Intestinal geotrichosis in a German shepherd

A 4-year-old sexually intact male German shepherd with a 3-month history of chronic watery diarrhea was referred to the Veterinary Medical Teaching Hospital. Dehydration and serum biochemistry revealed hypoalbuminemia, and fecal material contained a large quantity of round arthroconidia that were microscopically observed. A specimen was submitted for fungal culture and yielded a white to cream-colored subsurface colony. Microorganisms derived from the colony exhibited chains of smooth, septate hyaline hyphae that were producing 1-celled arthroconidia. Geotrichum candidum was thus identified. Colonoscopic features included erythema, edema, and loss of the usual fine vascular pattern, with granularity of the mucosa of the descending column. Treatment consisted of oral administration of ketoconazole and metronidazole for 3 weeks, while oral prednisolone was tapered after 1 week of therapy. The dog's feces gradually softened after the first treatment. Fecal smear examination revealed no trace of the yeast-like microbes 7 days after treatment was administered, and 2 weeks post-treatment the dog passed well-formed stools and had regained its normal body weight. The previously observed clinical signs did not reoccur, even after oral medication was withdrawn

Functional expression of the recombinant ATPase of orf virus

Nucleotide sequence analysis has indicated that the A32L gene of orf virus can encode an ATPase (Chan et al. in Gene 432:44-53, 2009). In this work, we cloned the A32L gene into a prokaryotic expression vector, and the recombinant protein was expressed and purified. The antigenicity of recombinant ATPase was examined by immunoblotting, and its identity was confirmed by mass spectrometry. The ATP hydrolysis function of the purified recombinant protein was examined, and our results showed that it exhibited the ATPase activity. Similar to other viral ATPases, the ATPase of orf virus remained active in the presence of different divalent ions; nevertheless, unlike other viral ATPases, our recombinant ATPase exhibited similar enzymatic activity in reaction buffers of different pH

Identification and phylogenetic analysis of orf virus from goats in Taiwan

An outbreak of contagious ecthyma in goats in central Taiwan was investigated. The disease was diagnosed by physical and histopathologic examinations, and the etiology of the disease was identified as orf virus by electron microscopy and polymerase chain reaction (PCR) and sequence of major envelope protein (B2L) gene. The entire protein-coding region of B2L gene were cloned and sequenced. Phylogenetic analysis of B2L amino acid sequences showed that the orf virus identified in this outbreak was closer to the Indian ORFV-Mukteswar 59/05 isolate. This is the first report on the molecular characterization of orf virus in Taiwan

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

Structure-Function Analysis of the Promoter Region of Influenza Virus RNAs

流行性感冒一直是公共衛生的重大問題。1918 年爆發的Spanish Flu 造成西方國家估計至少2-5 千萬人死亡。而近年來禽流感不僅造成畜牧業者重大損失，也逐漸威脅到人類族群。由於流感病毒基因型多變的特質，單靠施打疫苗並無法有效地控制此疾病。流感病毒的基因體是由八段負向(negative sense) 的RNA 所組成。調控病毒基因轉錄的啟動子(promoter) 位於每段RNA 的兩個末端，此序列在八段RNA之間具極高度的相似性（幾乎完全相同）。流感病毒RNA 的複製需藉由其本身的RNA 聚合.所啟始；病毒聚合.可專一性地結合到RNA 5』端的前11 個核.酸，進而啟動轉錄機序。因此，阻斷病毒RNA 聚合.和啟動子的結合可非常有效地抑制流感病毒複製。雖然流感病毒具有極高的基因變異機率，但由於此聚合.的氨基酸序列在各病毒亞型之間相似性很高(highly conserved)，且細胞中並無同源分子，因此可成為開發抗病毒藥劑的重要對象。此計畫將利用本實驗室改良的NAIM（Nucleotide Analog Interference Mapping;核.酸類似物干擾定位）技術，精確且細密地探討啟動子區段之序列和病毒RNA聚合.的互動關係。此實驗的設計原理：在試管中首先合成含病毒啟動子的RNA 片段；藉此反應可將個別的核.酸類似物質隨機嵌入RNA 片段中。這些核.酸分子上的功能基(functional group)已被特定地修飾，例如去除或增添不同的化學結構。再經由與RNA 聚合.結合的功能測試之後，選取出仍具功能的啟動子含類似物的RNA 族群﹔反言之，若因為特定核酸位置被改變，而失去功能的RNA 既無法被選取。最後與篩選前的總RNA 族群做比對，以確認參與病毒RNA 聚合.結合作用的核.酸位置以及功能基，可進而推測具有RNA 聚合.結合功能的最小RNA 片段。此結果除了可進一步瞭解流感病毒RNA 的複製機制，並且成為日後研發抗病毒製劑的重要訊息，例如: (1)合成RNA 片段來和病毒RNA驅動子競爭與RNA聚合.的結合位(2)有助於開發以阻斷病毒轉錄機制為訴求的病毒抑制劑篩選

