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Duck Plague Virus Promotes DEF Cell Apoptosis by Activating Caspases, Increasing Intracellular ROS Levels and Inducing Cell Cycle S-Phase Arrest.

ABSTRACT: BACKGROUND:Duck plague virus (DPV) can induce apoptosis in duck embryo fibroblasts (DEFs) and in infected ducks, but the molecular mechanism of DPV-induced apoptosis remains unknown. METHODS:We first used qRT-PCR and a Caspase-Glo assay to determine whether the caspase protein family plays an important role in DPV-induced apoptosis. Then, we used an intracellular ROS detection kit and the mitochondrial probe JC-1 to respectively detect ROS levels and mitochondrial membrane potential (MMP). Finally, flow cytometry was used to detect apoptosis and cell cycle progression. RESULTS:In this study, the mRNA levels and enzymatic activities of caspase-3, caspase-7, caspase-8, and caspase-9 were significantly increased during DPV-induced apoptosis. The caspase inhibitors Z-DEVD-FMK, Z-LEHD-FMK, and Q-VD-OphA could inhibit DPV-induced apoptosis and promote viral replication. Subsequently, a significant decrease in MMP and an increase in the intracellular ROS levels were observed. Further study showed that pretreating infected cells with NAC (a ROS scavenger) decreased the intracellular ROS levels, increased the MMP, inhibited apoptosis, and promoted viral replication. Finally, we showed that DPV infection can cause cell cycle S-phase arrest. CONCLUSIONS:This study shows that DPV causes cell cycle S-phase arrest and leads to apoptosis through caspase activation and increased intracellular ROS levels. These findings may be useful for gaining an understanding of the pathogenesis of DPV and the apoptotic pathways induced by ?-herpesviruses.

SUBMITTER: Zhao C

PROVIDER: S-EPMC6409732 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Duck Plague Virus Promotes DEF Cell Apoptosis by Activating Caspases, Increasing Intracellular ROS Levels and Inducing Cell Cycle S-Phase Arrest.

Zhao Chuankuo C Wang Mingshu M Cheng Anchun A Yang Qiao Q Wu Ying Y Jia Renyong R Zhu Dekang D Chen Shun S Liu Mafeng M Zhao XinXin X Zhang Shaqiu S Liu Yunya Y Yu Yanling Y Zhang Ling L Tian Bin B Rehman Mujeeb Ur MU Pan Leichang L Chen Xiaoyue X

Viruses 20190224 2

<h4>Background</h4>Duck plague virus (DPV) can induce apoptosis in duck embryo fibroblasts (DEFs) and in infected ducks, but the molecular mechanism of DPV-induced apoptosis remains unknown.<h4>Methods</h4>We first used qRT-PCR and a Caspase-Glo assay to determine whether the caspase protein family plays an important role in DPV-induced apoptosis. Then, we used an intracellular ROS detection kit and the mitochondrial probe JC-1 to respectively detect ROS levels and mitochondrial membrane potenti ...[more]

PMID: 30813500

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Project description:Duck plague virus (DPV) is a representative pathogen transmitted among aquatic animals that causes gross lesions and immune inhibition in geese and ducks. The mechanism of organ tropism and innate immune evasion of DPV has not been completely deciphered due to a lack of cell models to study the innate immune manipulation and pathogenicity of aquatic viruses. In the present study, we isolated five types of duck primary cells [duck embryo fibroblasts (DEFs), neurons, astrocytes, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages] to identify appropriate cell models for DPV, using tropism infection and innate immunologic assays. Cells responded differently to stimulation with DNA viruses or RNA virus analogs. DPV infection exhibited broad tropism, as the recombinant virulent strain (CHv-GFP) infected DEFs, neurons, astrocytes, and monocytes/macrophages, but not the PBMCs, as the expression of EGFP was negligible. The basal levels of innate immunity molecules were highest in monocytes/macrophages and lower in DEFs and astrocytes. Conversely, the titer and genomic copy number of the attenuated virus strain was higher in DEFs and astrocytes than in neurons and monocytes/macrophages. The titer and genomic copy number of the attenuated virus strain were higher compared with the virulent strain in DEFs, neurons, and astrocytes. The innate immune response was not significantly induced by either DPV strain in DEFs, neurons, or astrocytes. The virulent strain persistently infected monocytes/macrophages, but the attenuated strain did so abortively, and this was accompanied by the phenomenon of innate immune inhibition and activation by the virulent and attenuated strains, respectively. Blockage of IFNAR signaling promoted replication of the attenuated strain. Pre-activation of IFNAR signaling inhibited infection by the virulent strain. The selection assay results indicated that induction of innate immunity plays an essential role in controlling DPV infection, and monocytes/macrophages are an important cell model for further investigations. Our study provided practical methods for isolating and culturing duck primary cells, and our results will facilitate further investigations of organ tropism, innate immune responses, latent infection, and the effectiveness of antiviral drugs for treating DPV and potentially other aerial bird pathogens.

Dataset Information

Duck Plague Virus Promotes DEF Cell Apoptosis by Activating Caspases, Increasing Intracellular ROS Levels and Inducing Cell Cycle S-Phase Arrest.

Publications

Duck Plague Virus Promotes DEF Cell Apoptosis by Activating Caspases, Increasing Intracellular ROS Levels and Inducing Cell Cycle S-Phase Arrest.

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