Review Article

Brain Organoids as Emerging Platforms for Modeling CNS Infections: Neuropathogenesis, Therapeutic Discovery, and Drug Delivery

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DOI:

10.3791/70757

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August 7th, 2026

 ,  ,  , 
1Laboratory of Molecular Virology, George Mason University

Corresponding Authors: Fatah Kashanchi <fkashanc@gmu.edu>

* These authors contributed equally

In This Article

Summary

This review examines brain organoids as platforms for modeling selected neurotropic viral infections and highlights their applications in viral pathogenesis, antiviral discovery, innate immune responses, and central nervous system (CNS)-targeted drug delivery.

Abstract

Neurotropic viruses remain a persistent global health challenge, and the mechanisms by which they damage the human brain are not yet fully understood. Animal models are often limited by human-specific aspects of CNS biology, whereas two-dimensional (2D) cell cultures cannot recapitulate the complex three-dimensional (3D) cellular interactions that occur during viral infection of the brain. Over the past decade, brain organoids derived from human stem cells have emerged as physiologically relevant models that address these limitations. These 3D cultures self-assemble into structures containing neurons, astrocytes, and progenitor cells arranged in patterns that resemble early brain development. This review examines the application of brain organoids to the study of infections caused by Zika virus (ZIKV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), herpes simplex virus (HSV), and human immunodeficiency virus type 1 (HIV-1). Studies were screened from PubMed and shortlisted based on their relevance to organoid-based CNS infection modeling, antiviral drug screening, CNS-targeted drug delivery, and neuroinflammation. Organoid-based antiviral screening has identified promising compounds from libraries containing more than 1,000 candidates. Drug delivery strategies are also discussed, with particular emphasis on nanoparticles, polymer-based carriers, and extracellular vesicles (EVs) evaluated in organoid and spheroid models for their ability to cross the blood-brain barrier (BBB) and deliver therapeutic cargo to neural cells. The roles of damage-associated and pathogen-associated molecular patterns (DAMPs and PAMPs) in neuroinflammation, complement evasion, and chronic post-infection damage, including long COVID, are also examined. Current limitations, including the lack of functional vasculature, incomplete BBB components, and reproducibility challenges, are discussed. Despite these limitations, CNS organoids bridge the gap between basic research and clinical application, advancing the development of effective therapies for viral infections of the brain.

Introduction

The central nervous system (CNS) is a complex system comprising distinct functional regions of the brain and spinal cord. CNS organoids, often referred to as "mini-brains," are three-dimensional (3D) multicellular structures that mimic the developing human brain. They are primarily generated from human pluripotent stem cells (hPSCs), which can self-assemble into tissues that represent diverse brain regions. These mini-brains recapitulate the epigenetic patterns, structure, and transcriptional markers of the developing brain1,2,3.

The generation of cerebral organoids using patient-derived induced pluripotent stem cells (iPSCs) was first developed in 20134. Prior to this discovery, access to human brain tissue was limited because of the ethical issues associated with conducting experimental studies, particularly those involving fetal or in utero brain tissue2. The development of cerebral organoids demonstrated the ability to mimic human neurodevelopment, including the formation of distinct brain regions such as the cerebral cortex. Lancaster et al. provided the first experimental evidence that stem cells can form complex neural structures in vitro, offering a model that more accurately reflects human physiology than conventional two-dimensional (2D) cell cultures5. Importantly, certain human neurodevelopmental conditions, such as microcephaly, have proven difficult to replicate in rodent models, further emphasizing the need for human-derived systems6.

CNS organoids contain multiple cell types that differentiate and form multilayered structures resembling the developing brain7, making them well-suited for high-throughput studies of neurogenesis, neuronal migration, and developmental abnormalities8. The generation of CNS organoids begins with pluripotent stem cells (PSCs), which include iPSCs and embryonic stem cells. Embryonic stem cells are exposed to growth factors and signaling inhibitors to promote differentiation into neural cells and mimic early embryonic development2. Once the cells begin to aggregate, they undergo self-organization within a 3D environment and are often embedded in an extracellular matrix to provide structural stability. Organoids are then typically maintained under dynamic culture conditions, such as spinning bioreactors or orbital shaking, to enhance nutrient and oxygen delivery, and support continued growth9. Region-specific organoids of the forebrain, midbrain, and hindbrain can also be generated using guided protocols in miniaturized spinning bioreactors, with distinct brain-like regions typically emerging over several weeks to months9,10,11. CNS organoids capture many key structural features of the developing human brain, including ventricular regions containing radial glia that scaffold migrating neurons and intermediate progenitors that amplify neuronal populations12. Early neurons form cortical-like layers with emerging synaptic networks. Although organoids do not fully replicate the size or organization of the mature brain, they are more physiologically relevant than traditional monolayer cultures.

