The Porcine Circovirus Type 2 Nonstructural Protein ORF3 Induces Apoptosis in Porcine Peripheral Blood Mononuclear Cells

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. To analyze whether the PCV2 nonstructural protein ORF3 is able to induce apoptosis in nature target cells, transient expression of ORF3 in porcine peripheral blood mononuclear cells (PBMC) was performed, and apoptosis was confirmed by terminal dexoynucleotidyl transferase (TdT)-mediated BrdUTP-nick end labeling (TUNEL) assay. The apoptotic responses induced by the full length or the C-terminal half of ORF3 were significantly higher (p < 0.001) than that of cells transfected with the control plasmid. In contrast, the N-terminal half of ORF3 restrictively localized in the cytoplasm and remarkably reduced its ability to induce apoptosis, the apoptotic activity might be correlated with the nuclear localization of ORF3. Furthermore, two clusters of basic residues on the C-terminal half region at the amino acid residues 53-68 and 85-104 could mediate the nuclear localization of fusion protein, confirming their potential role as a nuclear localization signal

Mechanisms underlying Actinobacillus pleuropneumoniae exotoxin ApxI induced expression of IL-1β, IL-8 and TNF-α in porcine alveolar macrophages

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes fibrino-hemorrhagic necrotizing pleuropneumonia in pigs. Production of proinflammatory mediators in the lungs is an important feature of A. pleuropneumoniae infection. However, bacterial components other than lipopolysaccharide involved in this process remain unidentified. The goals of this study were to determine the role of A. pleuropneumoniae exotoxin ApxI in cytokine induction and to delineate the underlying mechanisms. Using real-time quantitative PCR analysis, we found native ApxI stimulated porcine alveolar macrophages (PAMs) to transcribe mRNAs of IL-1β, IL-8 and TNF-α in a concentration- and time-dependent manner. Heat-inactivation or pre-incubation of ApxI with a neutralizing antiserum attenuated ApxI bioactivity to induce cytokine gene expression. The secretion of IL-1β, IL-8 and TNF-α protein from PAMs stimulated with ApxI was also confirmed by quantitative ELISA. In delineating the underlying signaling pathways contributing to cytokine expression, we observed mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK) were activated upon ApxI stimulation. Administration of an inhibitor specific to p38 or JNK resulted in varying degrees of attenuation on ApxI-induced cytokine expression, suggesting the differential regulatory roles of p38 and JNK in IL-1β, IL-8 and TNF-α production. Further, pre-incubation of PAMs with a CD18-blocking antibody prior to ApxI stimulation significantly reduced the activation of p38 and JNK, and subsequent expression of IL-1β, IL-8 or TNF-α gene, indicating a pivotal role of β2 integrins in the ApxI-mediated effect. Collectively, this study demonstrated ApxI induces gene expression of IL-1β, IL-8 and TNF-α in PAMs that involves β2 integrins and downstream MAPKs

Mutations in the Salmonella enterica serovar Choleraesuis cAMP-receptor protein gene lead to functional defects in the SPI-1 Type III secretion system

Salmonella enterica serovar Choleraesuis (Salmonella Choleraesuis) causes a lethal systemic infection (salmonellosis) in swine. Live attenuated Salmonella Choleraesuis vaccines are effective in preventing the disease, and isolates of Salmonella Choleraesuis with mutations in the cAMP-receptor protein (CRP) gene (Salmonella Choleraesuis ∆crp) are the most widely used, although the basis of the attenuation remains unclear. The objective of this study was to determine if the attenuated phenotype of Salmonella Choleraesuis ∆crp was due to alterations in susceptibility to gastrointestinal factors such as pH and bile salts, ability to colonize or invade the intestine, or cytotoxicity for macrophages. Compared with the parental strain, the survival rate of Salmonella Choleraesuis ∆crp at low pH or in the presence of bile salts was higher, while the ability of the mutant to invade intestinal epithelia was significantly decreased. In examining the role of CRP on the secretory function of the Salmonella pathogenicity island 1 (SPI-1) encoded type III secretion system (T3SS), it was shown that Salmonella Choleraesuis ∆crp was unable to secrete the SPI-1 T3SS effector proteins, SopB and SipB, which play a role in Salmonella intestinal invasiveness and macrophage cytotoxicity, respectively. In addition, caspase-1 dependent cytotoxicity for macrophages was significantly reduced in Salmonella Choleraesuis ∆crp. Collectively, this study demonstrates that the CRP affects the secretory function of SPI-1 T3SS and the resulting ability to invade the host intestinal epithelium, which is a critical element in the pathogenesis of Salmonella Choleraesuis

