Wulong Liang

Classical swine fever virus and p7 protein induce secretion of IL-1β in macrophages.

Classical swine fever virus (CSFV) has a tropism for vascular endothelial cells and immune system cells. The process and release of pro-inflammatory cytokines, including IL-1β and IL-18, is one of the fundamental reactions of the innate immune response to viral infection. In this study, we investigated the production of IL-1β from macrophages following CSFV infection. Our results showed that IL-1β was upregulated after CSFV infection through activating caspase-1. Subsequent studies demonstrated that reactive oxygen species may not be involved in CSFV-mediated IL-1β release. Recently, research has indicated a novel mechanism by which inflammasomes are triggered through detection of activity of viroporin. We further demonstrated that CSFV viroporin p7 protein induced IL-1β secretion which could be inhibited by the ion channel blocker amantadine and also discovered that p7 protein was a short-lived protein degraded by the proteasome. Together, our observations provided an insight into the mechanism of CSFV-induced inflammatory responses.

A comparison of the impact of Shimen and C strains of classical swine fever virus on Toll-like receptor expression.

Classical swine fever is one of the most important swine diseases worldwide and has tremendous socioeconomic impact. In this study, we focused on the signalling pathways of Toll-like receptors (TLRs) because of their roles in the detection and response to viral infections. To this end, two classical swine fever virus (CSFV) strains, namely the highly virulent CSFV Shimen strain and the avirulent C strain (a vaccine strain), were employed, and the expression of 19 immune effector genes was analysed by real-time PCR, Western blot analyses, ELISA and flow cytometry analyses. In vitro experiments were conducted with porcine monocyte-derived macrophages (pMDMs). The results showed that the mRNA and protein levels of TLR2, TLR4 and TLR7 were upregulated in response to CSFV infection, but TLR3 remained unchanged, and was downregulated after infection with the C strain and the Shimen virus, respectively. Furthermore, TLR3-mediated innate immune responses were inhibited in Shimen-strain-infected pMDMs by stimulation with poly(I : C). Accordingly, comprehensive analyses were performed to detect TLR-dependent cytokine responses and the activation of TLR signalling elements. CSFV infection induced mitogen-activated protein kinase activation, but did not elicit NFκB activation, thereby affecting the production of pro-inflammatory cytokines. The Shimen strain infection resulted in a significant activation of IFN regulatory factor IRF7 and suppression of IRF3. These data provided clues for understanding the effect of CSFV infection on the TLR-mediated innate immune response and associated pathological changes.

Detection and differentiation of classical swine fever virus strains C and Shimen by high-resolution melt analysis

Differentiation of classical swine fever virus (CSFV) strains is crucial for the development of effective vaccination programs and in epidemiological investigations. Most of current detection methods do not discriminate between wild-type CSFV strains and those used in vaccines. In this study, method involving high-resolution melt (HRM) curve analysis for the simultaneous detection and differentiation of the C and Shimen strains of CSFV was developed. A specific fragment of the NS2 gene was amplified from various CSFV strains and subjected to HRM curve analysis. Analysis of the melt curve profile for the amplicons of each strain allowed the differentiation of CSFV strains in blood samples taken from the field, or from vaccinated commercial flocks. These findings indicate that HRM curve analysis is a rapid and practical technique for discriminating CSFV isolates/strains; it can contribute to epidemiological studies of CSFV and effective control of classical swine fever.

