# Characterization of a cerebral organoid model of astrovirus VA1 infection

> **NIH NIH R21** · WASHINGTON UNIVERSITY · 2024 · $194,375

## Abstract

Project Summary/Abstract
 Astroviruses have been recently recognized as an emerging cause of central nervous system infections
in humans, with astrovirus VA1 (VA1) as the most frequently identified astrovirus genotype. We previously
described the capacity of VA1 to infect primary human astrocytes and induce expression of inflammatory
cytokines like CXCL10, which can directly induce neuronal apoptosis. The histology from the VA1 cases of
encephalitis identified neuronal injury and apoptosis, but in cell culture, VA1 cannot replicate in primary human
neurons. These findings suggest that VA1 could cause neuronal apoptosis through an indirect mechanism,
including infection of astrocytes leading to CXCL10 expression and induction of apoptosis. It is also possible that
neuronal cell culture does not reflect necessary conditions for VA1 replication and a better model is needed.
 To further study the neuropathogenicity, we did not detect significant viral replication in brain tissue from
mice inoculated with VA1. In contrast, inoculation of human cerebral organoids (hCOs) with VA1 resulted in a
>500-fold increase in viral RNA 72 hours post-inoculation. These results demonstrate the capacity of hCOs to
support VA1 replication and now we can leverage this system to better understand cell-to-cell interactions that
are important during VA1 infection. hCOs consist of multiple cell types from the central nervous system, including
mature/immature neurons, astrocytes, and neural progenitor cells. Our protocol can generate hCOs with robust
populations of astrocytes and neurons, enabling cross-talk between cell types. In Aim 1, we will study the cellular
tropisms of VA1. We developed a novel fluorescent in situ hybridization (FISH) and immunohistochemical (IHC)
assays to detect VA1 that can co-localize with host markers of different cellular lineages. Using FISH and IHC,
we will define the cell lineages that support infection by VA1. Based on our previous results, we hypothesize
astrocytes but not neurons support VA1 replication. Nonetheless, the hCOs will allow us to determine if other
cell types, including neurons, support VA1 infection. In Aim 2, we will determine the consequences of VA1
infection. Given that VA1 induces neuronal apoptosis in vivo, we hypothesize that VA1 will also induce apoptosis
of neurons in hCOs. We will identify cells that are undergoing apoptosis after infection by staining for activated
caspase-3 and by TUNEL. Cells positive for apoptosis will be co-stained with cell lineage markers to identify the
cell types that are undergoing apoptosis. We will also perform single-cell RNA-seq to further describe the cellular
response to infection. This analysis will determine if VA1 infection induces expression of inflammatory cytokines,
like CXCL10, in hCOs. In addition, RNA-seq will identify if other signaling pathways, including cell death
pathways, that are activated in VA1 infected and uninfected cells. By dissecting the tropisms and consequenc...

## Key facts

- **NIH application ID:** 10896135
- **Project number:** 5R21AI168955-02
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Andrew Bok Seng Janowski
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $194,375
- **Award type:** 5
- **Project period:** 2023-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10896135

## Citation

> US National Institutes of Health, RePORTER application 10896135, Characterization of a cerebral organoid model of astrovirus VA1 infection (5R21AI168955-02). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10896135. Licensed CC0.

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