability at 24h **with or without infection** (Fig 4.A). The infectious virus is quantifiable only in the **luminal side** (below 40,000 infectious particles) (Fig 4.B).

* p < 0,05
** p < 0,01
*** p < 0,001
**** p < 0,0001

## CONCLUSION

The integrity of the **BBB-like model** generated *in vitro* by a co-culture of CD34+-derived ECs and pericytes displayed **limited alteration after EV-A71 infection**. Results provide evidence of a **low viral replication** in ECs and **limited crossing** through the *in vitro* BBB model even in an inflammatory environment. Future investigations will focus on the **transmigration** of EV-A71 through infected **leukocytes**.