A major advantage of CNS organoids is their ability to model developmental timing. Cultures progress from weeks to months and parallel human fetal brain development. This allows researchers to observe neurological processes such as neurogenesis, axon extension, and early neural circuit formation in real time. Organoids can also be programmed to include microglia, blood vessel-like cells, or even blood-brain barrier (BBB) components, such as astrocytes, endothelial cells, and pericytes. These organoids can support studies of immune responses and substance transport13. Additionally, organoids can be combined to create assembloids, which are self-organizing systems that model communication between different brain regions14. With these capabilities, CNS organoids offer a direct way to investigate how neurotropic viruses invade, replicate within, and damage the brain, questions that have long been difficult to address using conventional models.

There are a vast number and diversity of neurotropic viruses that can affect the human brain, including flaviviruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), herpes simplex virus (HSV), and human immunodeficiency virus type 1 (HIV-1). The development of effective antiviral therapies is crucial yet remains challenging due to the complexity of virus-brain interactions. The brain is considered an immune-privileged organ, supported by the BBB, which prevents large immune cells and molecules from entering the brain from the bloodstream.

Neurotropic viruses have evolved diverse strategies to breach this barrier, including transcellular migration, disruption of tight junctions, and the "Trojan horse" mechanism, whereby infected immune cells carry the virus across the endothelium4. As a result, immune surveillance in the brain is restricted, making viral clearance more difficult. Furthermore, the cellular components of the BBB, including endothelial cells, astrocytes, and pericytes, express pattern recognition receptors that detect viral pathogen-associated molecular patterns (PAMPs) and mount innate immune responses, although pro-inflammatory signaling can paradoxically worsen BBB disruption15. Additionally, limited access to human brain tissue hampers studies of viral interactions with neural cells and the delivery of potent antiviral agents to the CNS. These combined challenges hinder the discovery of new antiviral agents to treat viral infections at all stages of development and across the lifespan, making it essential to better understand how viruses interact with the brain and to develop new therapeutic strategies to combat these infections16. Overall, this review emphasizes the utility of CNS organoids for studying neurotropic viral infections, mechanisms of neuroinflammation, and antiviral drug discovery. Figure 1 provides an overview of the major applications of brain organoids in modeling neurotropic viral infections, investigating host-pathogen interactions, and advancing antiviral therapeutic development.

figure-introduction-1
Figure 1: Brain organoids as human-relevant platforms for modeling neurotropic CNS viral infections. The figure provides an overview of the key topics addressed, with a focus on three-dimensional (3D) brain organoids as experimental models. Neurotropic viruses discussed in this review include HIV-1, HSV-1/2, SARS-CoV-2, and ZIKV. Different brain organoid models enable studies of viral pathogenesis, host-pathogen interactions, blood-brain barrier (BBB) dysfunction, and neuroinflammatory responses involving neurons, astrocytes, microglia, and brain endothelial cells. Brain organoids provide physiologically relevant platforms for antiviral drug screening and evaluation of antiviral therapies. Created in BioRender. Kashanchi, F. (2026) https://BioRender.com/y7eenju Please click here to view a larger version of this figure.

Review and Perspective

Therapeutic discovery in virus-infected brain organoids

Antiviral screening

There is a lack of approved therapies for many neurotropic viral infections, limiting treatment options beyond supportive care. Brain organoids are a valuable tool for discovering novel antiviral compounds by enabling drug screening in a physiologically relevant, 3D environment that resembles human brain tissue. In an initial study, a high-content chemical screening of 1,120 FDA-approved drugs and drug candidates was performed against ZIKV in fetal-like brain organoids. Antiviral activity was assessed based on cell proliferation and suppression of ZIKV infection. Of all the compounds tested, nine showed potential, with two compounds—hippeastrine hydrobromide (HH) and amodiaquine dihydrochloride dihydrate (AQ)—standing out because of their strong antiviral effects, significantly reducing infection by suppressing viral RNA production17.