Effect of NF-kb inhibitors on influenza virus replication

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Effect of NF-kb Inhibitors on Influenza Virus Replication

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Effect of NF-kB Inhibitors on Influenza Virus Replication

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Development of a Recombinant Pseudorabies Virus Co-Expressing JEV Envelope Protein and Swine IL-2

假性狂犬病毒(pseudorabies virus, PRV)是疱疹病毒科的阿爾伐疱疹病毒亞科內之一員，可以感染許多的哺乳類動物，在台灣是重要的猪隻傳染病之一，影響本省農業經濟甚鉅。日本腦炎係由日本腦炎病毒(Japanese encephalitis virus, JEV)感染引起的急性腦膜腦炎，此病為一種病毒性人畜共通疾病，其感染途徑是藉由三斑家蚊與環紋家蚊等病媒蚊傳染。妊娠母猪感染日本腦炎後，胎盤受病毒侵害，引起流產和死產。台灣夏季常有孩童感染腦炎症的病例，本病在公共衛生上是不可忽視的重要疾病。而使養猪界煩惱的猪的死產及流產，主要是由於猪感染日本腦炎病毒或假性狂犬病毒所致。目前全世界已有推行假性狂犬病撲滅計劃的趨勢，因此，良好的疫苗研發，將是控制這些重要病毒性疾病的重要利器。本實驗室已完成了PRV的vhs缺損病毒及R23 (TK-/gE-)病毒的選殖及特性分析，根據in vitro及in vivo的實驗結果發現，vhs的刪除並不會影響PRV的複製生長，且其毒力也有明顯下降的趨勢。同時也發現vhs缺陷病毒感染細胞或小鼠後，能夠測到大量的TNF-α；且能延長小鼠的死亡時間及存活率，其臨床的感染症狀也比較輕微。因為TK、gE及vhs都和PRV的病毒毒力有關；但並不會直接影響PRV的複製；所以將這3種基因刪除後，會降低PRV的毒力使其成為一個安全的病毒載體。同時我們也完成了猪的IL-2及日本腦炎病毒的套膜蛋白E基因之選殖，也能利用原核系統表現出相對應之蛋白質。而套膜蛋白E具有3個抗原決定位，其中以第三個抗原決定位(ED3)為3個抗原決定區域中唯一能單獨折疊的抗原區域，可誘發宿主產生病毒專一性中和抗體反應及保護性免疫反應的產生。因此，我們擬將猪的介白素-2(IL-2)及JEV封套蛋白E的第三個抗原決定位E-D3基因一起植入假性狂犬病毒之基因體內，以開發具有佐劑特質及抗假性狂犬病與日本腦炎之多價活毒疫苗。Pseudorabies, caused by the alphaherpesvirus pseudorabies virus (PRV), is a serious infection in a wide range of mammals. Pig is the natural reservoir for the virus, and infection with PRV results reproductive and respiratory disorders, nervous symptoms and even death, dependent on the age of the animal and virus strain-specific virulence. Japanese encephalitis is a potentially lethal disease of the central nervous system caused by infection with Japanese encephalitis virus (JEV), a member of the mosquito-borne encephalitis complex of the family Flaviviridae. Swine are an important amplifier of Japanese encephalitis (JE) virus and PRV in the paradomestic environment. PRV and Japanese encephalitis virus (JEV) are major global health and growing medical problems. When piglets are infected with PRV or JEV have caused strillbirths and abortions in pregnant sows and cause significant losses in the pig industry worldwide. To date, eradication of PRV and control of JEV become important tasks in the pig industry to reduce economical losses. From our previous study, we constructed and characterized that R23 (gE-/TK-) and vhs mutant virus (Δ41). The inactivation of TK and gE synergistically reduces PRV replication and virulence in vivo. While the vhs mutant virus has similar growth kinetics to wild type PRV, absence of vhs activity significantly reduced PRV virulence; wild type is lethal, whereas vhs mutant is highly attenuated in mice model. Moreover, we found the lethality is correlated with the dissemination of virus in neural tissues where only wt PRV but not vhs deletion virus was detected. Hence, the gene-delected of TK, gE and vhs in PRV has been used as a live-viral vector to develop multivalent genetic engineering vaccine. The E protein of JEV contains 3 antigenic domains and plays important roles in receptor binding and cell fusion and possesses most of the virus-neutralizing epitopes. The major antigenic domain of E protein is domain III (ED3). In this study, we will develope and test a novel recombinant PRV, which could co-express protein of the swine IL-2 and ED3 protein of JEV using PRV TK-/gE- mutant (R23) as the vector. The immunogenicity of this recombinant virus will be compared to the currently used commercial JEV and PRV vaccine in animal model

