Mosquito- and tick-borne orthoflaviviruses cross an in vitro endothelial-astrocyte barrier

Abstract

Introduction: The genus Orthoflavivirus of the Flaviviridae family includes several notable pathogens such as mosquito-borne West-Nile virus (Orthoflavivirus nilense, WNV) and Tick-borne encephalitis virus (Orthoflavivirus encephalitidis, TBEV) that are highly neurotropic and may cause severe neurological disease leading to lifelong disabilities, coma and death. These viruses have developed mechanisms to breach the compact blood-brain barrier (BBB) and establish infection within the central nervous system (CNS). Nevertheless, neuroinvasive mechanisms of orthoflaviviruses remain poorly understood. Complex anatomy of the CNS and the organization of the BBB is a major challenge to study neuroinvasion of orthoflaviviruses in vivo. Therefore, in vitro BBB models are useful tools to study direct interaction of viruses with the endothelial barrier.

Methods: In this study, we employed an in vitro transwell BBB model comprising primary mouse brain microvascular endothelial cells and astrocytes to compare the ability of mosquito-borne and tick-borne orthoflaviviruses to cross a compact endothelial barrier and reach the basolateral compartment of the transwell system. The influence of virus inoculation on the barrier properties was determined by measuring transendothelial electrical resistance (TEER).

Results: The results demonstrate that while pathogenic WNV and TBEV cross the endothelial barrier the ability of low pathogenic Usutu virus (USUV) and Langat virus (LGTV) was inconsistent. All viruses tested display virus replication within the endothelial cells. Nevertheless, virus replication did not affect the barrier function of endothelial cells as demonstrated by sustained TEER and absence of leakage of high molecular weight dextran molecules through the endothelial barrier even at several hours post infection.

Discussion: Our findings indicate that orthoflaviviruses can infect the endothelial cells, replicate within them without affecting the cells and its barrier function. Nevertheless, only pathogenic WNV and TBEV showed the ability to cross the endothelial barrier and reach the basolateral compartment.

Keywords: astrocytes; blood-brain barrier; endothelial cells; neuroinvasion; orthoflavivirus; transendothelial electrical resistance.

Figure 1

Transendothelial electrical resistance (TEER) monitored over time after introduction of transwell inserts to the cellZscope the day after seeding of mouse brain microvascular endothelial cells (mBMEC) and four days after astrocyte seeding. Presented are the mean values of n=6 inserts (continuous line) with the standard deviation (SD; dotted lines and filled area) (A). TEER values obtained at 72h (C) and 120h (D) of incubation in the cellZscope were statistically tested with a repeated measures one-way analysis of variance (ANOVA), followed by Tukey’s test for multiple comparison (C, D). Barrier permeability assay of inserts after 120h of incubation in the cellZscope. Presented are the values of n=3 inserts with the SD. Horizontal dotted line indicates 1% of FITC-dextran stock solution, whereas horizontal dashed line indicates 0.1% of applied FITC-dextran concentration (B). FITC-dextran concentrations determined at 240 minutes after apical application were statistically tested with a repeated measures one-way ANOVA, followed by Tukey’s test for multiple comparison (E). Asterisks indicate statistical significance as follows: p < 0.05*, p < 0.01**, p < 0.0001****.

Figure 2

Relative transendothelial electrical resistance (TEER) monitored over time after inoculation of compact mBMEC-endothelial barriers on transwell inserts with West-Nile virus (WNV), Tick-borne encephalitis virus (TBEV), Usutu virus (USUV) and Langat virus (LGTV). Presented are the mean values of N=3 independent experiments (continuous line) with the standard deviation (SD; dotted lines) (A). Barrier permeability assay of inserts at 24 and 48 (B) hours post inoculation with TBEV, LGTV, WNV and USUV. Presented are the mean values of N=3 independent experiments with the standard deviation (SD). Horizontal dotted line indicates 1% of FITC-dextran stock solution, whereas horizontal dashed line indicates 0.1% of applied FITC-dextran concentration (B).

Figure 3

Quantification of viral particles in the apical (A) and basolateral (B) compartments via tissue culture infectious dose 50 (TCID₅₀) assays. Assays were performed on supernatants collected at 24 and 48 hours post inoculation with West-Nile virus (WNV), Tick-borne encephalitis virus (TBEV), Usutu virus (USUV) and Langat virus (LGTV). Presented are the mean values of N=3 independent experiments with the standard deviation (SD). Original data was log-transformed and tested for statistical significance using a repeated measure two-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparison. Asterisks indicate statistical significance as follows: p < 0.001***, p < 0.0001****.

Figure 4

Quantification of infectious virus in the supernatants of mBMEC monocultures inoculated with TBEV, LGTV, WNV and USUV. TCID₅₀ assays were performed on supernatants collected at different timepoints post inoculation. Presented are the mean values of N=3 independent experiments with the standard deviation (SD).