Classical swine fever virus induces oxidative stress in swine umbilical vein endothelial cells

BackgroundClassical swine fever virus (CSFV) infection causes significant losses of pigs, which is characterized by hemorrhage, disseminated intravascular coagulation and leucopenia. The swine vascular endothelial cell is a primary target cell for CSFV. The aim of this study was to determine the role of CSFV infection in inducing oxidative stress (OS) in vascular endothelial cells.ResultsWe demonstrated that CSFV infection induced oxidative stress in swine umbilical vein endothelial cells (SUVECs), characterized by the induction of reactive oxygen species (ROS) production and the elevations of porcine antioxidant proteins thioredoxin (Trx), peroxiredoxin-6 (PRDX-6) and heme oxygenase-1 (HO-1) expression. Furthermore, cyclooxygenase-2 (COX-2), a pro-inflammatory protein related to oxidative stress, was up-regulated while anti-inflammatory protein peroxisome proliferator-activated receptor-γ (PPAR-γ), an important mediator in vascular functional regulation, was down-regulated in the CSFV infected cells. In addition, antioxidants showed significant inhibitory effects on the CSFV replication, indicating a close relationship between CSFV replication and OS induced in the host cells.ConclusionsOur results indicated that CSFV infection induced oxidative stress in SUVECs. These findings provide novel information on the mechanism by which CSFV can alter intracellular events associated with the viral infection.

Discovering up-regulated VEGF–C expression in swine umbilical vein endothelial cells by classical swine fever virus Shimen

Infection of domestic swine with the highly virulent Shimen strain of classical swine fever virus causes hemorrhagic lymphadenitis and diffuse hemorrhaging in infected swine. We analyzed patterns of gene expression for CSFV Shimen in swine umbilical vein endothelial cells (SUVECs). Transcription of the vascular endothelial growth factor (VEGF) C gene (VEGF-C) and translation of the corresponding protein were significantly up-regulated in SUVECs. Our findings suggest that VEGF-C is involved in mechanisms of acute infection caused by virulent strains of CSFV.

Integrin β3 Is Required in Infection and Proliferation of Classical Swine Fever Virus

Classical Swine Fever (CSF) is a highly infectious fatal pig disease, resulting in huge economic loss to the swine industry. Integrins are membrane-bound signal mediators, expressed on a variety of cell surfaces and are known as receptors or co-receptors for many viruses. However, the role of integrin β3 in CSFV infection is unknown. Here, through quantitive PCR, immunofluorescence (IFC) and immunocytohistochemistry (ICC), we revealed that ST (swine testicles epithelial) cells have a prominent advantage in CSFV proliferation as compared to EC (swine umbilical vein endothelial cell), IEC (swine intestinal epithelial cell) and PK (porcine kidney epithelial) cells. Meanwhile, ST cells had remarkably more integrin β3 expression as compared to EC, IEC and PK cells, which was positively correlated with CSFV infection and proliferation. Integrin β3 was up-regulated post CSFV infection in all the four cell lines, while the CSFV proliferation rate was decreased in integrin β3 function-blocked cells. ShRNA1755 dramatically decreased integrin β3, with a deficiency of 96% at the mRNA level and 80% at the protein level. CSFV proliferation was dramatically reduced in integrin β3 constantly-defected cells (ICDC), with the deficiencies of 92.6%, 99% and 81.7% at 24 h, 48 h and 72 h post CSFV infection, respectively. These results demonstrate that integrin β3 is required in CSFV infection and proliferation, which provide a new insight into the mechanism of CSFV infection.

CSFV proliferation is associated with GBF1 and Rab2

The Golgi apparatus and its resident proteins are utilized and regulated by viruses to facilitate their proliferation. In this study, we investigated Classical swine fever virus (CSFV) proliferation when the function of the Golgi was disturbed. Golgi function was disturbed using chemical inhibitors, namely, brefeldin A (BFA) and golgicide A (GCA), and RNA interfering targets, such as the Golgi-specific BFA-resistance guanine nucleotide exchange factor 1 (GBF1) and Rab2 GTPases. CSFV proliferation was significantly inhibited during RNA replication and viral particle generation after BFA and GCA treatment. CSFV multiplication dynamics were retarded in cells transfected with GBF1 and Rab2 shRNA. Furthermore, CSFV proliferation was promoted by GBF1 and Rab2 overexpression using a lentiviral system. Hence, Golgi function is important for CSFV multiplication, and GBF1 and Rab2 participate in CSFV proliferation. Further studies must investigate Golgi-resident proteins to elucidate the mechanism underlying CSFV replication.

