Francisco Alvarez-leefmans (Committee Member), Nancy Bigley (Advisor), Barbara Hull (Committee Member)
Master of Science (MS)
To more closely mimic the in vivo progression of HSV-1 a novel in vitro method was created. In this study HEL-30 keratinocytes were infected with a 0.1 MOI of HSV-1 and plated unto a membrane with 8μm diameter pores. The membrane insert was then placed into culture wells that contained Neuro-2a cells (N2A). The neurites from the neuronal cells made cell-to-cell contact with the infected HEL-30 cells and the virus was transmitted into the neuronal cells. To determine if infection occurred in the N2A cells the cells were lysed and the lysate incubated with Vero cells to titrate virus plaque forming units. After incubation a virus plaque assay was performed and the results compared with those of uninfected control cells. Methylcellulose was used to protect against the HEL-30 cells lysing and releasing free virus particles into the medium and infecting the N2A cells without cell-to-cell contact. The methylcellulose is viscous enough to stop free virus particles from passage through the pore membrane in the chamber infecting the N2A cells. Therefore, the only way that the N2A cells were infected was if the neurites made direct contact with the infected keratinocytes, which would mimic the "real world" progression of the virus from primary site of infection to neuronal tissue infection. The use of methylcellulose in the co-culture system exerted a profound inhibitory effect on free-floating virus particles. Immunofluorescence microscopy was used to confirm cell-to-cell contact between the N2A and HEL-30 cells and to determine morphological differences that occur when the co-culture system is used versus monoculture. This model provides a new system for studying host/virus interactions.
Department or Program
Microbiology and Immunology
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