Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR) for diagnosis of natural infection with canine distemper virus

<p>Abstract</p> <p>Background</p> <p>Canine distemper virus (CDV) is present worldwide and produces a lethal systemic infection of wild and domestic <it>Canidae</it>. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR) of the matrix (M) gene to the fusion (F) gene (designated M-F UTR) in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions.</p> <p>Results</p> <p>Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains.</p> <p>Conclusions</p> <p>The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes.</p

The Effect of Classical Swine Fever Virus (CSFV) on Nitric Oxide (NO) in Porcine Aortic Endothelial Cells (PAECS)

猪瘟是一種在猪隻上常見的重要傳染病。具有很高的傳染率及死亡率。雖然目前大規模免疫計畫已被實施，但散發性的病例仍造成畜牧業重大的經濟損失。猪瘟病毒屬於黃病毒科的瘟疫病毒屬。在急性或亞急性病例中，激烈的炎症及免疫反應與過度活化的凝血活性會引起身體組織及臟器的點狀出血及多發性血栓。本病的致病機制主要與病毒對血管內皮細胞的傷害導致的血管炎有關。根據研究，由常在性及誘導性一氧化氮合成酶所製造的一氧化氮具有許多重要的生理功能包括做為血管壁張力的釋放因子、神經傳導物質及宿主對病原的防禦因子。尤其對病毒的感染具有保護及細胞毒殺兩種雙極效應。由感染細胞產生的一氧化氮可以抑制痘病毒及反轉錄病毒的複製。然而當細胞中一氧化氮合成酶因狂犬病毒及里奧病毒感染大量表現時，高濃度的一氧化氮會引起嚴重發炎而傷害組織。因此我們提出調查猪瘟病毒對血管內皮細胞一氧化氮濃度的影響。本計畫分為兩年期，在生物體外進行研究。在不同時間及劑量下將猪瘟病毒感染猪主動脈內皮細胞。首先利用共軛焦顯微鏡來觀察細胞中常在性及誘導性一氧化氮合成酶的表現程度及分布形式。同時並使用PCR及西方點墨法來定量常在性及誘導性一氧化氮合成酶的表達及代謝速率。細胞所分泌出的一氧化氮量和常在性及誘導性一氧化氮合成酶的起動子活性將被分析。更進一步，常在性一氧化氮合成酶的磷酸化狀態將被分析。利用選擇性抑制劑來觀察PI3-Akt信號路徑、Src蛋白家族和細胞內鈣離子在常在性一氧化氮合成酶的磷酸化所扮演的角色。MAPK信號路徑在iNOS表現上的影響也將被探討。基於我們過去在研究的經驗，本計畫預期將被順利執行。其成果將幫助我們對猪瘟病毒的分子致病機轉的了解。同時也提供未來防疫上的一個新方向。Classic swine fever (CSF) is a contagious disease of domestic pigs with high severity and mortality. It still causes a significant loss to pig industry in Taiwan even an extensive vaccination plan is implemented. Classic swine fever virus (CSFV) is a member of Pestivirus genus of Flaviridae. In acute and subacute cases, severe haemorrhagic diathesis and petechiation in multiple tissues and organs are derived from exaggerated inflammatory and immune responses and increased procoagulant activities. Vasculitis triggered by damages of endothelial cells when CSFV replicates inside constitutes the pathogenic basis of CSF. Nitric oxide (NO) identified as a vessel relaxing factor, a neurotransmitter, and a host defense mediator is generated from both a constitutive NO synthase (cNOS) and an inducible NO synthase (iNOS). Studies have indicated NO plays a role in both protective and cytotoxic responses in viral infections. NO produced by infected cells inhibited the replication of vaccinia virus and retroviruses. The highoutput NO from the up-regulated NO synthases followed cells infected with rabies virus and reovirus caused severe inflammations leading to tissue damages. A comprehensive study using culture cells for two years is proposed herein. Porcine aortic endothelial cells (PAEC) are infected with CSFV at different dosage and time dependent manners. The expression profiles and spatial localizations of cNOS and iNOS are checked by high resolution confocal laser scanning microscope. The expression levels and degradation rates of cNOS and iNOS are determined by semi-quantitative RT-PCR and Western blotting. The amount of nitric oxide and promoter activities of cNOS and iNOS are measured. Furthermore, the phosphorylation status of cNOS is examined. Selective inhibitors are used to determine the roles of PI 3-K/Akt pathway, Src protein, and intracellular calcium ions in eNOS phosphorylation. The expression of iNOS through MAPK pathway is analyzed. With past experiences in studies, this plan is surely to be conducted properly. This proposal will improve our understanding about molecular pathogenesis of CSFV and potentially contributes to develop a novel strategy for future prevention and therapy

