台灣地區鵝環狀病毒與鴨環狀病毒之PCR檢測、基因型分析與宿主特異性研究 台灣地區鵝環狀病毒與鴨環狀病毒之PCR檢測、 基因型分析與宿主特異性研究

Abstract

鵝環狀病毒 (goose circovirus, GoCV) 與鴨環狀病毒 (duck circovirus, DuCV) 為新興之水禽傳染病原體，此二病毒易侵犯淋巴組織造成免疫抑制，增加其他疾病之罹患率而造成水禽業的經濟損失。水禽環狀病毒尚無任何體外培養系統可應用，因此目前以聚合酶連鎖反應 (polymerase chain reaction, PCR) 為最重要的病毒檢測方法。本研究主題分為兩部分，第一部分是應用聚合酶連鎖反應檢測台灣水禽環狀病毒，方法為依據先前已發表之一組PCR引子 (P1762與P385)，與本研究中重新設計之一組引子 (P1813與P248)，進行台灣地區水禽環狀病毒盛行率之調查，並收集台中、彰化、雲林、台南、台東等地區之鴨華氏囊檢體67件與鵝華氏囊檢體29件進行PCR檢測，得到GoCV或DuCV之陽性率分別為58.6 %與58.2 %，此結果與匈牙利鵝環狀病毒之盛行率相似。另一方面，上述兩組引子可自GoCV及DuCV分別增幅出445 bp和415 bp，或255 bp和227 bp之PCR產物，藉此可直接區分GoCV與DuCV。分析此四種PCR產物之核酸序列發現DuCV相較於GoCV有約30個核苷酸缺損，進一步將病毒核酸序列以親緣樹圖分析後，可以將水禽環狀病毒分為三群，第一群為台灣GoCV分離株，第二群為台灣DuCV分離株，第三群為類似德國DuCV之台灣分離株，這是首度發現台灣地區存在有不同基因型別之DuCV分離株之報告。另外，本研究亦首度發現GoCV除感染鵝外，也可感染鴨，而DuCV除感染鴨之外，也可感染鵝。總結而言，利用此兩組引子不但可以快速檢測並區別出GoCV或DuCV之感染也可以經由定序結果區分來源病毒株之基因型。本研究之第二部分是應用水禽環狀病毒之重組複製蛋白(replication protein, Rep) 與重組外殼蛋白 (capsid protein, Cap)，建立以multiscreen之Western blot技術檢測水禽環狀病毒抗體，方法為以原核E. coli系統表現GoCV與DuCV之Rep及Cap，並以這些蛋白作為抗原，進行Western blot分析，對台灣各地共9場鵝血清與9場鴨血清進行調查，實驗結果發現有5場鵝血清和6場鴨血清呈現陽性反應。

Goose circovirus (GoCV) and duck circovirus (DuCV) are emerging infectious agents of waterfowls. These viruses could invade lymphoid tissues and lead to immunosuppression complicated with secondary diseases, which subsequently causes economic loss. There is no in vitro culture system for waterfowl circovirus, and thus polymerase chain reaction (PCR) is the most important method for the diagnosis of these viruses. This study was divided into two parts, the first part is application of PCR to detection of waterfowl circovirus in Taiwan. We used two primer sets, one was the published primer set (P1762f and P385r) and the other was designed in this study (P1813f and P248r), to conduct PCR analysis and investigate the prevalence of waterfowl circovirus infection in Taiwan. Of the 67 and 29 samples of bursa of Fabricius collected from ducks and geese farms in Taichung, Changhua, Yunlin, Tainan, and Taitung, 58.2 % of ducks and 58.6 % of geese flocks were found to contain GoCV or DuCV. This result is similar to that reported from Hungary. Moreover, the two primer sets amplified PCR products of 445 bp and 415 bp, or 255 bp and 227 bp, from GoCV and DuCV, so we could differentiate between these two viruses immediately. Sequences analysis of the four PCR products showed that DuCV has a deletion of about 30 nt when compared with GoCV. Phylogenetic analysis revealed that waterfowl circovirus could be divided into three distinct genetic group. Group I contains GoCV isolated in Taiwan, group II contains DuCV isolated in Taiwan, and group III contains Taiwanese DuCV isolates that are similar to Germany. This is the first report on the presence of different genetic groups of DuCV isolated in Taiwan, and also the first report showing that GoCV infects both geese and ducks, and so does DuCV. In conclusion we established a PCR procedure not only for the rapid identification and differentiation of GoCV and DuCV infections, but also differentiation of distinct genetic groups of these viruses. The second part of this study is application of recombinant replication protein (Rep) and capsid protein (Cap) of waterfowl circovirus to establishing multiscreen Western blot system for serological diagnosis. By using E. coli-expression system to produce both Rep and Cap of GoCV and DuCV, and using these recombinant proteins as antigens for Western blot assay, we found that geese sera from 5 out of the 9 farms and ducks sera from 6 out of the 9 farms were positive for anti-GoCV antibody or anti-DuCV antibody.