Characterization of the swine U6 promoter for short hairpin RNAexpression and its application to inhibition of virus replication

Expression of short hairpin RNAs (shRNAs) by the RNA polymerase type III U6 promoter is an effective and widely used strategy for RNA interference (RNAi) which is a sequence-specific gene silencing mechanism. The U6 promoters from human, mouse, and swine were cloned, respectively for constructing various shRNA expression vectors. The transcription efficiency of each U6 promoter was analyzed for its activity to drive expression of shRNA targeting enhanced green fluorescent protein (EGFP) mRNA in different mammalian cells. All three U6 promoters were functional and the swine U6 promoter demonstrated the most efficient knockdown of EGFP synthesis in all these three species of cell lines including porcine kidney (PK-15), human embryonic kidney (HEK293T), and mouse fibroblast (LM) cells. Furthermore, the antiviral effect of shRNA targeting the classical swine fever virus (CSFV) NS5B driven by the swine U6 promoter was confirmed by the significant reduction of virus replication

Effectiveness of Live-Attenuated Genotype III Japanese Encephalitis Viral Vaccine against Circulating Genotype I Viruses in Swine

Expansion of genotype I (GI) Japanese encephalitis viruses (JEV) has resulted in the replacement of the dominant genotype III (GIII) viruses, raising serious public health concerns for using GIII virus-derived vaccines to effectively control JEV epidemics. Therefore, this study used swine as the model to estimate the effectiveness of GIII live-attenuated vaccine against GI virus infection by comparing the incidence of stillbirth/abortion in gilts from vaccinated and non-vaccinated pig farms during the GI-circulation period. In total, 389 and 213 litters of gilts were recorded from four vaccinated and two non-vaccinated pig farms, respectively. All viruses detected in the aborted fetuses and mosquitoes belonged to the GI genotype during the study period. We thus estimated that the vaccine effectiveness of GIII live-attenuated vaccine against GI viruses in naive gilts based on the overall incidence of stillbirth/abortion and incidence of JEV-confirmed stillbirth/abortion was 65.5% (50.8&ndash;75.7%) and 74.7% (34.5&ndash;90.2%), respectively. In contrast to previous estimates, the GIII live-attenuated vaccine had an efficacy of 95.6% (68.3&ndash;99.4%) to prevent the incidence of stillbirth/abortion during the GIII-circulating period. These results indicate that the vaccine effectiveness of GIII live-attenuated JEV vaccine to prevent stillbirth/abortion caused by GI viruses is lower than that against GIII viruses

Characterization of the monoclonal antibody specific to the ORF72 protein of koi herpesvirus and cellular distribution analysis of the viral protein

