Publication Date

2018

Document Type

Thesis

Committee Members

Nancy Bigley (Committee Chair), Barbara Hull (Committee Member), Dawn Wooley (Committee Member)

Degree Name

Master of Science (MS)

Abstract

Keratinocytes and neurons cells are the main target for Herpes Simplex Virus Type 1 (HSV-1) invasion. Moreover, keratinocytes are the most abundant cell types in the epidermis layer in the skin. Therefore, they are the first cells to encounter HSV-1 in the primary infection. Next, the virus reaches the nerve endings and is transferred to neuronal cells as a result from the primary infection. In between these two events, innate immune cells including monocytes and macrophages response is activated and recruited to the infection site. In this study, keratinocytes (PAM-212) and murine macrophage (RAW 264.7) cell lines were utilized to investigate the response of macrophages (RAW 264.7 ) and keratinocytes (PAM-212) to HSV-1 infection and propagation in vitro. In this study, initially keratinocytes (PAM-212) and macrophages (RAW 264.7) and were studied either infected or uninfected with HSV-1 at MOI 0.1 in a monolayer model. Cell lines were co-cultured at ratio 1:5 (macrophages : keratinocytes) respectively in the co-culture model 1. The results showed some differences and similarities in cell viabilities, and viral replication between keratinocytes (PAM-212) and macrophages (RAW 264.7) at 24, 48, and 72 hours. In both the monolayer and the co-culture model, cells were exhibited morphological changes including irregularly shaped or rounded cells, enlargement of cell size, and cells appeared in different density compared to neighboring cells especially in the earlier phase of infection at 24 and 48 hours. In contrast, at 72 hours cells were degraded, detached, and more debris was observed in the medium. In like manner, our results of cell viability showed some variations after 24, 48, and 72 hours which confirms the HSV-1 infection. Furthermore, plaque assay showed a significant decrease in HSV-1 titers at 24 hours in the co-culture model compared to keratinocytes and macrophages monolayers. However, the keratinocyte monolayer appeared to tolerate HSV-1 replication and virus titers kept increasing in all time-points. On the other hand, the macrophage monolayer exhibited a noticeable decrease in the virus concentration at 48 hours, indicating the role of macrophages restricting viral replication. The effect of macrophages was diminished as time increased, but still exhibited a reduction in the virus titers compared to the keratinocytes and co-culture at 72 hours. That suggests a pivotal role of macrophages to induce immune response that limits viral replication as we proposed in our hypothesis. We still believe in the role of the cytokines produced by macrophages such as interferons IFN-[alpha/beta] and interleukin IL-1[alpha/beta] as antiviral therapy.

Page Count

74

Department or Program

Microbiology and Immunology

Year Degree Awarded

2018

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