# Three-dimensional human epithelial cultures as a model for evaluation of flavivirus-host interactions driving infection in the skin

> **NIH NIH R21** · UNIVERSITY OF PENNSYLVANIA · 2022 · $243,750

## Abstract

PROJECT SUMMARY
Zika virus (ZIKV), dengue virus (DENV), and West Nile virus (WNV) all cause severe human disease as a result
of their widespread mosquito-borne transmission. While these flaviviruses share a common primary site of
infection in the dermis, they disseminate to varying secondary sites of infection with highly divergent clinical
outcomes. We propose that discrepancies in early virus-host interactions among ZIKV, DENV, and WNV are
responsible for these differences in disease; however, there is a gap in our knowledge of the molecular
mechanisms responsible. Elucidation of distinct virus-host interactions occurring at the initial infection site is thus
key to understanding how these viruses establish productive infections in the skin and elsewhere. The proposed
research will establish organotypic epithelial cultures as a genetically tractable and immunocompetent
3D human skin model of flavivirus infection, to be used for virus-host interaction studies. We will expand on
the current model commonly used to study DNA viruses, typically comprised solely of human fibroblasts and
keratinocytes, with the addition of human skin-resident dendritic cells and macrophages, to accurately recreate
natural flavivirus infection in the skin. Additionally, we will incorporate mosquito saliva, which includes
immunomodulatory and anti-inflammatory proteins, into the virus inoculum to further recapitulate the molecular
events occurring at the primary site of flavivirus infection. We will determine individual infection conditions for
ZIKV, DENV, and WNV, and evaluate cell tropism and host responses with each virus. Once established, we
will employ this 3D human skin model to evaluate the role of the host ribonuclease L (RNase L) protein during
ZIKV infection in the skin, which will validate this system for flavivirus-host interaction studies. While antiviral
RNase L activity is well described, we have recently discovered proviral RNase L activity during ZIKV infection.
In contrast, we observed canonical antiviral RNase L activity during DENV and WNV infections. These studies
were performed in 2D monolayer culture systems with immortalized cell lines, therefore we will use our 3D skin
model containing primary cells to test the hypothesis that RNase L plays a role in ZIKV cell tropism and
spread in the skin in a more relevant system. We will use CRISPR-Cas9 gene editing to delete RNase L from
the different skin cells comprising epithelial cultures, and subsequently generate RNase L-deficient skin cultures
for infections with ZIKV, DENV, or WNV. We will assess effects of RNase L deletion on infection and spread of
the different flaviviruses in the skin, as well as how host responses are impacted. The proposed studies will
characterize the role of RNase L as an important host factor repurposed by ZIKV during infection in the skin.
Furthermore, this project will establish organotypic epithelial cultures as an exciting new human model amenable
to gene editing which...

## Key facts

- **NIH application ID:** 10416065
- **Project number:** 5R21AI157147-02
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Susan R Weiss
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $243,750
- **Award type:** 5
- **Project period:** 2021-06-02 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10416065

## Citation

> US National Institutes of Health, RePORTER application 10416065, Three-dimensional human epithelial cultures as a model for evaluation of flavivirus-host interactions driving infection in the skin (5R21AI157147-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10416065. Licensed CC0.

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