Catechin inhibition of transmissible gastroenteritis coronavirus in swine testicular cells is involved its antioxidation

ABSTRACT Transmissible gastroenteritis virus (TGEV) causes transmissible gastroenteritis (TGE), especially in newborn piglets, which severely threatens the worldwide pig industry. In this study, (+)-catechin was evaluated for its antiviral effect against TGEV in vitro. Viability assays revealed that (+)-catechin treatment exerted a dose-dependent rescue effect in TGEV-infected ST cells, and this result was only obtained with the post-treatment application of (+)-catechin. The viral yields in (+)-catechin-treated cultures were reduced by almost three log10 units. Quantitative real-time PCR analysis of the TGEV genome revealed that TGEV RNA replication was restricted after (+)-catechin treatment. Intracellular reactive oxygen species (ROS) detection showed that (+)-catechin alleviated ROS conditions induced by TGEV infection. Our results showed that (+)-catechin exerts an inhibitory effect on TGEV proliferation in vitro and is involved its antioxidation.

FKBP8 interact with classical swine fever virus NS5A protein and promote virus RNA replication

The non-structural 5A (NS5A) protein of classical swine fever virus (CSFV) is proven to be involved in viral replication and can also modulate cellular signaling and host cellular responses via to its ability to interact with various cellular proteins. FKBP8 is also reported to promote virus replication. Here, we show that NS5A specifically interacts with FKBP8 through coimmunoprecipitation and GST-pulldown studies. Additionally, confocal microscopy study showed that NS5A and FKBP8 colocalized in the cytoplasm. Overexpression of FKBP8 via the eukaryotic expression plasmid pDsRED N1 significantly promoted viral RNA synthesis. The cells knockdown of FKBP8 by lentivirus-mediated shRNA markedly decreased the virus replication when infected with CSFV. These data suggest that FKBP8 plays a critical role in the viral life cycle, particularly during the virus RNA replication period. The investigation of FKBP8 protein functions may be beneficial for developing new strategies to treat CSFV infection.

Rab5 Enhances Classical Swine Fever Virus Proliferation and Interacts with Viral NS4B Protein to Facilitate Formation of NS4B Related Complex

Classical swine fever virus (CSFV) is a fatal pig pestivirus and causes serious financial losses to the pig industry. CSFV NS4B protein is one of the most important viral replicase proteins. Rab5, a member of the small Rab GTPase family, is involved in infection and replication of numerous viruses including hepatitis C virus and dengue virus. Until now, the effects of Rab5 on the proliferation of CSFV are poorly defined. In the present study, we showed that Rab5 could enhance CSFV proliferation by utilizing lentivirus-mediated constitutive overexpression and eukaryotic plasmid transient overexpression approaches. On the other hand, lentivirus-mediated short hairpin RNA knockdown of Rab5 dramatically inhibited virus production. Co-immunoprecipitation, glutathione S-transferase pulldown and laser confocal microscopy assays further confirmed the interaction between Rab5 and CSFV NS4B protein. In addition, intracellular distribution of NS4B-Red presented many granular fluorescent signals (GFS) in CSFV infected PK-15 cells. Inhibition of basal Rab5 function with Rab5 dominant negative mutant Rab5S34N resulted in disruption of the GFS. These results indicate that Rab5 plays a critical role in facilitating the formation of the NS4B related complexes. Furthermore, it was observed that NS4B co-localized with viral NS3 and NS5A proteins in the cytoplasm, suggesting that NS3 and NS5A might be components of the NS4B related complex. Taken together, these results demonstrate that Rab5 positively modulates CSFV propagation and interacts with NS4B protein to facilitate the NS4B related complexes formation.