Date of Award

Spring 5-7-2016

Document Type

Thesis

Degree Name

Master of Science (MS)

Programs

Pathology & Microbiology

First Advisor

Nora Chapman

Abstract

Coxsackievirus B3 (CVB3) is an enterovirus with no known form of latency. However, assays designed to detect enteroviral RNA have shown that CVB3 RNA can persist for weeks beyond the acute infection both naturally and experimentally. Our previous work with coxsackievirus revealed an inhibited version of enteroviral replication where the progeny virus, termed terminally deleted (TD) virus, was missing a maximum of 49 nucleotides from the beginning of the 5’ non-translated region (NTR). The largest terminally deleted virus, TD50, effaced stem a, stemloop b, and stemloop c from the secondary structure, the cloverleaf. We hypothesized that further deletion beyond those first 49 nucleotides (nt) would disrupt stemloop d and prevent alternative binding of replicative machinery to the point of lethality. Plasmid DNA designed to create functional wildtype CVB3, was used as a template to create two novel constructs: TD57 and TD78. The mutant plasmids would transcribe infectious viral RNA missing up to 56nt and 77nt from the start of the 5’ NTR respectively. After transcription, the mutant RNA was transfected into HeLa cells and incubated for three days. Using RT-PCR we prove that despite the loss of stemloop d, these CVB3-TDs replicated and produced infectious encapsidated viral RNA.

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