Liqi Zhu

The epidermal growth factor receptor regulates cofilin activity and promotes transmissible gastroenteritis virus entry into intestinal epithelial cells

Transmissible gastroenteritis virus (TGEV), a coronavirus, causes severe diarrhea and high mortality in newborn piglets. The porcine intestinal epithelium is the target of TGEV infection, but the mechanisms that TGEV disrupts the actin cytoskeleton and invades the host epithelium remain largely unknown. We not only found that TGEV infection stimulates F-actin to gather at the cell membrane but the disruption of F-actin inhibits TGEV entry as well. Cofilin is involved in F-actin reorganization and TGEV entry. The TGEV spike protein is capable of binding with EGFR, activating the downstream phosphoinositide-3 kinase (PI3K), then causing the phosphorylation of cofilin and F-actin polymerization via Rac1/Cdc42 GTPases. Inhibition of EGFR and PI3K decreases the entry of TGEV. EGFR is also the upstream activator of mitogen-activated protein kinase (MAPK) signaling pathways that is involved in F-actin reorganization. Additionally, lipid rafts act as signal platforms for the EGFR-associated signaling cascade and correlate with the adhesion of TGEV. In conlusion, these results provide valuable data of the mechanisms which are responsible for the TGEV pathogenesis and may lead to the development of new methods about controlling TGEV.

Mitophagy in TGEV infection counteracts oxidative stress and apoptosis

The intestinal epithelial cells contain a large number of mitochondria for persisting absorption and barrier function. Selective autophagy of mitochondria (mitophagy) plays an important role in the quality control of mitochondria and maintenance of cell homeostasis. Transmissible gastroenteritis virus (TGEV) is a porcine enteropathogenic coronavirus which induces malabsorption and lethal watery diarrhea in suckling piglets. The role of mitophagy in the pathological changes caused by TGEV infection is unclear. Here, we report that TGEV induces mitophagy to suppress oxidative stress and apoptosis induced by viral infection in porcine epithelial cells (IPEC-J2). We observe that TGEV infection induce mitochondrial injury, abnormal morphology, complete mitophagy, and without obvious apoptosis after TGEV infection. Meanwhile, TGEV also induces DJ-1 and some antioxidant genes upregulation to suppress oxidative stress induced by viral infection. Furthermore, silencing DJ-1 inhibit mitophagy and increase apoptosis after TGEV infection. In addition, we demonstrate for the first time that viral nucleocapsid protein (N) is located in mitochondria and mitophagosome during virus infection or be expressed alone. Those results provide a novel perspective for further improvement of prevention and treatment in TGEV infection. These results suggest that TGEV infection induce mitophagy to promote cell survival and possibly viral infection.

Transmissible gastroenteritis virus does not suppress IFN-β induction but is sensitive to IFN in IPEC-J2 cells.

Abstract Coronaviruses tend to efficiently evade innate immune sensing. Alpha-coronaviruses interfere with the type I interferon (IFN) response in various ways, ensuring the limited activation of IFN responses. Transmissible gastroenteritis virus (TGEV), an Alphacoronavirus genera virus, is an important pathogen that mainly infects piglet, but little is known about the activation of the host immune response. We show that TGEV induces a delayed activation of the IFN response in intestinal epithelial cells. Briefly, IFN-β expression induced by TGEV infection is delayed with respect to that induced by poly(I:C) transfection. In addition, some of the IFN-stimulated genes (ISGs) were up-regulated in the early infection stage without obvious expression of IFN-β. Moreover, we show that activation of IFN responses induced by poly(I:C) could inhibit viral replication in the early infection stage, but failed in the late infection stage in IPEC-J2 cells. Finally, the activation of IFN responses induced by TGEV infection cannot inhibit viral replication. Taken together, this study provides a preliminary analysis of an interaction between TGEV and IFN-β responses of intestinal epithelial cells.

Functional Analyse of GLUT1 and GLUT12 in Glucose Uptake in Goat Mammary Gland Epithelial Cells

Glucose transport, mediated by glucose transporters, is necessary for mammary gland development and lactation. GLUT1 and GLUT12 could both be expressed in the pregnant and lactating mammary gland to participate in the glucose uptake process. In this study, the goat GLUT1 and GLUT12 genes were cloned from Saanen dairy goats and transfected into goat mammary gland epithelial cells to assess their biological functions and distributions. The results showed that both goat GLUT1 and GLUT12 had 12 predicted membrane-spanning helices. Goat GLUT1 and GLUT12 each influenced the mRNA expression of the other transporter and increased the glucose consumption and lactose yield in GLUT1- and GLUT12-transfected goat mammary gland epithelial cells, respectively. The overexpression of GLUT1 or GLUT12 also increased the expression of amino acid transporters SLC1A5, SLC3A2 and SLC7A5 and affected genes expressions in GMGE cells. Using immunofluorescence staining, GLUT1 was detected throughout the cytoplasm and localized to the Golgi apparatus around the nuclear membrane, whereas GLUT12 was mainly distributed in the perinuclear region and cytoplasm. This study contributes to the understanding of how GLUT1 and GLUT12 cooperate in the incorporation of nutrient uptake into mammary gland epithelial cells and the promotion of milk synthesis in the goat mammary gland during lactation.

