Jin Hyang Kim

Document Type

Thesis - University Access Only

Award Date

2001

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

Abstract

Infection with Influenza A virus can be detected by several well-established methods. These assays are designed to recognize the presence of individual, specific viral components. However, a way to evaluate the direct interactions between virus and host cells is needed to demonstrate virulence-related activities on the function and physiology of cells. Cells in culture may be used to model the interactions between the virus and host cells in vivo. Such a model will help to establish the virulence of individual viral isolates in the clinical setting. In this thesis, a simple, rapid colorimetric assay was evaluated to verify its function in measuring virulence related physiological changes related to swine influenza virus (SIV) infection of MDCK cell cultures. The tetrazolium salt 3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MIT) assay is based on the mitochondiral dehydrogenase enzyme system of metabolically active cells. These enzymes convert MIT into a bluish-purple, insoluble formazan. In tum, the formazan product can be measured spectrophotometrically. The MTT assay was found to be sensitive in the assessment of viable and physiologically active Mardin-Darby Canine Kidney (MOCK) cells. It clearly discriminated live MDCK cells from medium background. The MTT signal of cells treated with Triton X-100 correlated with viability measured using trypan blue exclusion. In addition, the level of MTI signal was demonstrated to be directly proportional to the number of cells placed in culture. Therefore, MTT was demonstrated to be appropriate for measurement of MOCK cells in cultures. The MTT assay was utilized to assess viral activity in MDCK cell cultures. Two-fold and 10-fold serial dilutions of swine influenza virus [A/Swine/Indiana/1726/88 (HlNl)] stocks were used to infect MOCK cultures, in 24 wellplates, over a wide range of initial virus exposure. MTT was used to determine virusinduced changes in cellular activity. MTT conversion in :MDCK cells was reduced by swine influenza virus in an input dose-dependent manner. About 20% reduction in MTT conversion was observed at the highest input concentrations of virus after 24 hours and a more linear reduction relative to input dose was observed after 48 hours of incubation. When MTI conversion was compared to the fraction of cells in culture demonstrating cytopathic effect (CPE), a clear inverse correlation was obtained (correlation coefficients were between ?0.7 and ?0.9). This inverse correlation was significant when analyzed over 11 trials (p~ 0.02). The cellular damage done by virus was determined using both MTT 50 % viral effect on physiological activity (MTT50) and the point at which 50 % of the cells in culture demonstrated CPE (CPE50) by extrapolation. The 50% cell damage dose obtained using each of these methods increased with the duration of exposure to virus in MOCK cell cultures. The most consistent and sensitive results for assessment of viral damage were observed between 36 hr and 48 hr p.i .. At 36 and 48 hours, similar "fractions of damaged cells" were observed using MTT so and CPEso estimations. The utility of MTT assay in demonstrating diminished physiological activity related to viral virulence of swine influenza provides the basis of a cost-effective and reliable bioassay for assessment of the virulence of field isolates. This model remains to be tested with a series of field isolates of known virulence to be validated.

Library of Congress Subject Headings

Swine influenza Virus diseases -- Diagnosis

Format

application/pdf

Number of Pages

60

Publisher

South Dakota State University

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