Several subsequent studies explored compounds targeting different stages of the viral life cycle. In a study by Watanabe et al., 25-hydroxycholesterol (25HC), a compound that blocks viral entry by inhibiting membrane fusion, reduced ZIKV mRNA levels in cortical organoids by ~74% and decreased ZIKV-positive cells by ~72%. Furthermore, the same study evaluated antiviral compounds targeting the AXL receptor expressed on neural progenitor cells. A small-molecule inhibitor of the AXL tyrosine kinase domain, R428, produced a modest reduction in ZIKV mRNA levels (~41%). However, treatment with an anti-AXL antibody did not significantly reduce ZIKV mRNA levels18. Finally, three anti-flaviviral drugs, duramycin, ivermectin, and azithromycin, were tested in organoids. Duramycin and ivermectin significantly reduced ZIKV mRNA levels, whereas azithromycin showed no protective effect against ZIKV, as determined by RT-qPCR and immunostaining18.

In a separate study, Xu et al. screened 173 compounds as suppressors of ZIKV-induced caspase-3 activity and identified emricasan as the most potent neuroprotective candidate without affecting cell proliferation in brain organoids. Two additional compounds, niclosamide and PHA-690509, demonstrated significant anti-ZIKV activity by inhibiting viral replication at a post-entry stage in a dose-dependent manner. Furthermore, co-treatment with emricasan and PHA-690509 produced an additive effect and preserved astrocyte viability following ZIKV infection19. In another study, enoxacin, an RNA interference (RNAi) enhancer, demonstrated direct antiviral activity in RNAi-competent cells and completely prevented ZIKV-induced microcephalic phenotypes in brain organoids, suggesting that enhancing intrinsic antiviral pathways in neural progenitor cells may represent a viable therapeutic strategy20.

Pettke et al. subsequently screened 110 structural analogues of TH3289 at multiple doses in ZIKV-infected organoids21. Two compounds, TH3289 and TH6744, showed the strongest antiviral activity, and the most active hits shared a benzimidazolone core. This study was significant because the organoid system enabled not only rapid compound screening but also mechanistic follow-up. For example, TH6744 was found to interfere with late stages of the ZIKV life cycle21.

Brain organoids have also been used to identify therapeutic candidates for SARS-CoV-2 infection. An FDA-approved anti-hepatitis C drug, sofosbuvir, improved neuronal survival and rescued impaired synaptogenesis in SARS-CoV-2-infected brain organoids22. Brain organoids have also contributed to understanding HSV-1-mediated CNS infection; however, antiviral drug screening for HSV-1 in organoid models remains limited compared with studies of ZIKV and SARS-CoV-2. A landmark study using iPSC-derived neurons demonstrated that TLR3 deficiency renders human neurons susceptible to HSV-1 infection, revealing a human-specific phenomenon that rodent models had failed to capture23. More recently, Bellizzi et al. demonstrated that clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 targeting of HSV-1 genomic sequences suppressed both primary infection and viral reactivation in 3D organoid cultures, establishing gene editing as a feasible antiviral strategy for herpesvirus CNS infection24. Given that acyclovir resistance is an emerging clinical problem and no curative therapy exists for HSV encephalitis25, the absence of large-scale antiviral compound screening against HSV-1 in brain organoids represents a significant knowledge gap.

In addition to small molecules, antisense oligonucleotides (ASOs), which target RNA and modify gene expression, are emerging therapeutic tools for treating various diseases, and cerebral organoids can be employed to evaluate novel ASO therapies26. Given the utility of organoids for ASO studies, important characteristics such as biodistribution, toxicity, and therapeutic efficacy could be investigated in virus-infected brain organoids.

One of the most important applications of brain organoids is assessing the neurotoxicity of existing antiviral drugs. This is particularly important because commonly used HIV treatments have been shown to be toxic to brain cells both in vitro and in vivo27. Akay et al. demonstrated the neurotoxicity of antiviral drugs in primary cortical cultures containing microglia, astrocytes, and neurons. MAP2-positive neurons showed a dose-dependent decrease following exposure to ritonavir and saquinavir. Treatment with combinations of antiretroviral drugs (cART) resulted in acute neurotoxicity, and cultures treated with zidovudine (AZT) exhibited increased production of reactive oxygen species (ROS)28. Although these findings were obtained using 2D cultures, brain organoids could expand this research by providing a more physiologically relevant platform for high-throughput neurotoxicity screening of individual or combination antiretroviral therapies. To investigate the mechanisms underlying antiviral-induced neurotoxicity in these 3D models, techniques such as morphological analysis, functional assays, and transcriptomic profiling can be employed29.

