HSV-1 Fluorescent Viral Dynamics

We used a genetically engineered HSV-1 construct incorporating enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (RFP) as reporter genes whose expression is driven by the viral promoters ICP0 and Glycoprotein C, respectively.^{25 (link)} EGFP expression in infected cells indicates that HSV-1 has entered lytic cycles, while RFP expression indicates commitment to viral DNA replication. Cells were incubated with HSV-1 for 2 h at specified multiplicity of infection (MOI). To inhibit viral replication, cells were preincubated with antivirals used in animal models: acyclovir (50 μM), or (E)-5-(2-bromovinyl)-2′-deoxyuridine (5BVdU, 30 μM) along with interferon-alpha (IFN-α, 125 U/ml).^{22 (link)} The proportion of cells expressing EGFP and RFP was determined by flow cytometry (FC). Images were acquired using a Leica IL MD LED inverted fluorescence microscope and a Leica DM5500B fluorescence microscope.

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D’Aiuto L., Prasad K.M., Upton C.H., Viggiano L., Milosevic J., Raimondi G., McClain L., Chowdari K., Tischfield J., Sheldon M., Moore J.C., Yolken R.H., Kinchington P.R, & Nimgaonkar V.L. (2014). Persistent Infection by HSV-1 Is Associated With Changes in Functional Architecture of iPSC-Derived Neurons and Brain Activation Patterns Underlying Working Memory Performance. Schizophrenia Bulletin, 41(1), 123-132.

Publication 2014

IL MD LED inverted fluorescence microscope DM5500B fluorescence microscope

2 deoxyuridine Acyclovir Animal models Antivirals Enhanced green fluorescent protein Flow cytometry Fluorescence microscope Glycoprotein c Hsv 1 Ifn α Infection Inhibit Red fluorescent protein Reporter genes Viral replication

Corresponding Organization : University of Pittsburgh

Other organizations : University of Bari Aldo Moro, Johns Hopkins Medicine, Johns Hopkins University, Rutgers, The State University of New Jersey

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Variable analysis

independent variables

Antiviral treatments: acyclovir (50 μM), or (E)-5-(2-bromovinyl)-2′-deoxyuridine (5BVdU, 30 μM) along with interferon-alpha (IFN-α, 125 U/ml)
Multiplicity of infection (MOI)

dependent variables

Proportion of cells expressing EGFP (indicative of HSV-1 lytic cycles)
Proportion of cells expressing RFP (indicative of commitment to viral DNA replication)

control variables

Incubation time of cells with HSV-1 (2 h)

controls

Positive control: Cells infected with HSV-1 without any antiviral treatment
Negative control: Uninfected cells

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