Koi herpesvirus (KHV) is an emerging pathogen of koi and common carp that causes a severe disease and mass mortality of infected fish. The KHV ORF72 protein is an important capsid protein that has been suggested to be a candidate for the development of diagnostic reagents and KHV vaccines. The purpose of this study was to clone and express the KHV ORF72 gene for further preparation of a specific monoclonal antibody (mAb) and to analyse cellular distribution of the viral protein. The mAb 3E1 could specifically recognize the expressed ORF72 protein of transfected cells by indirect immunofluorescence, and the antigenic site recognized by the mAb 3E1 was mapped to the region of N-terminal 124 residues of KHV ORF72. This mAb was further demonstrated to specifically detect the KHV-infected fish tissue by immunohistochemistry, thereby suggesting its high diagnostic potential. In addition, the cellular distribution analysis of the KHV ORF72 protein revealed that the region of amino acid residues 125-247 was related to mitochondrial localization and proliferation. Furthermore, a putative nuclear export signal (NES) of ORF72 at the residues 201-212 was confirmed on the basis of its function associated with NES activity

Characterization of the monoclonal antibody against classical swine fever virus glycoprotein E(rns) and its application to an indirect sandwich ELISA

Classical swine fever virus (CSFV) E(rns) is an envelope glycoprotein possessing RNase activity. The E(rns)-based enzyme-linked immunosorbent assay (ELISA) has been considered a discriminating diagnostic test for differentiating infected from vaccinated animals. The purpose of this study was to produce a specific monoclonal antibody (MAb) to E(rns) for further developing an indirect sandwich ELISA. The MAb CW813 was shown to specifically recognize both the monomer and dimer forms of Pichia pastoris yeast-expressed E(rns) (yE(rns)). The antigenic site recognized by MAb CW813 was mapped to the region of amino acid residues 101-160 of E(rns) where it was neither a neutralizing epitope nor essential to RNase activity. Furthermore, MAb CW813 was utilized as a capture antibody to develop a yE(rns)-based indirect sandwich ELISA for detecting swine antibody to E(rns). The assay demonstrated a high sensitivity and specificity that may provide an alternative method for developing a diagnostic kit with easy manipulation and low cost

PORCINE CIRCOVIRUS TYPE 2 CAPSID PROTEIN SUBUNITS AND THE USE THEREOF

本發明係關於，具有良好特異性及敏感性之豬環狀病毒二型(Porcine circovirus type 2，PCV2)外殼蛋白(capsid protein，Cap)次單位片段，以及利用其診斷豬隻體內PCV2病毒感染之用途。本發明尤其是關於，利用大腸桿菌表現系統大量製備該等Cap次單位片段。本發明亦進一步提供用於檢測豬隻體內PCV2感染之血清診斷方法及套組

Exploring the surface epitope and nuclear localization analysis of porcine circovirus type 3 capsid protein

Abstract Porcine circovirus 3 (PCV3) is a newly emerging virus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders, impacting global pig populations. Porcine circoviruses contain two major open reading frames (ORFs), and the ORF2 encodes the viral capsid protein (Cap). Cap is the most antigenic structural protein and an ideal candidate for the development of vaccines and diagnostic reagents. This study generated a monoclonal antibody (MAb) specific to PCV3 Cap, MAb CCC160, for diagnosis and pathogenesis studies of this novel virus. The MAb specifically recognized PCV3-infected swine lymph node tissue in an immunohistochemical analysis confirming its clinical diagnostic potential. In addition, a novel linear B-cell epitope recognized by MAb CCC160 was identified at the amino acid region 120–134 of Cap. Nuclear localization analysis of PCV3 Cap revealed a potential nuclear localization signal (NLS) in the middle region (aa 131–143) in addition to the dominant N-terminal NLS that is already known. A cell viability assay further demonstrated that the cytotoxicity of PCV3 Cap is correlated with its nuclear localization, indicating a crucial role of Cap in the pathogenic mechanism of PCV3. A full-length construct of PCV3 Cap was successfully expressed using a baculovirus expression system and purified recombinant proteins self-assembled into virus-like particles (VLPs). The protein constitution of the VLPs was confirmed by MAb CCC160 recognition, indicating the correct conformation and specificity of VLP and exhibiting the linear epitope aa 120–134 on the VLP surface. These results provide insights for developing diagnostic tools and potential VLP vaccines for PCV3, revealing its pathogenesis and antigenic properties