Date of Award

Winter 2014

Project Type

Thesis

Program or Major

Microbiology

Degree Name

Master of Science

First Advisor

Aaron B Margolin

Second Advisor

Elise R Sullivan

Third Advisor

David J Spiro

Abstract

Enteroviruses are pathogens which exist in water sources, and are passed via the fecal-oral pathway. These viruses are of public health concern due to the high occurrence globally of human infections and potential for serious illness or death. Current methods employed by the EPA for the detection of enteroviruses involve using both cell culture and quantitative PCR (qPCR). Laboratories working with samples independent of the EPA often use a detection method that integrates cell culture and qPCR (ICC/qPCR). Substituting suspension cultures for traditional monolayers has been proposed as a less expensive alternative method with higher sensitivity than traditional monolayers while using ICC/qPCR. Therefore, the ICC/qPCR method was investigated to determine the relationship between incubation periods of cultures post infection as a function of the initial virus concentration as needed for detection using both monolayer and suspension cultures.

Varying concentrations of poliovirus were added to polypropylene tubes or cell culture flasks containing 5 ml of cells and media. Samples were incubated in 24 hour intervals for 6-7 days prior to extraction and qPCR. Later, incubation intervals were reduced to 8 hour intervals for 48 hours. Using 100 PFU/ml, it was determined no significant improvement in detection was obtained after incubating cells in either suspension or monolayer cultures after 24 hours. Seeded surface water samples, both treated and untreated, were subjected to the suspension culture protocol. Virus was detected in all samples, with no significant increase in enterovirus nucleic acid detected after 32 hours. The suspension ICC/qPCR protocol is as effective as the monolayer protocol in detecting enteroviruses in water, and can therefore be used as opposed to using tradition cell culture materials. In addition, as there is no increase in viral concentration after 24-32 hours post infection a reduction in overhead costs is obtainable by eliminating extended incubation periods.

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