Drummond, Coyne
(2017)
The Development of Physiologically Relevant Cell Culture Models for Intestinal Enterovirus Infection.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
This is the latest version of this item.
Abstract
Enteroviruses are small, single stranded RNA viruses that are spread via the fecal-oral route and encounter the small intestinal epithelium as their primary site of infection. This family of pathogens, including poliovirus, coxsackieviruses, and echoviruses, is responsible for pediatric and neonatal infections with severe and often fatal outcomes. Echovirus 11 can cause particularly devastating neonatal infections, resulting in enteroviral sepsis, meningitis, and hepatic failure. In order to gain access to sites where severe virally-induced disease occurs, enteroviruses must first overcome the defenses of the epithelial barrier of the small intestine. The human small intestine is a complex organ made up of a variety of specialized, differentiated sub-cell types. Research of enterovirus infection at this important primary barrier to infection is limited by the fact that no accurate in vitro model of infection exists, nor does an animal model that recapitulates the natural gastrointestinal (GI) route of infection.
Here, I utilize two recently developed cell culture models designed to better represent the human small intestinal epithelium as it exists in vivo, in order to characterize enterovirus infection at this important entry portal. First, I describe the use of a bioreactor and microscaffold beads for culturing intestinal cell lines 3-dimensions (3-D). The resulting polarized cell cultures, with enhanced brush borders and upregulated expression of intestinal genes, were used to model coxsackievirus B (CVB) infection. Secondly, human intestinal enteroid cultures, derived from primary intestinal crypts, were used as an ex-vivo cell model. These cells proliferate and differentiate into the repertoire of epithelial cell sub-types found in the human small intestine in-vivo. Human enteroids were used to study epithelial infection by different enteroviruses. In differentiated cultures, we find that the absorptive enterocyte and enteroendocrine cell lineages are highly permissive to echovirus 11 infection, while goblet cells were restrictive to infection. Contrary to infection in traditional cultures of immortalized intestinal cell lines, we find that enteroids derived from the primary intestinal stem cells produce robust antiviral signaling in response to echovirus 11 challenge. In summary, the models for intestinal infection presented in this dissertation will allow for novel enterovirus studies not previously possible in undifferentiated cell lines.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
11 May 2017 |
| Date Type: |
Publication |
| Defense Date: |
16 February 2017 |
| Approval Date: |
11 May 2017 |
| Submission Date: |
19 March 2017 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
153 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Virology and Microbiology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
culture model enteorvirus virus gastronitestinal infection |
| Date Deposited: |
11 May 2017 15:52 |
| Last Modified: |
11 May 2017 15:52 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/31830 |
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The Development of Physiologically Relevant Cell Culture Models for Intestinal Enterovirus Infection. (deposited 11 May 2017 15:52)
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