Year of Publication

2015

Season of Publication

Spring

Paper Type

Master's Thesis

College

College of Arts and Sciences

Degree Name

Master of Science in Biology (MS)

Department

Biology

NACO controlled Corporate Body

University of North Florida. Department of Biology

First Advisor

Dr. Doria F. Bowers

Second Advisor

Dr. Gregory Ahearn

Third Advisor

Dr. Paul Linser

Department Chair

Dr. Daniel Moon

College Dean

Dr. Barbara Hetrick

Abstract

Arthropod-borne-viruses (arboviruses) pose a global threat due to their ability to be transmitted by hematophagous insects to vertebrate hosts resulting in a range of serious infectious diseases. Sindbis virus (SINV) is the prototype arbovirus of the genus Alphavirus in the family Togaviridae. The purpose of this study was to investigate the use of a fluorescent tagged reporter virus in both in vitro and in vivo environments. The fluorescent protein GFP was inserted between the Capsid and PE2 in the genome of TR339; SINV TaV-GFP (Wm. Klimstra Lab). This virus construct should have the same infectivity and virulence as wild type TR339, leaving a fluorescent ‘path’ in infected cells that may reveal virus transit. Virus stocks were grown in BHK-21 vertebrate cells and C7-10 mosquito cells. Two Aedes albopictus mosquito cell lines, C7-10 and C6/36, were then challenged with vertebrate and mosquito grown reporter virus. Evidence of GFP were seen as early as 6 hours post infection (p.i.) in all samples. Infected C7-10 cells with the vertebrate grown reporter virus were fixed for 1 hour in chilled 4% buffered paraformaldehyde; GFP was shown to be resilient to both fixation and light quenching. Ultimately, Ae. aegypti mosquitoes were challenged with a viremic bloodmeal at a titer of 107 PFU/ml and midguts were dissected over several days. The presence of GFP was observed in midgut columnar epithelial cells as early as day 3 p.i. and remained localized even at day 30 p.i. This is in agreement with published work on the interaction of TR339 in Ae. aegypti gut, signaling this viral construct as a means to visualize wild-type infection.

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