In addition to neurotoxicity screening, brain organoids have been developed to study HIV-associated neurocognitive disorder (HAND), which continues to affect 30%–50% of people living with HIV despite effective antiretroviral therapy (ART). Microglial cells are the primary reservoir of HIV-1 in the CNS, and organoid models have been instrumental in elucidating how microglial infection drives neuronal damage. Kong et al. used iPSC derived cerebral organoids containing microglia and observed a marked increase in CCL2 and CXCL10 chemokine gene expression, together with activation of multiple type I interferon-stimulated genes (MX1, ISG15, ISG20, IFI27, IFITM3, and others) following HIV-1 infection30. The inflammatory gene expression profile was propagated to bystander cells, including uninfected neurons. These neurons exhibited a marked decline in neurotransmitter transporter expression together with increased expression of genes associated with cellular senescence and cell death. The use of brain organoids for HIV research was pioneered by Dos Reis et al., who incorporated HIV-infected microglia into human brain organoids31. This model reproduced the low-level viral replication typically observed in the human brain. Incorporation of HIV-infected microglia triggered a neuroinflammatory response associated with astrogliosis, neurodegenerative pathology, and degeneration and loss of neuronal synaptic processes. Donadoni et al. subsequently used human induced pluripotent stem cell (hiPSC)-derived cerebral organoids containing microglia and astrocytes to model NeuroHIV and evaluate the effects of combination antiretroviral therapy (cART) in a 3D organoid system32. Collectively, these organoid-based studies provide important mechanistic insights into HAND and establish a valuable model system for investigating neuroprotective therapies.

In conclusion, brain organoids provide a unified model system for screening antiviral compounds while simultaneously assessing neurotoxic effects early in the drug development process. Identification of effective antiviral compounds is only the first step toward clinical translation (Table 1). Equally important is the development of drug-delivery strategies capable of delivering therapeutic agents to infected cells within the CNS.

VirusOrganoid ModelCell TypesKey FindingsLimitations Reference
ZIKVhPSC-derived fetal-like organoidsNeural progenitors, neuronsHH and AQ reduced viral RNA from 1120-compound screenNo immune cells; fetal stage17
Cortical organoidsNeural progenitors25HC reduced infection by ~74%; R428 reduced ZIKV mRNA by ~41%No vasculature18
Brain organoidsNeurons, astrocytesEmricasan neuroprotective; niclosamide and PHA-690509 inhibit replicationNo microglia19
Brain organoidsNeural progenitorsEnoxacin prevented ZIKV-induced microcephaly via RNAiNo immune cells20
Brain organoidsNeuronsTH3289 and TH6744 (benzimidazolone core) interfere with late ZIKV life cycleNo immune cells21
SARS-CoV-2Cerebral organoidsNeuronsSofosbuvir rescued synaptogenesis and neuronal survivalNo BBB22
HSV-1iPSC-derived neuronsNeuronsTLR3 deficiency leaves human neurons susceptible; human-specific phenomenon2D neurons only23*
3D organoid culturesNeuronsCRISPR-Cas9 suppressed primary infection and reactivationLimited maturation24
HIV-1iPSC cerebral organoidsNeurons, microgliaCCL2, CXCL10 upregulation; type I interferon gene activation; neuronal senescenceNo vasculature30
Human Brain ORGanoids with microglia Neurons, microgliaNeuroinflammation, astrogliosis, synaptic degenerationNo vasculature31
hiPSC cerebral organoidsNeurons, microglia, astrocytescART effects evaluated in 3D NeuroHIV modelEarly stage model32

Table 1: Brain organoid models used to study neurotropic viral infections. This table summarizes the virus studied, organoid model, cell types included, key findings, limitations, and reference for representative brain organoid-based studies of ZIKV, SARS-CoV-2, HSV-1, and HIV-1 infections.

Drug delivery platforms

Finding the most effective antiviral compounds for treating CNS infections is essential, but without efficient delivery to the brain, these compounds may fail to achieve therapeutic benefit. Brain organoids can be used to evaluate drug delivery systems for their ability to penetrate neural tissue and deliver antiviral payloads.

Lipid-based nanoparticles (LNPs) are widely used as drug carriers. These drug carriers were shown to increase drug bioavailability and enable targeted drug delivery to the disease site, especially across the BBB33. LNP-based mRNA formulations were successfully utilized in COVID-19 vaccination34,35. Brain organoids can be used to evaluate the effectiveness and safety of new LNP-based formulations. LNPs can also be vectorized to the CNS by surface modification with targeting ligands. For example, transferrin or lactoferrin were conjugated to LNPs to facilitate receptor-mediated transcytosis and enhance transport across the BBB36,37. Collectively, the potential drug delivery efficacy, neurotoxicity, and changes in nanoformulation stability after BBB penetration can be investigated in brain organoids38.

