Publication Date

2013

Document Type

Thesis

Committee Members

F.Javier Alvarez-leefmans (Committee Member), Nancy Bigley (Advisor), Barbara Hull (Advisor)

Degree Name

Master of Science (MS)

Abstract

Herpes simplex type 1 (HSV-1) quiescent infection was established in L929 cells, murine fibroblasts, and in Neuro-2A (N2A) cells, mouse neuroblastoma, by treating them with the nucleoside analogue acyclovir (ACV) 24 hours before infection. Subsequent release of virus from the non-productive state was accomplished by treating the cells with the histone deacetylase inhibitor trichostatin A (TSA). Treatment of both L929 and N2A cell lines with ACV 24 hours before infection induced protection from HSV-1 cytopathic effect. A quiescent state was confirmed by absence of virus plaques when supernatant fluids from ACV-treated HSV-1 infected cultures were titered on Vero cell monolayers. Acyclovir was maintained in the cell culture medium for one day before infection and two days post infection. Removal of ACV from the culture medium did not permit reactivation of virus as determined by virus plaque assays. Virus reactivation was confirmed in ACV- treated HSV-1 quiescent cells by examining cell lysates for virus plaque forming units. Four days post infection of ACV-treated cells, culture medium was removed and replaced with medium containing TSA. Supernatant fluids from cultures treated with TSA showed virus production in plaque forming assays on Vero cells. Media from both lytically infected cultures and TSA reactivated cultures contained productive virus; however, media from latently infected cultures did not show the presence of infectious virus. Treatment of L929 cells with ACV 2 hours after infection with HSV-1 induced a quiescent effect better than treatment of the cells with AVC 24 hours before infection, because higher cell densities survived. As expected quantitative real-time polymerase chain reaction (qPCR) analysis of HSV-1 transcripts showed about one fold increase in Latency Associated Transcript (LAT) and a decrease in the lytic cycle infected cell protein (ICP0) in ACV-treated HSV-1 infected L929 cells at 16 hours post infection as compared with the untreated HSV-1 infected L929 cells. At the same time, lytic cycle infected cell protein (ICP27) showed a one and half fold increase in ACV-treated HSV-1 infected L929 cells compared with the untreated HSV-1 infected L929 cells. At 48 hours post infection, both infected cell proteins (ICP27 and ICP0) and LAT were observed at low levels in ACV-treated HSV-1 infected L929 cells compared with the lytically infected control cells. In N2A cells, LAT was noted at low level in ACV-treated HSV-1 infected N2A cells as compared with the untreated HSV-1 infected N2A cells at 16 hours post infection. At the same time, ICP0 decreased and ICP27 showed approximately one fold increase in ACV-treated HSV-1 infected N2A cells as compared with the untreated HSV-1 infected N2A cells. At 48 hours post infection, both lytic cycle transcripts (ICP27 and ICP0) and LAT were observed at low levels in ACV-treated HSV-1 infected N2A cells as compared with untreated HSV-1 infected control N2A cells. LAT was present but not significantly increased in both cell lines although they were latently infected and maintained with the ACV treatment.

Page Count

75

Department or Program

Microbiology and Immunology

Year Degree Awarded

2013

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