Molecular cloning and nucleotide sequence of 3 '-terminal region of classical swine fever virus LPC vaccine strain

A cDNA of the 3'-terminus of classical swine fever virus (LPC vaccine strain) was cloned and sequenced. The 3431 nucleotides and deduced amino acid sequences were compared with those of other pestiviruses, and the similarity of nucleotide sequences and deduced amino acid sequences were found to be 84-95% and 95-98%, respectively. Similar to other isolates of classical swine fever virus, the sequenced region included the non-structural gene p58 (NS5A) and part of p76 (NS5B) gene. The p76 gene of LPC vaccine strain also contained a highly conserved motif G-D-D (Gly-Asp-Asp) that is present in the RNA replicase of positive-stranded RNA viruses. With the sequence data currently available, we carried out a phylogenetic analysis and obtained a genealogical relationship among members of the classical swine fever virus. The sequence has been submitted to GeneBank with an accession number AF001986

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 Delta 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 Delta 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 Delta 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 Delta 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 Delta 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 and Functional Analysis of the Classical Swine Fever Virus Structural Glycoproteins and Their Applications on Diagnostics

猪瘟病毒（classical swine fever virus﹔CSFV）為引起猪隻高度傳染性疾病-猪瘟之病原，造成經濟上相當大的損失。猪瘟病毒表面封套包含三種結構性醣蛋白Erns、E1及E2，其中Erns及E2可誘發宿主產生中和抗體，而Erns除了具有RNase之酵素活性亦被認為與感染動物引起淋巴細胞凋亡（apoptosis）現象有關，故猪瘟病毒結構性醣蛋白在致病機制上扮演相當重要的角色。然而有關E1之抗原性及Erns之蛋白結構與RNase酵素活性在病毒的life cycle中所參與的功能目前則尚未清楚。本實驗室已建立利用酵母菌Pichia pastoris真核表現系統來表現Erns醣蛋白之技術，且Erns重組表現蛋白具有RNase活性及正確之抗原性。本計畫之目的為應用酵母菌表現系統進行猪瘟病毒結構性醣蛋白Erns、E1及E2之大量製備並建構出一系列Erns缺損突變重組表現蛋白，以進一步進行結構性醣蛋白之功能及抗原性分析與Erns誘發淋巴細胞凋亡機制之探討。本計畫將分三年完成，第一年度主要的工作包括猪瘟病毒醣蛋白Erns、E1及E2表現蛋白之建構與大量生產，特異性單源抗體的製備及抗原決定位分析。第二年度則針對猪瘟病毒Erns蛋白之相關重要功能區(functional domains)包括N-glycosylation、RNase活性、dimerization及translocation等之分析與Erns誘發老鼠淋巴細胞凋亡作用途徑與次族群標的細胞之測定。而第三年度則持續完成猪瘟病毒neutralizing epitopes之變異性分析，建立分別針對猪瘟病毒Erns及E2醣蛋白之特異性ELISA抗體檢測方法，並完成大規模野外猪隻血清之實際測試及評估。本計畫所得結果將可提供做為猪瘟病毒致病機制相關研究與診斷試劑及疫苗研發上之材料與參考，有助於對猪瘟的防治與監控。Classical swine fever virus (CSFV) is the causative agent of classical swine fever, which is highly contagious in pigs, and this disease causes large economical losses. The viral envelope contains three structural glycoproteins, Erns, E1, and E2, of which Erns and E2 are capable of inducing the production of neutralizing antibodies in host. The glycoprotein Erns has been identified to posses RNase activity and is associated with the induction of lymphocytes apoptosis in infected animals. Thus these structural glycoproteins play an important role in CSFV pathogenesis. The purpose of this project is to produce these structural glycoproteins in large scale by the yeast Pichia pastoris eukaryotic expression system for further functional analysis and applications. The major works in the first year of this 3-year project are the construction and expression of glycoproteins Erns, E1, and E2 as well as a series Erns deletion mutant proteins, and preparation of specific monoclonal antibodies against CSFV Erns, E1, and E2 respectively. In the second year, the functional domains of Erns responsible for N-glycosylation, dimerization, RNase activity as well as translocation, and the mechanism related to the induction of lymphocytes apoptosis by Erns will be investigated in great insight. In the third year, the variation of neutralizing epitopes in the CSFV Erns and E2 will be continuously analyzed and ELISA tests for detecting swine antibody against Erns and E2 will be set up. All the results obtained from this project will provide useful materials and information for future studies on CSFV pathogenesis and developing diagnostic reagents and vaccines

Study on the Protein Strtucture and Functions and Pathogenesis of the Classical Swine Fever Virus Glycoprotein Erns