The roles of topoisomerases in the replication and expression of pseudorrabies virus

假性狂犬病為一種重要的豬的傳染病。假性狂犬病毒為此病的病原，其基 因体為線性雙股DNA其大小約為145kbp，病毒粒子的大小約 150-180nm， 其內部核心由DNA與蛋白質結合而成，包覆核心的蛋白殼由162個 capsomers 組成，為正二十面体結構(icosah- edron)，最外圍封套是醣 蛋白與酯蛋白組成，介於封套與蛋白殼之間的不定形結構稱之為 Tegument.其功能仍不清楚。假性狂犬病毒DNA可分為Us(Unique short region),Ul(Unique long region)，及包夾住Us的兩個IR(Inverted repeat),和(Terminal repeat) DNA複製的機制為rolling-circle模式， 分別由位於Ul和Us上的兩個複製起點開始，病毒本身的DNA合成酵素(DNA polymerase)及一些輔助因子如DNA binding protein一起擔任合成的工作 。本計畫探討假性狂犬病毒DNA複製及基因表達與拓樸異構酵素的關係， 拓樸異構酵素廣泛存在原核細胞及真核細胞中，控制DNA的拓樸結構構也 影響基因的表達。由理論推測與實驗數據得知，拓樸異構酵素對於基因體 為封閉環狀DNA的病毒(如SV40)之複製十分重要。近來的實驗報告也支持 拓樸異構酵素對線性雙股DNA的病毒的複製與基因表現非常重要。在本研 究中分別利用第一型拓樸異構酵素抑制劑camptothecin和第二型拓樸異構 酵素抑制劑VP-16及 ellipt- icine，發現拓樸異構酵素與假性狂犬病毒 的繁殖有關，更進一步証明假性狂犬病毒DNA的複製需要第一型拓樸異構 酵素參與，我們的結果也顯示在病毒基因表現上的轉錄部分都受第一和第 二型拓樸異構酵素之影響。Pseudorabies disease is an important infectious disease of swine, and pseudorabies virus is the causative agent. The pseudorabies virus genome is a linear double-stranded DNA(about 145kbp).The icosahedral capsid approximately 150-180nm in diameter contains 162 capsomeres , sometimes asymmetric material surrounding the capsid designated as the tegument, and an envelope containing viral glycoprotein spikes on its surface.The pseudorabies virus genome consists of two covalently linked components , designated as unique L(long) and unique S(short), which were bracketed by inverted repeat and terminal repeat.The viral DNA is synthesized by a virus-encoded DNA polymerase and a rolling circle replicating mechanism has been proposed. Our research goal is to elucidate the roles of topoisomerases in the replication and expression of pseudorabies virus. Both type I and II DNA topoisomerases have been found in prokaryotes and eukaryotes. They have important roles in cellular DNA replication and transcription. In animal viruses, topoisomerases also involve in the life cycle of viruses containing closed circular DNA( such as SV 40 virus) as well as viruses possessing double- stranded linear DNA(such as adenovirus and herpesviruses). Our results reveal that type I topoisomerase inhibitor (camptothecin) and type II topoisomerase inhibitors (VP-16 and ellipticine) could inhibit the multiplication of pseudorabies virus. The type I topoisomerase activity is essential for the DNA synthesis of pseudorabies virus.Our findings also suggest that both type I and type II topoisomerases are required for the gene expression of the pseudorabies virus genome

Classical swine fever virus down-regulates endothelial connexin 43 gap junctions

Classical swine fever is a contagious disease of pigs characterized by fatal hemorrhagic fever. Classical swine fever virus (CSFV) induces the expression of pro-inflammatory and pro-coagulant factors of vascular endothelial cells and establishes a long-term infection. This study aimed to understand the effect of CSFV on endothelial connexin 43 (Cx43) expression and gap junctional intercellular coupling (GJIC). Porcine aortic endothelial cells were infected with CSFV at different multiplicity of infection for 48 h. Semi-quantitative RT-PCR, immunoconfocal microscopy, and Western blotting showed that the transcription and translation of Cx43 were reduced, and this was associated with an attenuation of GJIC. This decrease occurred in a time-dependent manner. An ERK inhibitor (PD98059), a JNK inhibitor (SP600125), and proteasome/lysosome inhibitors all significantly reversed the reduction in Cx43 protein levels without any influence on the titer of progeny virus. In addition, CSFV activated ERK and JNK in a time-dependent manner and down-regulated Cx43 promoter activity, mainly through decreased AP2 binding. This effect was primarily caused by the replication of CSFV rather than a consequence of cytokines being induced by CSFV infection of endothelial cells