In addition to LNPs, dual-targeted polypeptide nanocarriers were also studied as a drug delivery system in human brain organoids. Targeted poly(L-glutamic acid) nanoparticles (NPs) showed excellent cytocompatibility and the ability to enter midbrain-like organoids. It was demonstrated that alanine and glutathione could efficiently direct NPs to cerebral endothelial cells and penetrate cerebral organoids, resulting in drug delivery to the brain39. The permeability of polymer-based nanoparticles was investigated in human 3D microvasculature organoids, focusing on the effects of their size and surface chemistry. The generated organoids consisted of human iPSC-derived endothelial cells, astrocytes, and primary brain pericytes. Confocal images revealed that conjugation of the NPs with the brain-associated ligand holo-transferrin (Tf) significantly increased their permeability40.

Next, the absorption, penetration, and distribution of gold nanoparticles (AuNPs) were investigated in BBB brain spheroids. These spheroids are composed of six brain cell types: neurons, astrocytes, pericytes, endothelial cells, microglia, and oligodendrocytes. The authors reported that transient hypoxia led to BBB opening and increased transport of AuNPs into the spheroid interior. The nanoparticles were able to enter cells and nuclei easily41. In a similar study using the same spheroid model system, concurrent tracking of nanocarrier penetration kinetics and their therapeutic payload release was evaluated by confocal laser scanning microscopy42. In another study, Park et al. demonstrated that brain-derived neurotrophic factor (BDNF)-conjugated AuNPs successfully entered cerebral organoids without causing significant toxicity43.

Beyond targeting moieties, the shape and size of nanoparticles can also play a significant role in a nanoformulation's ability to deliver its therapeutic payload to the brain44. Polystyrene 200 nm spheres vectorized with antibodies against VCAM-1 accumulated 1.2- to 1.5-fold greater than the control non-vectorized NPs. Interestingly, rod-shaped particles exhibited even greater accumulation of 2.1- to 2.5-fold over corresponding controls, suggesting that rod-shaped particles offer an advantage over spheres in terms of cell binding. Cerebral organoids can provide detailed insights into how particle geometry can affect brain penetration. Additionally, the effects of hypoxia and other biologically active compounds on BBB permeability could be further investigated in neurovascular brain organoids45,46.

Stem cell-derived extracellular vesicles (EVs) represent a biological alternative to synthetic drug carriers such as LNPs. Branscome et al. explored the reparative potential of EVs against HIV-1 infection in human neurospheres47. In this study, it was demonstrated that EVs produced from mesenchymal stem cells (MSCs) and iPSCs can naturally penetrate neurospheres, rescue cellular viability, and reduce the expression of inflammatory cytokines in HIV-1-infected neurospheres, highlighting the neuroprotective and anti-inflammatory properties of stem cell EVs47. In another study by Kim et al., small extracellular vesicles (sEVs) were engineered for antiviral delivery to treat SARS-CoV-2 infection48. The nanocarriers were loaded with soluble ACE2 (sACE2) via genetic modification of the donor cells with a plasmid encoding eGFP-CD9ΔTM4-sACE2(WT), resulting in the expression of sACE2 on the sEV surface. It was demonstrated that sACE2-vectorized sEVs protected against WT and mutant S protein pseudotyped virus48. The protection mechanism could be tested in SARS-CoV-2-infected brain organoids. Additionally, another study showed that EVs released from HSV-1-infected cells carry innate immune components such as STING, which can activate innate immune responses in recipient cells, resulting in a potent inhibition of HSV-1 replication. A crucial separation of EVs from infectious viral particles was achieved by density gradient centrifugation49.

Cerebral organoids can also be used to test the potential neurotoxicity of new drug nanoformulations. For instance, when pegylated AuNPs were injected into brain organoids, significant inflammation, mediated by ROS production, was observed. In contrast, polymeric poly-lactic acid NPs exhibited efficient drug delivery and low cytotoxicity due to their slow uptake and degradability, enabling penetration into the brain organoid without eliciting an immune response50. The neurotoxicity of AuNPs, graphene, and carbon dots was also assessed in 3D neurovascular organoids. These organoids consisted of neural cells, microglia, and an endothelial cell surface. These studies revealed that microglia could play a pivotal role in the permeability and toxicity of these formulations51. Furthermore, high concentrations of ZnO NPs have also been shown to cause significant neuronal death. The mechanism of toxicity was linked to intracellular accumulation of Zn ions, which decreased LC3B protein levels, suggesting defective autophagy52. Overall, these studies show that 3D brain organoids can capture how nanoformulations interact with neural tissue, from uptake and penetration to immune activation and toxicity. The success of CNS-targeted drug delivery strategies ultimately depends not only on reaching target cells but also on modulating the inflammatory pathways activated during infection, many of which are initiated by DAMPs and PAMPs signaling.

