Lab-Grown Blood-Brain Barrier Model Screens Antivirals That Reach the Brain

Neurotropic viruses that invade the central nervous system can leave patients with meningitis, encephalitis and permanent cognitive impairment, yet for many of them, including tick-borne encephalitis virus (TBEV), no clinically approved antiviral drugs exist. A study published in Fluids and Barriers of the CNS now describes a laboratory system designed to close part of that gap: an in vitro model of the human blood-brain barrier (BBB) combined with antiviral testing into a single screening platform the authors call BASA, the BBB-permeable antiviral screening assay. Using the platform, the researchers confirmed that known CNS-penetrating anti-HIV drugs behave as expected, showed that TBEV can cross an intact barrier model to infect astrocytes, and identified a compound that crosses the barrier while retaining antiviral activity against TBEV.

The core of the model is a transwell insert system built from primary human brain microvascular endothelial cells (HBMEC) seeded on the apical side and primary human brain microvascular pericytes (HBMVP) on the basal side. Microscopy and flow cytometry confirmed that the two cell types expressed their characteristic markers, CD31 in the endothelial cells and neural/glial antigen 2 (NG2) in the pericytes, allowing clear distinction between the populations. Barrier function was assessed with two standard readouts: leakage of 70-kilodalton FITC-conjugated dextran and transendothelial electrical resistance (TEER). Co-cultures of endothelial cells and pericytes allowed significantly less dextran passage and developed significantly higher TEER than endothelial monocultures, indicating that the pericytes contribute materially to barrier tightness, consistent with earlier in vitro findings.

The screening concept is a two-stage assay. In stage one, a candidate compound is added to the apical, blood-side compartment of transwell inserts that either contain the endothelial-pericyte barrier or are empty. The empty inserts serve as a control for maximal permeation. After 24 hours, the basal supernatant is collected. In stage two, that supernatant is applied together with virus to a target cell line, and infection is measured after 72 hours by viral antigen detection, viral RNA quantification, or a reporter readout. Because the assay tests the drug that has actually crossed the barrier, it captures not only penetration but also whether the compound remains functionally active after passage, an important consideration since the BBB is a metabolically active interface that can modify drug activity.

To validate the platform, the team turned to anti-HIV drugs, one of the few antiviral classes with well-characterized CNS penetration. They selected nevirapine and abacavir, which carry high CNS penetration effectiveness (CPE) scores, and saquinavir and enfuvirtide, which carry low scores. In the assay, basal supernatants from nevirapine- or abacavir-exposed barrier inserts reduced HIV infection of a reporter cell line to levels similar to those seen with the empty inserts, indicating that both drugs permeated the model. In contrast, infection was significantly higher in cells receiving supernatants from saquinavir- or enfuvirtide-exposed barrier inserts than from empty inserts, and direct drug measurements confirmed substantially lower concentrations behind the barrier. The results tracked the published CPE rankings, and cytotoxicity testing showed the effects were not due to cell death.

With the assay validated, the researchers characterized how TBEV, specifically the highly pathogenic Hypr strain, interacts with the cells of the barrier. In monoculture, fewer than 5 percent of HBMEC stained positive for the Flavivirus envelope protein even at the highest multiplicity of infection tested, and viral RNA remained low, confirming earlier reports of limited TBEV infection of brain endothelial cells. No envelope protein or viral RNA was detected in the pericytes. Notably, TBEV did not disrupt the integrity of endothelial monolayers even at high inocula, in contrast to the transient disruption caused by exogenous TNF-α.

The picture changed for astrocytes. The team differentiated an immortalized human neural stem cell line, HNSC.100, over 14 days into astrocyte-enriched cultures and confirmed upregulation of the astrocytic markers GFAP and AQP4. These cells proved clearly permissive: a significant percentage became envelope-protein positive by two days post-infection across all tested multiplicities, viral RNA rose significantly, and infectious virus was released into the supernatants, indicating productive infection. Cell viability was not significantly reduced despite the high infection levels, and the findings align with published work on primary and induced pluripotent stem cell-derived astrocytes.

