Puzio, Maria Valeria (2017) “Caprine Herpesvirus 1 (CpHV-1) a potential candidate for oncolytic virotherapy”. [Tesi di dottorato]

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Oncolytic virotherapy is a promising avenue of cancer therapy. It is a therapeutic approach to cancer treatment that utilizes native or genetically modified human viruses that selectively replicate in tumor cells, or nonhuman viruses, that are naturally oncotropic and preferentially replicate in tumor cells, displaying minimal adverse effects in normal healthy cells. To date, a wide variety of viruses have been evaluated for their oncolytic potential, including DNA viruses as well as RNA viruses. CpHV-1 is a species-specific herpesvirus closely related to Bovine herpesvirus type 1 (BHV-1). Previuos data have demonstrated that CpHV 1 is able to induce apoptosis in goat peripheral blood mononuclear cells, moreover we have investigated on the pro apoptotic potential of CpHV 1 in Madin Darby bovine kidney cells, evaluating apoptotic profiles like chromatin condensation and DNA laddering. Recently, the intracellular pathway through which CpHV 1 has the capacity to induce apoptosis has been characterized in more details, by analyzing the gene expression response during the apoptotic phase of CpHV 1 infection in a murine neuroblastoma cell line (Neuro 2a). The aim of the present research was to investigate the ability of CpHV-1 to replicate, cause cell death and affect cellular viability in a panel of human cancer cells lines. Human breast adenocarcinoma (MDA-MB-468), Human cervical adenocarcinoma (HeLa), Human osteosarcoma (U2OS), Human prostatic adenocarcinoma (PC3), Human lung carcinoma (A549) and Chronic Myelogenous Leukemia (K562) cell lines were used. Madin Darby bovine kidney (MDBK) cell line was used as control. In a first series of experiment we have analyzed the effect of CpHV‐1 infection on cell viability by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at different time post infection (p.i.) and several multiplicity of infection (MOI). All cell lines, except K562 cells, showed a marked cytopatic effect (CPE), demonstrating an oncolytic potential of CpHV-1 in tested human cancer cells. The reduction of cells viability was associated with significant levels of viral production as assayed by TCID50 after 24 h p.i. for MDBK, MDA-MD468, A549, U2OS, and after 48 h post infection (p.i.) for PC3, HeLa and K562 cell lines. Then, we investigated virus induced cytotoxicity, viability, and apoptosis within a single assay well, by using the ApoTox‐Glo Triplex assay. Analysis of virus‐infected cells revealed activation of caspase‐3, a marker of apoptosis at 24 h post‐infection. Analysis of virus‐infected, cells by western blot assay, revealed activation of caspase‐3 and cleaved caspase 3 at 24 h p. i. in MDBK, PC3, MDA-MD-468 and U2OS cell lines. Our findings demonstrate the effect of CpHV‐1 infection in neoplastic cell lines in terms of caspase activation and apoptosis modulation, thus CpHV-1 could be a novel candidate oncolytic virus.

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