DAMPs and PAMPs in viral infections

Brain organoids can be used to study DAMPs and PAMPs that are released by stressed, injured, or dying cells in the CNS. DAMPs include heat shock proteins, ATP, and uric acid, whereas PAMPs include bacterial, viral, fungal, and parasitic macromolecules. Hence, these molecular patterns alert the innate immune system and activate several signal transduction pathways in response to foreign antigens or any sign commonly associated with infection. The mechanisms underlying their protective effects in response to viral infections are not fully understood, and detailed investigations of these processes can be carried out in brain organoids53. Released DAMPs and PAMPs bind to a large group of receptors, including Toll-like receptors (TLRs), NOD-like receptors (NLRs), AIM2-like receptors, RIG-I-like receptors (RLRs), and C-type lectin receptors (CLRs), which are expressed on the plasma membrane or intracellularly54. These receptors are crucial for cytokine and interferon production in response to neurotropic viral infections, including EV-71, HIV-1, HSV-1, flaviviruses, West Nile virus (WNV), and others54.

Slowikowski et al. investigated how DAMPs, chemokines, and adhesion molecules released by damaged neural cells coordinate leukocyte entry into the CNS during flavivirus encephalitis55. In particular, it was revealed that WNV increases the expression of highly inflammatory necroptosis and pyroptosis cell death markers that promote the release of DAMPs in the infected brain. When such molecules, for example, high mobility group box 1 (HMGB1) protein, are released, they induce multiple signaling pathways, leading to CXCR4-dependent migration of activated monocytes, macrophages, and dendritic cells to infected tissues56,57. Furthermore, it was shown that DAMPs can be released in response to infection, followed by upregulation of TLRs and exacerbation of CNS inflammation and tissue damage58. Overall, these studies indicate that viral infections appear to trigger overlapping DAMP-PAMP signaling pathways, leading to neuroinflammation and tissue damage. Collectively, brain organoids containing microglia and astrocytes could serve as a model for tracing immune cascade responses in human neural tissue, especially the interplay between DAMP release and TLR-mediated neuroinflammation.

The complement system is a major element of innate immunity that responds rapidly to infection. However, many viruses have developed mechanisms to overcome this defense system59. Certain viruses evade the complement system by producing proteins that bind to complement components and either inhibit or sequester them. This has been demonstrated in a variety of viruses, including human T-lymphotropic virus-1 (HTLV-1), human cytomegalovirus (HCMV), HIV-1, simian virus 5 (SV5), and mumps virus (MuV), which incorporate host complement proteins into their virions (i.e., CD55/DAF, CD59, CD64, and MCP). These proteins inhibit the innate immune response, promoting viral replication and persistence60. Thus, brain organoids can be used to identify the specific mechanisms by which viruses evade the complement system. Growing evidence suggests that DAMPs and PAMPs not only shape the initial antiviral response but also serve as key mediators of the persistent inflammation that underlies chronic post-viral conditions.

Role of DAMPs/PAMPs in persistent inflammation caused by viral infection

Beyond the acute inflammatory phase, viral infections can cause chronic exposure to DAMPs/PAMPs, resulting in long-term inflammatory consequences, particularly in the brain. Cerebral organoids can become a powerful tool to study the underlying mechanisms of syndromes related to the post-acute phase of infection.

Most currently known human non-persistent viruses activate immune responses during the acute phase of infection, followed by pathogen elimination. However, in some cases, patients experience persistent symptoms that may differ from those observed during the acute phase and can linger for months or even years. Specifically, patients with long COVID-19 report chronic fatigue, "brain fog," fever, pain, and other neurological complications61. Moreover, chronic inflammation may increase mortality and morbidity, as well as the risk of atherothrombosis, cancer, and cognitive impairment62,63.

