Human Assembloid Model of Emergent Neurotropic Enteroviruses
Posted on: 1 April 2026
Preprint posted on 19 November 2025
We might be close to obliterating poliovirus, but non-polio enteroviruses are on the horizon. No pressure tho: Stanford’s hSpO-hSkM assembloid might also arm us to fight these new villains.
Selected by Mitchell Sarmie
Categories: immunology, microbiology, neuroscience
Why I pick this preprint:
I love learning about infectious diseases and their evolutionary abilities to subvert and invade host immune barriers. Studying these mechanisms at the molecular and cellular levels is intricate; thus, I have chosen to highlight this preprint’s usage of an in-vitro assembloid model which provides a clear and concise insight into emerging neurotropic enteroviruses host-pathogen interactions and how we can leverage this information to study other neurotropic viruses like herpes and West Nile viruses.
Introduction:
Emerging and re-emerging RNA viruses have shown to be one of mankind’s greatest foes: the SARS-CoV-2 pandemic, HIV/AIDS, Influenza, etc1, just to mention a few. Every time the scientific community makes progress to decipher the underlying cellular and immunological mechanisms of these viral agents, they evolve and outsmart us2. One rapidly rising concern involves meningitis in children caused by an unknown viral agent3. Meningitis leads to paralysis, mainly poliomyelitis and polio-like acute flaccid myelitis. As a result of global mass vaccination campaigns, science has never been so close to eliminating poliovirus (poliomyelitis) than now. But as these stories always go, there are new villains. Non-polio enteroviruses, EV-A71 and EV-D68 (causing polio-like acute flaccid myelitis), are now the center of attention, given the limited understanding of these viruses’ host-pathogen interactions4,5. To address these villains, this preprint presents a miniature 3D assembloid model that mimics EV-A71 and EV-D68 infections and treatment in humans.
The Assembloid Architecture
In 2020, the authors of this preprint designed a human spinal cord/hindbrain (hSpO) organoid from induced pluripotent stem cells (iPSCs). This basically means they constructed a model that includes all the necessary cells to study diseases associated with the spinal cord. In addition to this, they blended this promising hSpO organoid model with another differentiated skeleton myoblast-specific organoid (hSkM) that forms a functional neuromuscular model (hSpO-hSkM) to study the paralysis mechanisms of Poliovirus, EV-A71 and EV-D68.
Key Findings
Neurotropic Enteroviruses Warfare
Poliovirus, EV-A71 and EV-D68, come alive once inside a host neurotropic cell. All three viruses productively showed infectivity in the hSpO model. They also suppressed muscle contractions in the hSpO-hSkM assemboids, which rupintrivir prevented, revealing that active replication of these viruses causes paralysis (Preprint Figs. 1A-J). Next, a time-course test was conducted to determine the susceptibility and permissiveness of each virus separately. The results showed that Polio virus replicates more robustly than EV-D68 and EV-A71, reaching higher intracellular RNA levels and producing higher extracellular viral titers earlier in the infection (Preprint Figs. 2A-G).
Innate responses and Tropism in the Human Spinal Cord/Hindbrain Organoid (hSpO)
To evaluate the hSpO model’s immune response to the enteroviruses, the multi-analyte luminex-based assay was used. Regardless of the absence of microglia, the model secreted high levels of pro-inflammatory cytokines (e.g. IL-6, IL-8, CCL5, etc.), but exhibited a low induction of interferons (I, II, and III), which validates the viral permissiveness in the model because a system with high interferon activity has a complete immune arsenal against viruses and vice versa (Preprint supplementary figure 1A). Next, single-cell RNA-seq was performed to investigate the viral tropism. The results showed that all three viruses infect motor neurons, but with distinct tropisms (Preprint Figure 3). More specifically, Polio virus and EV-D68 infected motor neurons, whereas EV-A71 had a greedy preference for both glial lineages (astroglia and cycling progenitors) and motor neurons (Preprint Figs. 4A–D).
Post-Infection Features
Curiosity might be the thief of joy but it is also the fuel that drives science. In this case, the scientists were curious about the cellular impact of the three viruses in neurons and glia. The result was analysed based on this simple rubric: After infection, did the cells remain intact, or did they become disrupted? The live imaging experiments held the answers. It revealed that approximately 50% of the Polio virus-infected cell bodies retained their shape, 60% of EV-D68 cells showed subtle fragmentation, and EV-A71 cells rounded up (Preprint Figs. 5A–B). To measure the damage beyond the individual cells (= to the entire hSpO organoid), lactate dehydrogenase (LDH) release was tracked to quantify the cytotoxicity of each virus. To bring you up to speed to what LDH is: it is a cytoplasmic enzyme that leaks out when the cell membrane is not intact. Polio virus and EV-A71 showed a strong cytotoxicity (high LDH release), while EV-D68 showed lower LDH release despite cellular fragmentation. Similarly, the programmed cell death (fancily known as apoptosis) was highly associated with Polio virus and EV-A71. Specifically, Polio virus apoptosis occured primarily in virus-positive cells, while EV-A71 triggered apoptosis in both infected and nearby cells (Preprint Figs. 5G–H).
Conclusion and Future Direction
Poliovirus, EV-A71, and EV-D68 use different tropisms (neuronal vs glial cells) and apoptotic cascades (cell-autonomous vs non-cell-autonomous). Regardless of these cellular differences in infection mechanisms, they can all result in paralysis. Non-polio enteroviruses (EV-A71 and EV-D68) are emerging, and we need to develop effective antivirals and vaccines against these threats. Fortunately, the hSpO-hSkM in-vitro assembloid model has demonstrated prominence in connecting the mechanistic dots of the host-viral interaction in neurons and glia cells; filling in the critical gaps of limited human models. However, there are more gaps to address:
- During the immune response, there were no microglia/macrophages present limiting the full innate immunity response regardless of the high cytokines response revealed in this study.
- The model has a short lifespan which cannot sustain chronic infection in longer experiments.
- While this model mimics the cells of the spine, it only contains neuromuscular cells and therefore doesn’t fully represent spinal cord physiology which opens a pandora’s box (missing system level effects), leaving more questions about the complexities of how viruses relies extensively on a diverse collection of cells for sustained infection, and how the host immune system may either persevere or get cold feets during these infections.
References
- Miranda, M. N. S. et al. A Tale of Three Recent Pandemics: Influenza, HIV and SARS-CoV-2. Front. Microbiol. 13, 889643 (2022).
- Aw, D. Z. H., Zhang, D. X. & Vignuzzi, M. Strategies and efforts in circumventing the emergence of antiviral resistance against conventional antivirals. npj Antimicrob Resist 3, 54 (2025).
- Pons‐Espinal, M. et al. Trends in Paediatric Viral Meningitis and Encephalitis With Unconfirmed Aetiology: A Spanish Population‐Based Study, 2016–2020. Acta Paediatrica 114, 2298–2305 (2025).
- Sooksawasdi Na Ayudhya, S., Laksono, B. M. & Van Riel, D. The pathogenesis and virulence of enterovirus-D68 infection. Virulence 12, 2060–2072 (2021).
- Cox, J. A., Hiscox, J. A., Solomon, T., Ooi, M.-H. & Ng, L. F. P. Immunopathogenesis and Virus–Host Interactions of Enterovirus 71 in Patients with Hand, Foot and Mouth Disease. Front. Microbiol. 8, 2249 (2017).
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