| Microfilament, as the skeleton in host cell, plays an essential role in the intracellular material transport, cell migration, the formation of the synapse, mitosis and other activities. As a strictly intracellular parasite, Virus evolves a variety of interactions with the host cytoskeleton mechanism in order to create a beneficial environment for its invasion, replication and release. The microfilament also interferes intracellular viral activity. The generalized actin cytoskeleton includes tight junction proteins. Porcine epidemic diarrhea is a contagious acute gastrointestinal infection, which has brought huge economic losses to the pig industry in recent years. Therefore, this research was focused on the interaction between porcine epidemic diarrhea virus (PEDV) and the actin cytoskeleton, as well as the impact to the tight junction proteins, in order to provide some basement works for the further study of PEDV.1The pathological studies of porcine epidemic diarrhea virus infected IPEC-J2cellsTCID50=105.5/mL PEDV was applied for trial. PEDV was concentrated by sucrose density gradient centrifugation for electron microscopy negative staining to study the virus morphology. Many scattered virus particles were found, the virus particles were round, oval, kidney-shaped, and polygonal-shaped. Infected IPEC-J2cells were observed with swelling round, refraction strengthen, small vacuoles; then vacuoles gradually merged to large vacuoles, swollen cells started to shed; finally a large cell debris floated in the culture medium. RT-PCR showed PEDV proliferation peaked at12h, and then the amount of progeny viral gradually declined. Electron microscopy observed the uitrastructurai changes of infected IPEC-J2cells. Scanning electron microscope (SEM) images showed that a large number of microvilli shedded, the apical of microvilli were enlargement and rounded; some of the apical of microvilli fused to mushroom-like blebs. Transmission electron microscope (TEM) images showed the internal microfilament of the microvilli disappeared, the microfilament skeleton of the host cell disordered, severely mitochondrial vacuolization, nuclear membranes that virus particles contacted with were gradually dissolved, nuclear membranes that without virus particles contacted showed clear membrane boundaries. The results declared that PEDV replicated in IPEC-J2cells, accompanied with typical vacuolization, microvilli shedding and microfilament cytoskeleton disarrangement, suggesting that PEDV caused the remodeling of cell microfilament cytoskeleton.2The deregulation of tight junction protein expressions in IPEC-J2cells induced by porcine epidemic diarrhea virusWe try to figure the effect PEDV induce to intestinal epithelial barrier and tight junction proteins. Transepithelial electrical resistance (TEER) and paracellular pathway rate revealed the integrity of intestinal epithelial barrier, then the expression of tight junction proteins were also detected. The TEER of infected IPEC-J2monolayer cell cultures went through an instantly rise, and then drop sharply at60min. The paracellular pathway rate showed that PEDV increased the penetration of IPEC-J2monolayer cell cultures. Tight junction proteins Claudin-1and ZO-1altered significantly post infection, E-cadherin expression stabilized in a normal level, the expression levels of the other three proteins dropped to about85%of the normal expression. The results proved that PEDV infection showed less affection to the tight junction expressions. Compared to the significant affection to the microfilament cytoskeleton, we speculated that PEDV infection caused microfilament cytoskeleton reorganization and perijunctional ring contraction, and then resulted in the alteration of tight junction protein expressions.3The interaction between porcine epidemic diarrhea virus and microfilamentImmune-fluorescence images showed different periods of actin remodelling of PEDV infected IPEC-J2cells, we artificially divided the procedure into five periods: depolymerization phase I (0-40min), membrane-annulus phase (60min), depolymerization phase II (80-100min), nucleus-annulus phase (6-48h) and apoptotic phase (72h). Depolymerization phase I, F-actin depolymerised into misty; membrane-annulus phase, actin repolymerized into ring-like chains under IPEC-J2cell edge; depolymerization phase II, the ring-like chains depolymerized again; nucleus-annulus phase, F-actin accumulated nearby cell nucleus; apoptotic phase, F-actin aggregated staining showed apoptosis and shedding, which is a typical apoptotic cells characteristics. Jasplakinolide (Jas) and cytochalasin D (Cyto D), which are able to promote actin polymerization and induce destabilization of the actin filament respectively, were applied to testify whether actin skeleton interfere PEDV infection. Progeny virus particles were notably decrease by Cyto D and Jas in virus invasion, proliferation and and release, while the suppression of Cyto D during virus proliferation and release was significant (P<0.01). These findings suggest that PEDV interfered microfilament cytoskeleton and the amount of F-actin, meanwhile microfilament cytoskeleton affected PEDV invasion, replication and release. |