Additionally, the persistence of viral proteins and RNA can lead to elevated levels of pro-inflammatory cytokines, such as TNF-α, IL-1α, IL-1β, IFN-γ, and IL-6, thereby causing tissue damage, premature aging, and immune system exhaustion64. Slow replication or traces of viral components could trigger the release of DAMPs/PAMPs that interact with their specific receptors, causing persistent low-grade inflammation65. In some cases, HSV-1 infection of the brain can lead to neuronal damage followed by encephalitis and may eventually contribute to the development of neurodegenerative disorders such as Alzheimer's disease66. Disrupted autophagy mechanisms can be a major trigger of chronic inflammation in patients with HSV-2, in which defective protective mechanisms lead to persistent viral replication, the continuous presence of viral components in the brain, and neurodegeneration67. These viral components activate innate immune sensors through PAMPs and can manipulate signaling pathways to bypass the antiviral effects of innate immune activation. Importantly, HCMV and Epstein-Barr virus (EBV) have been shown to establish chronic viral infections that promote immune dysfunction68,69. Sustained infection-induced senescence may also contribute to the long-term inflammation associated with various viral infections. 3D brain organoids with integrated immune components provide relevant platforms for understanding the complex mechanisms underlying chronic exposure to DAMPs/PAMPs and their role in the long-term complications of viral infections in the CNS.

Limitations and perspective:

Despite their advantages over traditional in vitro models, brain organoids have significant limitations that must be addressed to support their broader application. Specifically, most organoids lack resident immune cells, which are major contributors to viral disease modeling and to understanding innate and adaptive immune responses during viral infection70. In particular, standard organoid protocols lack incorporated microglia, the primary CNS immune cells and reservoir of HIV-1, which limits the modeling of neuroinflammation and viral persistence71. Another shortcoming is incomplete tissue complexity, including the absence of vasculature, which plays an important role in nutrient and oxygen delivery, as well as viral dissemination72. Thus, organoid growth is limited because cells in the center cannot receive adequate nutrients. Additionally, excretion of metabolic waste is impaired, often leading to hypoxic or necrotic cores in large organoids. Moreover, organoids do not fully reproduce the interactions among neurons, endothelial cells, and stromal cells that can influence viral infection and pathogenesis. Furthermore, at this stage of development, organoids cannot recapitulate systemic infection, interorgan spread, and whole-body immune responses73. Another limitation is that many epithelial organoids have limited accessibility for viral inoculation due to their inward-facing apical surface74. As a result, viral inoculation requires microinjection, leading to variability and low throughput. Thus, inoculation of organoids with different diameters makes the multiplicity of infection difficult to control. Additionally, many organoids resemble fetal tissue rather than adult organs, which can affect viral tropism, replication kinetics, and host responses73. This is particularly evident in the case of HSV-1, as neuronal maturation impacts latency establishment and reactivation processes that fetal-stage organoids may not fully recapitulate23. Next, organoids may vary considerably in composition and function, leading to varied outcomes, low reproducibility, and the need for numerous experimental replicates. For example, organoids may differ in size, cellular composition, architecture, and maturation state. This makes standardization difficult, if not impossible, across laboratories7,75. Finally, the high cost and limited scalability of organoid generation and maintenance remain significant barriers. As a result, a major challenge is the development of high-throughput platforms compatible with organoid technology74.

Despite these shortcomings and limitations, organoids hold significant potential for modeling viral infections, toxicology, and drug discovery. Overcoming these limitations could significantly reduce reliance on animal models and facilitate fundamental mechanistic studies76. One of the notable shortcomings of existing organoid models is their limited physiological complexity and lack of diverse cellular composition. Future developments will need to focus on incorporating immune, neuronal, vascular, and stromal components into organoid models. This could be achieved using co-culture models that integrate organoids with the aforementioned cell types77,78. Specifically, incorporating immune cells will enable modeling of systemic immune responses and inflammation during viral infections. Furthermore, vascularized organoids can be further developed to improve physiological complexity and better recapitulate in vivo conditions79,80. Thus, cerebral organoids incorporating endothelial tissues will support investigations of blood-brain barrier function and drug delivery during viral infections13,81. Additionally, 3D brain organoids can replicate several key aspects of viral infection in the brain. In fact, several organoid models with intricate structures that closely represent various brain regions are currently under investigation82. Finally, inclusion of neuronal and stromal components would further increase the fidelity of organoids to native organ systems. Reproducibility remains another serious barrier to the broader application of organoid technologies. Therefore, generating robust, highly reproducible organoids is crucial for the further translation of organoid applications. Various methods for cell isolation and seeding, as well as the selection of matrices and soluble factors, should be standardized across the field83. Of note, the composition of the culture medium, cell density, and culture time may affect the outcomes of these models. Therefore, strategies to reduce matrix variability, including synthetic hydrogels composed of fully defined polymer networks with precisely controlled chemical and mechanical characteristics, have been introduced as a promising alternative84. These organoids exhibit minimal batch-to-batch variability, enabling controlled studies. Furthermore, the creation of organoid biobanks representing diverse patient populations would become a valuable resource for drug discovery and biomarker identification. Emerging artificial intelligence (AI) technologies are becoming important tools in organoid research85. Further application of these technologies may accelerate analyses and reduce bias in data interpretation. Nevertheless, AI applications in this field remain in their early stages. Incorporation of AI approaches into organoid development, such as AI-driven image analysis86 or predictive optimization of culture conditions87, would significantly facilitate organoid research.

