Interaction Between Porcine Circo Virus TYPE 2 And Intestinal Porcine Epithelial Cell Microfilament

Porcine circovirus type 2 (PCV2) can cause granulomatous enteritis which spreads through digestive tract, representing diarrhea, and bring certain economic losses to pig industry in recent years. Microfilament is an important component of cytoskeleton and in a highly dynamic state. They generally form fasciculation or a network in the plasma membrane, crucial for many kinds of important cellular processes including cell division, adhesiveness, motility, uptake of nutrients and intracellular transport. The life process of virus depends on cells strictly. It is therefore not surprising that many viruses have evolved highly varied strategies to manipulate microfilament system in order to facilitate their invasion, replication, release and spread. Previous studies showed that virus could utilize vesicular transport and/or involve in the regulation of actin cytoskeleton signaling pathways to complete their life processes. In this study, intestinal porcine epithelial cell line (IPEC-J2) was used for the infection of PCV2. We tentatively provide a cell intestinal infection model of PCV2 for subsequently studying PCV2 pathogenesis. Meanwhile, we preliminarily explore the interaction between PCV2 and its host cell microfilament, laying the foundation for future in-depth research on the PCV2 infection mechanism for digestive tract through the interaction between PCV2 and microfilament.1. Preliminary construction of porcine circovirus type 2 infection model on intestinal porcine epithelial cell lineIn this study, PCV2 of TCID50=1045/mL was used to infect the intestinal porcine epithelial cell line (IPEC-J2). Under light microscope, PCV2 caused no lesions in the infected IPEC-J2 cells. However, cells ultrastructure changed observed by transmission electron microscope, such as the mitochondria (changed into big and round, and its matrix became shallow and cristae turned into few and short, and a trend of transforming into vacuole was existed at 48 h p.i.). Meanwhile, compared with uninfected cells, virions appeared round the microvilli of differing lengths and widths at 1 h p.i. and in the cells at 48 h p.i.. In terms of the IPEC-J2 susceptibility for PCV2, there was a upward trend from 1 to 72 h p.i. detected by flow cytometry method (FCM). Real-time PCR showed PCV2 proliferated from 6 to 96 h p.i. steadily, and then the PCV2 copy numbers declined. In order to detect the sustained susceptibility of PCV2 on IPEC-J2 cells, the IPEC-J2 cells after infection was passaged continuedly, and we found that PCV2 could be associated with cell passages to the 6th generation, showing a sharp growth infection trend. The results above reveals that PCV2 can infect IPEC-J2 cells and proliferates in the cells, and IPEC-J2 cells are thus proved to be a cell intestinal infection model of PCV2 in vitro preliminarily.2. Interaction between porcine circovirus type 2 and intestinal porcine epithelial cell line microfilamentThis study explored the interaction between PCV2 and IPEC-J2 microfilament preliminarily. The immune-fluoresence images revealed that the uninfected IPEC-J2 cells emerged as cobblestone shape with clear boundaries, and intracytoplasmic filaments were scattered in a parallel distribution. In comparison with the uninfected cells, microfilaments were polymerized at 1 h p.i., many short F-actin gathering and forming small lumps. At 24, 48 and 72 h p.i., the stress fibers reduced, F-actin network presenting cavitary, and cell boundaries became clearer. The data of quantifying total cellular F-actin by FCM showed that the total F-actin of PCV2-infected group was higher than that of control group. Meanwhile, microfilament depolymerizing agent (Cytochalasin D, CytD) or microfilament stabilizer (Cucurbitacin E, CuE) was used to detect the changes of PCV2 proliferation after microfilament reorganization. The results showed that actin polymerization accelerates invasion of PCV2 on IPEC-J2 cells, and the dissolution of cortical actin was necessary in PCV2 egress. To further study the interaction of PCV2 with the microfilament, we explored the related signaling pathways. The cellular total RNA was extracted after PCV2 infection, and then the changes of some actin-related proteins were detected in the levels of mRNA expression by real-time quantitative PCR. Results showed that, there was no significant difference in the expression of selected genes between uninfected cells and PCV2-infected cells at 1 h p.i.; while the expression of gelsolin which associated with actin nucleation significantly reduced (P<0.01), and the actin upstream regulatory proteins RhoA (P< 0.01), ROCK1 (P<0.05) and ROCK2 (P<0.05) which were concerned with the formation of stress fibers all increased in different degrees at 48 h p.i., suggesting that the influence on actin cytoskeleton signaling pathways by PCV2 may be achieved through the RhoA-ROCK pathway, accompanied by actin nucleation during infection.