To ask whether TBEV can traverse an intact barrier, the researchers assembled a tri-culture: the endothelial-pericyte inserts placed above astrocyte cultures, with virus added to the blood side. In the absence of a barrier, astrocytes were significantly infected by two days post-infection. With the barrier present, infection was delayed but still significant by three days, with similar trends in viral RNA and infectious titers in the basal compartment. Crucially, dextran permeability of the barrier inserts showed no significant changes, suggesting that barrier disruption is not the main route TBEV uses to cross this model. The authors interpret this as pointing toward transcellular passage, an interpretation supported by prior HBMEC monoculture studies and by mouse models indicating TBEV entry into the CNS does not require barrier breakdown. They caution, however, that multiple passage mechanisms may operate during human infection, and that the observed delay could reflect either the barrier’s physical restriction or induced host responses such as interferon-stimulated antiviral genes.

The screening application focused on compound 3d, a nucleoside analog previously reported to inhibit Zika virus replication, with ribavirin as a comparator since it had shown activity against TBEV. In glioblastoma U251 MG target cells, compound 3d displayed a lower half-maximal inhibitory concentration than ribavirin. When applied through the assay, supernatants from ribavirin-exposed barrier inserts produced significantly higher TBEV infection than supernatants from ribavirin-exposed empty inserts, indicating poor permeation, a result consistent with ribavirin’s hydrophilicity and published evidence of limited CNS penetrance. Compound 3d, by contrast, showed no significant difference in infection levels between barrier and empty-insert supernatants, and it retained activity against Zika virus after barrier passage as well. Permeability assays confirmed neither compound disrupted barrier integrity. The authors describe this as the first demonstration that compound 3d crosses the BBB model and remains active against TBEV.

The study’s authors frame BASA as a modular, early-stage preclinical tool. Because the target cell line in stage two can be swapped, the assay could in principle be adapted to other neurotropic viruses with distinct cellular tropisms. The authors also note that quantitative drug titration through the barrier may allow estimation of compound retention, and that the system could reduce reliance on animal experiments by filtering out candidates unlikely to reach and act within the brain, in line with the replacement, reduction and refinement principles of animal research. They acknowledge limitations: every in vitro BBB model has technical and physiological constraints, the model has not yet been applied to mechanisms of barrier disruption or neuroinflammation, and incorporating immune cells and inflammatory factors would be needed to study the interplay of infection and neuroinflammatory signaling at the barrier.

For a field in which drug candidates fail roughly nine times out of ten during clinical development, and in which CNS access adds a further hurdle, the value of a functional screen lies in what it excludes early. By testing whether a compound both crosses a human barrier model and still inhibits viral replication on the other side, BASA offers a way to prioritize antiviral candidates for neurotropic viruses before costlier stages of development, while providing an experimental system in which the passage of viruses such as TBEV across the blood-brain barrier can be studied directly.

Subject of Research: An in vitro human blood-brain barrier model for screening BBB-penetrating antivirals against neurotropic viruses such as TBEV

Article Title: Establishment of an in vitro human blood-brain barrier model for antiviral screening against neurotropic viruses

Article References: Zuniga, M., Herrmann, A., Rizzato, M., Himl, M., Fontes, A., Zischka, H., Silvestri, R., Brancale, A., Bugert, J. J., & Brack-Werner, R. (2026). Establishment of an in vitro human blood-brain barrier model for antiviral screening against neurotropic viruses. Fluids and Barriers of the CNS, 23(1), Article 111. https://doi.org/10.1186/s12987-026-00860-z

Image Credits: AI Generated

DOI: 10.1186/s12987-026-00860-z

Keywords: blood-brain barrier, tick-borne encephalitis virus, antiviral screening, in vitro model, astrocytes, pericytes, brain endothelial cells, nucleoside analogs, HIV drugs, CNS drug delivery, neurotropic viruses, transwell model