Survey and Prevention of Parasites and Leptospira of Bovine and Chicken Farms Suffering from Flood

本計畫目標為調查泛洪區災後草食動物及家禽場可能發生潛在之疾病種類，並訂定疾病防治的措施。重要工作項目包括：泛洪區牛雞畜牧場環境土樣採集及分析、牛畜牧場之動物檢體採集及分析、雞畜牧場之動物檢體採集及分析。於期末統計泛洪區災後草食動物及家禽場已發生疾病，並參考國外文獻訂定泛洪區災後草食動物及家禽場可能爆發疾病的標準處理流程及防治措施。The target of the program was surveying the outbreak of potential diseases in herbivore and poultry farm after suffering from flood damage and setting disease preventing procedure. The main contents of this program including collecting environmental soil from bovine and poultry farms suffered from flood, specimens of infected animal from bovine and poultry farms and conducting specific pathogen analysis. At the end of this program, summarize and classify of the diseases happened in bovine and poultry farms suffered from flood; at the same time, standard disease preventing and treating procedures was set according to the references of foreign countries and the analyzed result in this program

Novel post-translational modifications of the hemagglutinin and neuraminidase proteins of avian influenza virus expressed by Kluyveromyces lactis

Avian influenza virus (AIV) is an enveloped virus with segmented RNA that belongs to the Orthomyxoviridae. Recently, avian influenza virus isolates have not only posed a significant threat to the poultry industry but also serious public health concerns. The full-length viral hemagglutinin (HA), neuraminidase (NA) or both genes were inserted into the yeast Kluyveromyces lactis genome to allow for secreted expression. Both hemagglutinin and neuraminidase activities were demonstrated for the expressed proteins. Based on PNGase F digestion and immunoassays, N-glycosidically linked high mannose or hybrid-type carbohydrate chains on the HA protein are predominant. It is noteworthy that when co-expression of the HA and NA proteins was carried out, the NA protein was able to react with the HA protein, resulting in deglycosylation in a manner similar to PNGase F digestion. Such post-translational modifications in the HA and NA proteins of AIV are described for the first time. (C) 2011 Elsevier B.V. All rights reserved

Differential diagnosis of orf viruses by a single-step PCR

The complete nucleotide sequence of the A32L gene (named after vaccinia virus, corresponding with open reading frame 108 of the orf virus and encoding an ATPase) of the orf virus was studied using samples of orf virus from infected goats, which were collected from six outbreaks in central Taiwan. DNA sequence analysis of the A32L genes of these and isolates from other countries showed sequence heterogeneity (base pair variation and deletion) in the 3'-terminal regions. This finding led to the development of a polymerase chain reaction (PCR) method for the rapid differential diagnosis of orf virus infections, and the results demonstrated that this was an easy and reliable method for genotyping of orf viruses. (C) 2009 Elsevier B.V. All rights reserved

Development of an antigen-capture ELISA for beak and feather disease virus

Psittacine beak and feather disease (PBFD) is characterised by degenerative feather, feather dystrophy, and beak deformity. Sometimes acute forms can lead to fatal cases in nestlings. The worldwide distribution of this disease affects numerous species of parrots with an average prevalence of 40%, including in Taiwan. The pathogen of PBFD is beak and feather disease virus (BFDV), which is a single-stranded circular DNA virus, circovirus. To date, hemagglutination and PCR assays have been routinely used to detect this virus. In this study, both the replication-associated protein (Rep) and the structural capsid protein (Cap) were expressed and then used as antigens for the production of monoclonal antibodies. Conserved epitopes recognised by the anti-Cap and anti-Rep monoclonal antibodies were determined to be NFEDYRI and LSALKKM, respectively. Clinical samples collected from different species of parrots were tested by hemagglutination, PCR, and anti-Cap antigen-capture ELISA assays and the positive rates were the same at 49%. Thus, this anti-Cap antigen-capture ELISA is able to be used for the rapid identification of BFDV-infected birds in a non-invasive manner