Bacillus subtilis and surfactin inhibit the transmissible gastroenteritis virus from entering the intestinal epithelial cells

Intestinal epithelial cells are the targets for transmissible gastroenteritis (TGE) virus (TGEV) infection. It is urgent to develop a novel candidate against TGEV entry. Bacillus subtilis is a probiotic with excellent anti-microorganism properties and one of its secretions, surfactin, has been regarded as a versatile weapon for most plant pathogens, especially for the enveloped virus. We demonstrate for the first time that B. subtilis OKB105 and its surfactin can effectively inhibit one animal coronavirus, TGEV, entering the intestinal porcine epithelial cell line (IPEC-J2). Then, several different experiments were performed to seek the might mechanisms. The plaque assays showed that surfactant could reduce the plaque generation of TGEV in a dose-dependent manner. Meanwhile, after incubation with TGEV for 1.5 h, B. subtilis could attach TGEV particles to their surface so that the number of virus to bind to the host cells was declined. Furthermore, our data showed that the inhibition of B. subtilis was closely related to the competition with TGEV for the viral entry receptors, including epidermal growth factor receptor (EGFR) and aminopeptidase N (APN) protein. In addition, Western blotting and apoptosis analysis indicated that B. subtilis could enhance the resistance of IPEC-J2 cells by up-regulating the expression of toll-like receptor (TLR)-6 and reducing the percentage of apoptotic cells. Taken together, our results suggest that B. subtilis OKB105 and its surfactin can antagonize TGEV entry in vitro and may serve as promising new candidates for TGEV prevention.

Inhibition of H9N2 Virus Invasion into Dendritic Cells by the S-Layer Protein from L. acidophilus ATCC 4356

Probiotics are essential for the prevention of virus invasion and the maintenance of the immune balance. However, the mechanism of competition between probiotics and virus are unknown. The objectives of this study were to isolate the surface layer (S-layer) protein from L. acidophilus ATCC 4356 as a new antiviral material, to evaluate the stimulatory effects of the S-layer protein on mouse dendritic cells (DCs) and to verify its ability to inhibit the invasion of H9N2 avian influenza virus (AIV) in DCs. We found that the S-layer protein induced DCs activation and up-regulated the IL-10 secretion. The invasion and replication of the H9N2 virus in mouse DCs was successfully demonstrated. However, the invasion of H9N2 virus into DCs could be inhibited by treatment with the S-layer protein prior to infection, which was verified by the reduced hemagglutinin (HA) and neuraminidase (NA) mRNA expression, and nucleoprotein (NP) protein expression in the DCs. Furthermore, treatment with the S-layer protein increases the Mx1, Isg15, and Ddx58 mRNA expressions, and remits the inflammatory process to inhibit H9N2 AIV infection. In conclusion, the S-layer protein stimulates the activation of mouse DCs, inhibits H9N2 virus invasion of DCs, and stimulates the IFN-I signaling pathway. Thus, the S-layer protein from Lactobacillus is a promising biological antiviral material for AIV prevention.

Proteomic Analysis of IPEC-J2 Cells in Response to Coinfection by Porcine Transmissible Gastroenteritis Virus and Enterotoxigenic Escherichia coli K88

Piglet diarrhea causes large economic losses to the swine industry. Epidemiological investigations show that piglet diarrhea is often caused by mixed infections, but the mechanisms by which multiple microorganisms cause disease are unclear.

Immune Responses Induced by Recombinant Bacillus Subtilis Expressing the Hemagglutinin Protein of H5N1 in chickens.

To develop an effective, safe, and convenient vaccine for the prevention of highly pathogenic avian influenza (HPAI) H5N1, we have constructed a recombinant Bacillus subtilis strain (B.S.-HA) expressing the hemagglutinin (HA) protein. Then we evaluated the immune function in chicken bone marrow derived dendritic cells (BM-DCs), and the immune response after oral immunization. Our results show that the recombinant Bacillus subtilis B.S.-HA could be sampled by BM-DCs in vitro and increase the BM-DCs major histocompatibility complex (MHC) II phenotype. The weight, height of the small intestine villus, and lymphoid tissue area of the ileum increased significantly in B.S.-HA immunized chickens (P < 0.05 or P < 0.01). B.S.-HA induced the secretion of cytokines and the expression of Toll-like receptors in the trachea and small intestine (P < 0.05 or P < 0.01). In addition, B.S.-HA elevated the specific IgA titers in the trachea, IgG and HI antibody titers in serum (P < 0.05 or P < 0.01). Therefore, B.S.-HA provides a potential novel strategy and approach for developing an H5N1 vaccine.