Study of the Anti-Influenza Virus Mechanism of Ferruginol, Indirubin , and Herb Extracts

流行感冒一直是公共衛生的重大課題。 回溯估計1 918 年爆發的 「Spanish Flu」造成西方國家至少2-5 千萬人死亡，而後亞洲地區亦傳出嚴重疫情，例如1957 年 「AsianFlu」及1968 年 「Hong Kong Flu」 死亡人數高達百萬人。 自從1997 年以來禽流感不僅造成畜牧業者重大損失，也嚴重威脅到人類族群。由於流感病毒基因型多變的特質，單靠施打疫苗並無法有效地控制此疾病。 目前兩類抗流感病毒製劑，分別以抑制病毒結構蛋白M2 (如：amantadine) 及NA (如：oseltamivir, zanamivir )之功能而達療效。 臨床結果顯示現今流行之流感病毒對於M2抑制劑敏感度大幅下降；針對近年的禽源流感疫情，Oseltamivir 及Zanamivir 為主要治療製劑，但已有研究發現此高病原性流感病毒逐漸演化出抗Oseltamivir 作用的新病毒型。因此實有研究開發新抗流感病毒藥物之必要性。近年來許多研究發現植物成份在病毒感染中參與重要反應：例如，靛玉紅(indirubin)衍生物會抑制細胞的轉錄調控因子NF-κB 活化，而此反應為維持流感冒病毒感染所必須；台灣肖楠外皮萃取物Ferruginol(松香烷類)可抑制老鼠巨噬細胞分泌細胞素IL-1β，具有抑制發炎的活性，但此二物質是否會直接影響流感病毒感染則尚待証實。 因此，本計劃將深度探討indirubin、ferruginol，以及數種由本實驗室初步篩選可抑制流感病毒增殖之植物萃取物(大花咸豐草、山素英)之抗病毒機制。 希冀藉由研究三個病毒複製過程：(1)與細胞接受體之接觸；(2)與endosome 之膜融合；(3)病毒基因體RNA 複製與 mRNA 合成，以及探究細胞抗病毒細胞素(如，干擾素)之合成，或是與病毒感染有關的細胞訊號傳遞（signal transduction），以瞭解植物成分的抑制流感病毒機制。此結果可作為日後開發抗流感病毒配方之重要參考