The Molecular Mechanisms Of Spring Viraemia Of Carp Virus Infection Epithelioma Papulosum Cyprini Cells In Inducing Autophagy And Proteomic Analysis

Spring viraemia of carp virus (SVCV) is a bullet-shaped RNA virus. Outbreaks of SVCV in several carp species and other cultivated fish can cause significant mortality and jeopardize billion-dollar fish industry world wise. Currently the host response of SVCV is still largely unknown. As an eukaryotic cellular degradation pathway, autophagy involves the delivery of cytoplasmic cargo to the lysosome. And it is essential for intracellular homeostasis in eukaryocytes. On the other hand, many microbes have also evolved mechanisms to evade host autophagy. Some viruses can inhibit host autophagy by their self-encoded proteins.Our study focused on two projects that was to prove that the autophagy could promote SVCV replication and the proteomic analysis of epithelioma papulosum cyprini cells infected with spring viraemia of carp virus. These studies provide dynamic and useful protein-related information to further understand the underlying pathogenesis of SVCV infection and reveal a previously undescribed connection between autophagy and SVCV replication, and propose autophagy suppression as a novel means to restrict SVCV viral replication.1. The molecular mechanisms of autophagy and SVCV(1) We observed that autophagy is activated in SVCV infected Epithelioma Papulosum cyprinid (EPC) cells through TEM.(2) We demonstrated that SVCV glycoprotein, rather than viral replication, activates the autophagic pathway.(3) SVCV utilized the autophagic pathway to facilitate its own genomic RNA replication as well as to enhance its titers in the supernatants. Autophagy promoted the survival of SVCV infected cells by eliminating damaged mitochondria DNA generated during virus infection.(4) We further showed that SVCV induces autophagy in EPC cells involved in ERK/mTOR signaling pathway.2. Proteomic analysis of epithelioma papulosum cyprini cells infected with spring viraemia of carp virus.(1) To better understand molecular responses to SVCV infection, two dimensional electrophoresis (2-DE) and MALDI-TOF/TOF were performed to investigate altered proteins in epithelioma papulosum cyprini cells (EPCs). Differentially expressed proteins in mockinfected EPCs and SVCV-infected EPCs were compared. A total of54differentially expressed spots were successfully identified (33up-regulated spots and21down-regulated spots) which include cytoskeleton proteins, macromolecular biosynthesis-associated proteins, stress response proteins, signal transductionproteins, energy metabolism, and ubiquitin proteasome pathway-associated proteins.(2)7corresponding genes of the differentially expressed proteins were quantified using real time RT-PCR to examine their transcriptional profiles. The presence of four selected cellular proteins (beta-actin,gammal-actin, heat shock cognate71kDa protein and annexin A2) associated with the spring viremia of carp virus (SVCV) particles was validated by Western blot assay.These studies provide dynamic and useful protein-related information to further understand the underlying pathogenesis of SVCV infection.