台灣家禽網狀內皮症病毒分離鑑定與序列分析

Isolation, Identification and Sequence Analysis of Avian Reticuloendotheliosis Viruses in Taiwan

鄭宛芯(Wan-Hsin Cheng)

指導教授 ： 王金和

國立臺灣大學/生物資源暨農學院/獸醫學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.02657

摘要

家禽網狀內皮症病毒 (avian reticuloendotheliosis virus, REV)，在台灣家禽疾病中較不被重視，但現場感染雞隻可發現雞隻出現生長不良、羽毛生長異常、胸腺及華氏囊萎縮等症狀發生，但是尚無有效的預防控制方法，所以目前種雞場最重要的是區分感染及未感染的雞隻，以避免病毒傳播的可能；並檢測現場使用之疫苗是否有REV污染，以釐清現今台灣所使用的疫苗在病毒傳播上所扮演的角色。因此本研究以ELISA方式調查台灣種雞及土雞REV抗體陽性率，同時由血液中抽取DNA檢測病毒核酸，並由臨床送檢病例的血液樣本與腫瘤乳劑中進行病毒分離，此外也從動物用藥品檢定分所取得禽痘疫苗與馬力克疫苗以進行REV污染檢測。在抗體陽性率結果顯示在種雞場之抗體陽性率為83％，土雞場之抗體陽性率為8％，而病毒核酸檢測結果在土雞的感染率為29％，由結果可知台灣雞隻REV感染情形相當普遍。病毒分離方面由現場病例中分離到兩株病毒，編號分別為3122/03與3295/04。而疫苗檢測結果方面，則從18種禽痘病毒疫苗中分離出一株且此分離株具有細胞病變 (cytopathic effect, CPE) 現象；在馬立克病毒疫苗方面，17種疫苗都未分離到REV病毒。由於目前商用ELISA套組檢測方式以偵測抗體為主，所以本研究欲想發展偵測抗原之ELISA，以便現場診斷之大量運用，所以本研究則利用REV分離株3122的部分envelope基因選殖出來，進行重組蛋白3122env的表現並針對選殖部分的胺基酸片段發展單株抗體，結果共獲得3株單株抗體mAb1、mAb2及mAb3，進一步利用單株抗體來偵測REV抗原，結果可順利偵測到重組蛋白與REV病毒，顯示這些單株抗體具有應用在偵測抗原型ELISA之潛力。綜和本實驗之結果，由血清學、病毒核酸檢測及病毒分離方面，皆充分顯示出台灣REV感染情形相當普遍，此外亦從疫苗株分離到REV，更代表此疾病在雞群中之潛在危險性。

關鍵字

家禽網狀內皮症病毒 ； 封套基因

並列摘要

Avian reticuloendotheliosis virus (REV) infection is common, but not ubiquitous. The REV-infected chickens showed stunted, pale, and abnormal feather development. The principal lesions include runting, and atrophy of the thymus and bursa of Fabricius. No procedures have been applied in commercial practice for the control of RE, so the most important thing in the field is to distinguish the infected and uninfected chickens. In this study, we used commercial ELISA kits to detect REV-specific antibodies in color-breed chickens, breeder chickens, and broilers; meanwhile we detected the viral DNA in blood by polymerase chain reaction (PCR). Clinical materials, including whole blood, plasma, tumor tissues, and litter, were collected for virus isolation. We collected fowl pox virus (FPV) vaccines and Marek’s disease (MDV) vaccines provided by branch institute of animal drugs inspection animal health research institute for detection of REV contamination, and tried to isolate REV from these vaccines. The result of prevalence investigation showed 83% antibody positive rate in breeder chickens and 8% in color-breed chickens. The result of detection of REV in native color hybrid blood samples by PCR showed 29% positive rate, which meant that the infection of REV was popular in Taiwan. Two REVs, named 3122/03 and 3295/04, were isolated from clinical materials. Among eighteen FPV vaccines we collected, eleven of them showed REV LTR sequences integration and one REV was isolated from these vaccines. This isolate had cytopathic effect in DF1 cell line. No REV was isolated from seventeen MDV vaccines. We cloned partial envelope gene for the expression of recombinant protein used for the production of monoclonal antibody to develop antigen-capture ELISA. The results showed that we had selected three monoclonal antibodies which could recognize the recombinant protein and REVs. These monoclonal antibodies have potential for developing of antigen-capture ELISA. Based on the results of serology, REV DNA detection, and virus isolation, the REV infection in Taiwan is common. Specially, the isolation of REV from FPV vaccines shows a potential danger in the field.

並列關鍵字

avian reticuloendotheliosis virus ； envelope gene

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