Conclusions

This review has examined how brain organoids, 3D neural cultures that closely mimic the human neurovascular unit, are being used to study the invasion, replication, and CNS damage caused by neurotropic viruses. Although each virus has unique genetic and structural features, many of the pathological processes triggered by viruses in the brain are shared, and organoid models have proven well-suited for dissecting these common and different mechanisms. Excessive inflammation can worsen nerve cell damage; however, when the innate immune system is properly controlled, it can limit viral spread and protect brain tissue.

3D organoids enable the study of both effects in a more physiologically relevant setting, where animal models and 2D cell cultures have failed to capture these dynamics. This information is critical for combating viral pathogenesis in the brain and for identifying treatments that target the right pathways without increasing inflammation. Brain organoids have advanced understanding of neurotropic viral infections, but some technical gaps remain to be addressed. For instance, slow maturation, limited vasculature, and the absence of BBB components remain common challenges. Variability in organoid differentiation techniques and batch-to-batch inconsistencies also hamper research involving brain organoids. Moreover, quantification of viral entry, replication, cell-type tropism, and host responses in a controlled 3D context can be improved. The work reviewed in the manuscript has shown that the brain organoids are among the most relevant systems available for studying how neurotropic viruses interact with the human brain, testing antiviral candidates, and evaluating drug delivery to the CNS. As technology matures with improved vascularization, immune cell integration, and the adoption of standardized protocols, organoid-based studies will be better positioned to identify compounds and strategies with genuine potential for clinical translation.

Abbreviations: ASOs, antisense oligonucleotides; BBB, blood-brain barrier; CLRs, C-type lectin receptors; CNS, central nervous system; CRISPR, clustered regularly interspaced short palindromic repeats; CXCL10, C-X-C motif chemokine ligand 10; DAMPs, damage-associated molecular patterns; EVs, extracellular vesicles; hPSCs, human pluripotent stem cells; HSV-1/2, herpes simplex virus type 1/2; HIV-1, human immunodeficiency virus type 1; IFNs, interferons; IL-6, interleukin-6; ISGs, interferon-stimulated genes; LNPs, lipid nanoparticles; NLRs, NOD-like receptors; PAMPs, pathogen-associated molecular patterns; PRRs, pattern recognition receptors; RNAi, RNA interference; RT-PCR, reverse transcription polymerase chain reaction; sACE2-EVs, soluble ACE2-decorated extracellular vesicles; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; scRNA-seq, single-cell RNA sequencing; TLRs, Toll-like receptors; TNF-α, tumor necrosis factor-alpha; ZIKV, Zika virus; 3D, three-dimensional. ZIKV, Zikavirus; hPSC, human pluripotent stem cell; HH, hippeastrine hydrobromide; AQ, amodiaquine dihydrochloride dihydrate; 25HC, 25-hydroxycholesterol, BBB, blood–brain barrier; cART, combination antiretroviral therapy; CRISPR, clustered regularly interspaced short palindromic repeats; hiPSC, human induced pluripotent stem cell; HIV-1, human immunodeficiency virus type 1; hPSC, human pluripotent stem cell; HSV-1, herpes simplex virus type 1; RNAi, RNA interference; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TLR3, Toll-like receptor 3.* iPSC-derived 2D neuron study included for comparison.

Disclosures

ChatGPT (OpenAI; GPT-5.5) was used in a limited capacity to assist with literature screening and language refinement. All literature selection, fact-checking, reference verification, and scientific interpretation were performed independently by the authors.

Acknowledgements

The authors gratefully acknowledge all members of the Kashanchi Laboratory, with special appreciation to Gwen Cox for her invaluable support. This study was also supported by National Institutes of Health (NIH) Grants R01MH134389, AI078859, and AI074410 R21DA057887 and Department of Defense HT9425-24-1-0876 to F.K.

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Neurotropic VirusesAntiviral Drug ScreeningBlood Brain BarrierNeuroinflammationExtracellular VesiclesNanoparticle Delivery

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