台灣鵝環狀病毒感染及其病原之研究

Abstract

2001年9月間，台灣雲林縣之鵝群發生有羽毛脫落、毛囊壞死等疑似鵝環狀病毒(GCV)感染症狀出現，經採取其臟器檢體進行聚合酶連鎖反應(Polymerase Chain Reaction, PCR)檢測，結果呈鵝環狀病毒陽性反應，此一388bp的PCR產物經定序後亦證實此環狀病毒與基因庫上國外分離株之序列相似性為95％，此為首度在台灣乃至亞洲證實有GCV存在。此期間由台灣地區分離到的11株鵝環狀病毒經全長定序，其基因體分別為1820nt.(9/11株)和1821nt.(2/11株)；經和基因庫上德國株(1394/97)一起進行親緣樹分析，結果可將之分為三個群，第一群為德國株，第二群及第三群分別包括三個和八個台灣株，其中第二群比較接近第一群，同群間病毒株的差異性在0.2~1.0 %，不同群間病毒株的差異性則為7.0~7.7 %。所有台灣GCV分離株的基因體經開放閱讀區分析(Open reading frame analysis) 顯示包含四個蛋白質：V1 (Rep, nt 73~954, 293 amino acids)、V2 (nt 1613~1726, 37 amino acids)、C1 (capsid, nt 1761~1009, 250 amino acids) and C2 (nt 427~128, 99 amino acids)。其中V1、C1及C2和德國株相似，但V2則台灣株的37個胺基酸較德國株的120個胺基酸為短。GCV的基因間區域(nt 1762~72)序列比對顯示台灣株和Todd等所確認在GCV複製上極為重要的 stem-loop/nonanucleotide (nt 1800~13)以及 heptamer repeats (nt 11~17 and 18~24)仍高度保留。此外在stem-loop region 左側的nt 1762~1798亦呈高度保留, 推測可能為GCV欲轉錄和複製時作為其和宿主蛋白質的接合位置。鵝環狀病毒 (goose circovirus, GCV)迄今並無任何體外培養系統可供應用，最重要的診斷方法仍為PCR。文獻中已發表二對引子在實驗室測試結果皆未臻理想，因此依據GCV序列之高度保留區設計一對新引子，P1762f,P385r，增幅產物為445bp，進行PCR之測試並與先前已發表之引子進行比較。結果此對引子不論在檢出率與敏感性上皆優於其它二組因子，而且對於GCV具有極高度之特異性，因此以此對引子所建立之PCR程序很適合用於檢測本省之GCV，值得推廣應用。另外此組引子亦能自正番鴨中檢測出環狀病毒之存在，這是首度證實環狀病毒亦能感染鴨隻之報導。核酸序列之比對發現，在正番鴨中所發現之環狀病毒有兩種，一種在序列上極類似GCV，其序列相似性為92.7~99.0%之間；另一種則與GCV相距較遠，相似性只有73.5~79.6%。為瞭解鵝環狀病毒台灣之感染狀況，本研究以PCR技術偵測台灣不同地區鵝隻屠宰場屠體華氏囊之鵝環狀病毒核酸存在陽性率，2002年間採取150個樣本中有49個樣本為陽性，陽性率為32.7﹪，2003年間採取237個樣本中有224個樣本為陽性，檢出陽性率高達94.5﹪，顯示台灣地區鵝隻感染環狀病毒之比率有逐年提升現象且已甚為普遍。2002~2003年間自不同地區所分離鵝環狀病毒株經全長定序及比對後發現，其序列相同性皆在97%以上，顯示台灣各地鵝環狀病毒株之基因體序列甚為穩定。由於無法在體外細胞複製病毒，因此應用定量PCR技術檢測，發現從個別鵝隻所採集華氏囊所含之病毒套數約在5 ×106~ 5 ×1010/g華氏囊之間。另外將華氏囊所含之病毒以蔗糖梯度離心純化後，病毒套數可達109/μL以上，這些純化之病毒可供進一步研究如病毒致病性分析等之用。

In September 2001, adult geese (White Roman geese) in many farms located at southwest coast of Taiwan exhibited feathering disorder and stunted growth. According the symptoms observed, circovirus or circovirus-like virus infection was suspected, and this suspicion was verified by PCR amplification of circovirus-specific sequences from the tissue homogenates of affected geese. Sequence analysis of the 388 bp fragment showed that this sequence matched the genomic sequence of goose circovirus(GCV)deposited in GenBank (95% identity) . This result showed that the geese we examined were infected by GCV, and this is the first report on GCV infection in Asia. We reported the complete nucleotide (nt) sequences of eleven goose circovirus (GCV) isolated in Taiwan. Nine out of the eleven isolates have a genome size of 1821nt, whereas the remaining two isolates have a size of 1820 nt. Pair-wise sequence comparison of the eleven Taiwanese GCV isolates and a German isolate reveals that these viruses could be divided into three distinct genetic groups. Group I contains the German isolate, group II contains three Taiwanese isolates, and group III contains eight Taiwanese isolates. Nucleotide variations between viruses of different genetic groups range from 7.0~7.7 %, whereas the differences within the same group are only 0.2~1.0 %. The most diversified sequences were found at a region between nt 27~72 of the viral genome, which corresponds to the right one third of the intergenic region. Open reading frame analysis shows that the genome of all Taiwanese GCV isolates might encode four proteins: V1 (Rep, 293 amino acids), V2 (37 amino acids), C1 (capsid, 250 amino acids), and C2 (99 amino acids). The sizes of V1, C1 and C2 proteins of all Taiwanese and German GCV isolates are identical. However, the size of V2 protein (37 amino acids), though identical in all Taiwanese isolates, were much smaller than that of the German isolate (120 amino acids). Because of the lack of in vitro culture system for GCV, the diagnosis of GCV could not be verified by the method of virus isolation. The observation of clinical symptoms and gross necropsy, as well as the detection of virus by PCR, are important for the diagnosis of this disease. This study designed a set of PCR primers (P1762 and P385) based on the published sequences of GCV, and then tested the efficacy of this set of primers in the detection of GCV collected from different areas of Taiwan. The result of PCR showed that primers P1762 and P385 performed better than all PCR primers published previously; moreover, PCR by using P1762 and P385 could also detect circovirus from Muscovy ducks. This is the first report on the presence of circovirus in ducks. Nucleotide sequences analysis showed that the circovirus found in ducks fell into two groups; the first group had 92.7~99.0% nucleotide sequence identity to GCV whereas the second group had only 73.5~79.6% identity to GCV. In conclusion, the PCR by using primers P1762 and P385 could serve as a rapid method for the detection of circovirus in geese and ducks. The application of this PCR method to the epidemiological study of waterfowl circovirus in Taiwan should contribute to the prevention and treatment of this disease. The goose circovirus present in bursa of Fabricius of geese collected in slaughter houses in Taiwan was detected by PCR. Among the 150 samples collected in 2002, 49 (32.7%) were found to be positive, and among the 237 samples collected in 2003, 224 (94.5%) were found to be positive. This result showed that the majority of geese in Taiwan were infected by goose circovirus and the prevalence of infection was increasing in 2002~2003. Sequence analysis of viruses isolated in 2002~2003 showed that these viruses exhibited a high degree of sequence identity (>97%) to each other, indicating a high genomic stability of this virus. Quantitative PCR assay showed that the virus could be purified by sucrose gradient centrifugation to 109 copies/μL. The purified virus could be used in the further studies including the pathogenicity assay of goose circovirus.