豬瘟病毒（classical swine fever virus﹔CSFV）為引起豬隻高度傳染性疾病-豬瘟之病原，造成經濟上相當大的損失。豬瘟病毒表面兩種結構性醣蛋白Erns及E2皆可誘發宿主產生中和抗體，而Erns除了具有RNase之酵素活性亦被認為與感染動物引起淋巴細胞凋亡（apoptosis）現象有關，故Erns在豬瘟病毒致病機制上扮演相當重要之角色。然而有關Erns之蛋白結構與RNase酵素活性在病毒的life cycle中所參與的功能目前則尚未清楚。本實驗室已建立利用酵母菌Pichia pastoris真核表現系統來表現Erns醣蛋白之技術，且Erns重組表現蛋白具有RNase活性及正確之抗原性。本計畫之目的為建立Erns表現蛋白大規模生產技術並建構出一系列Erns缺損突變重組表現蛋白，以進一步進行其功能分析與其誘發淋巴細胞凋亡機制之探討。本計畫將分兩年完成，第一年度主要的工作包括全長與一系列缺損突變豬瘟病毒醣蛋白Erns表現蛋白之建構與大量生產純化及特異性單源抗體的製備及抗原決定位分析。而第二年度則包括豬瘟病毒Erns表現蛋白之相關重要功能區(functional domains)包括N-glycosylation、RNase活性、dimerization及translocation等之分析與Erns誘發老鼠淋巴細胞凋亡作用途徑與次族群標的細胞的測定。本計畫所得結果將可提供做為豬瘟病毒致病機制相關研究與診斷試劑研發上之材料與參考，有助於對豬瘟的防治與監控

Preparation of Monoclonal Antibody against Porcine Circovirus Type 2 (PCV2) and Development of an ELISA Antibody Diagnostic Kit

豬環狀病毒二型(porcine circovirus; PCV2)與離乳豬系統性消耗症(postweaning multisystemic wasting syndrome; PMWS)具密切關連性，對養豬產業造成重大威脅。本計畫目標為利用大腸桿菌表現系統大量製備特定病毒外殼蛋白(capsid protein; Cap)重組片段，進一步製備出特異性之單源抗體並研製ELISA檢測套組應用於野外血清的檢測。豬環狀病毒二型之特異性單源抗體的製備可提供檢測PCV2的病原或抗體之診斷試劑，並可應用於對病毒之特性及致病機制的相關研究。而豬環狀病毒二型ELISA抗體檢測套組的開發將可應用於例行大規模豬隻血清之檢測，可藉由進行PCV2的調查、診斷及血清流行病學的調查，對豬環狀病毒感染之監控與防治將有所助益。Postweaning multisystemic wasting syndrome of swine associated with porcine circovirus type 2 (PCV2) is a recently reported and economically important disease. The viral structural protein, capsid protein (Cap), consists of major antigenic domains. The purpose of this project is to produce the viral Cap protein subunit in large amount by E. coli expression system for preparing monoclonal antibodies and developing ELISA diagnostic reagents. The results obtained from this project will provide insight information on viral pathogenesis and will assist the control of this disease. The recombinant Cap subunits possessing defined antigenicity are easy to produce and the subunit-based ELISA was developed with a high specificity and sensitivity that may provide a useful method for routine serodiagnosis of PCV2 infection

Study of the Pseudorabies Virus Immediate-Early Gene

假性狂犬病毒(pseudorabies virus；PRV)是一種阿爾法皰疹病毒(alphaherpesvirus)。該病毒在細胞內進行複製時，因其基因階段性的控制表現，可將其基因分為立即早期(immediate-early；IE)，早期(early)及晚期(late)三群。而其中立即早期基因在病毒感染細胞之後並不需要其他病毒蛋白的產生即可立刻開始進行轉錄。此外，IE基因產物即立即早期蛋白(IE蛋白)對病毒的早期及晚期基因具有激活作用(trans-activation)的功能且是這些病毒基因進行轉錄時所必需，故在整個病毒感染過程中扮演相當重要的角色。本專題計畫乃擬對PRV國內分離株(TNL株)之立即早期基因之功能與其對病毒的早期或晚期基因激活作用之影響做更深入的探討。故下一年度計畫將分為下列三個工作項目進行：(一)持續表現假性狂犬病毒立即早期蛋白細胞株之建立；(二)假性狂犬病毒立即早期基因對病毒早期基因或晚期基因激活作用之分析；(三)假性狂犬病毒立即早期基因缺損病毒株之開發。本研究計畫主要是對PRV立即早期基因之功能做更深入的研究，將可對整個病毒之感染複製過程與致病機序(pathogenesis)有更清楚的了解。而假性狂犬病毒立即早期基因缺損病毒株可進而發展成為一安全性病毒載體，在疫苗的開發應用上將具有相當之潛力與貢獻