牛冠狀病毒(BCoV)具外套膜及正股單鏈RNA，可引起牛隻下痢及呼道感染症，雖為世界性重要牛病，台灣地區迄今卻仍無正式確診報告，亦無本土疫苗可使用，主要因為培養困難。本研究將臨床疑似本病之病牛檢體，先以RT-PCR偵測病毒之核蛋白衣(N)基因，呈陽性者再以人類直腸腫瘤細胞株(HRT-18G cell line)培養之，已成功分離3株本地病毒命名為BCoV-TW1、BCoV-TW2及BCoV-TW3。病毒純化後進行RT-PCR，亦可偵測到病毒之核酸聚合酶(P)、核蛋白衣(N)、血球凝集素-酯解酶蛋白(HE)及突棘蛋白(S)基因，其增幅產物純化後予以定序，再由其解譯之胺基酸序列，與美國參考病毒株(Mebus strain)進行比對，其相似性分別為98.6~99.3％、98.5~99.3％、96.4~99.4％及98.7~99.6％。電子顯微鏡觀察可見到典型皇冠樣形態，BCoV-TW1表面亦可觀察到HE及S蛋白，直接螢光抗體試驗偵測檢體或培養病毒之細胞亦皆呈病毒陽性反應。以上述P基因片段序列與其他冠狀病毒作親緣樹狀圖分析，則台灣分離株可歸類為冠狀病毒第二群，與人類SARS病毒不同群。S與HE基因序列分別具有細胞蛋白酶切割序列-KRRSRR- 及酯解酶(esterase)活性胺基酸序列-FGDS-，其紅血球凝集作用與破壞紅血球受器酵素生物活性亦證實存在。三株分離病毒皆對濕黴素(hygromycin B)具感受性，本研究已成功分離及確診台灣地區牛冠狀病毒之感染，並對其基因序列與活性作完整的分析。初步以BCoV-TW1病毒大量增殖後，添加 0.002 M binary ethyleneimine 予以不活化處理，再添加磷酸鋁膠、MVP乳膠與Montanide雙相油劑等三種不同佐劑，再分別免疫接種實驗動物，以血球凝集抑制試驗測定其血清中和抗體力價幾何平均值，分別測得為1:130、1:144、1:176。由此結果顯示，Montanide油劑組疫苗可以達到國際規定保護力價標準(1:160)，試製疫苗亦可以使天竺鼠達到具保護力價之抗體，且具動物安全性，中和抗體對另外二株病毒BCoV-TW2及BCoV-TW3之細胞感染亦具中和作用，顯示BCoV-TW1可作為疫苗候選株。另外，鑑於病毒細胞培養力價仍低(105.5 TCID50 / 100 ml )，故本研究另行調配無血清培養基培養之，結果可提高病毒力價達107.1 TCID50 / 100 ml ，此法可供病毒初次分離方法改良之參考及製備疫苗時提高病毒效價用。

Bovine coronavirus (BCoV) is an enveloped, single stranded and positive-sense RNA virus. It can cause cattle diarrhea and respiratory tract infection. Although it is a worldwide important cattle disease, there was still no formal identified case report in Taiwan. No local vaccine is available either. Difficulty in virus isolation is the major problem. In this study, we picked up the suspected stool specimens, pre- identified with detection of BCoV N gene by RT-PCR. Specimens were inoculated into the HTR-18 cell line. Serial blind passages were done. Three virus strains were successfully isolated, naming BCoV-TW1、BCoV-TW2 and BCoV-TW3. The viral N、P(polymerase)、HE(haemagglutinin-esterase) and S(spike protein) genes were detected in the purified virus preparations. The RT-PCR products were further purified and sequenced. The equivalent amino acid sequences were deduced and compared with the American BCoV strain, Mebus strain. Similarities for these four genes ranged from 98.6~99.3％、98.5~99.3％、96.4~99.4 ％ and 98.7~99.6％, respectively. Typical corona-like morphology was found with electron microscope. HE and S proteins on the surface of virion were distinguishable in BCoV-TW1. Viral antigens were also detected in the intestine specimen and inoculated HRT-18 cell by direct immunofluorescence assay. Phylogenetic tree based on the nucleotide sequence alignment of coronavirus P gene was constructed. These three Taiwan isolates belong to coronavirus group 2 and genetically distinct from human SARS coronary virus. Amino acid sequences of S and HE genes were deduced and shown to contain the susceptiable cellular protease cleavage site(K-R-R-S-R-R) and esterase active site (F-G-D-S), respectively. Haeagglutination and erythrocytes receptor destroying enzyme activities were demonstrated too. Three virus isolates were all susceptible to hygromycin B. BCoV isolation and identification in Taiwan were thus successful. BCoV-TW1 isolate was selected and enriched in cell culture, then inactived by 0.002 M binary ethyleneimine. Alumium phosphate gel、MVP Emulsigen or Montanide two phase oil adjuvant was added. Guinea pigs immunized with these 3 different vaccine preparations can produce neutralinzing HAI antibody with titers of 1:130、1:144 and 1:176, respectively. The Montanide -adjuvanted vaccine immunization can reach the international standard of protective potency of 1:160. This vaccine preparation was further shown that it has animal safety and inducing neutralinzing antibody against both the BCoV-TW2 and BCoV-TW3 infection in tissue culture. Therefore, BCoV-TW1 can be the candidate of vaccine. To promote the low viral titer in cell culture (105.5 TCID50 / 100 ml), we formulated a serum-free medium for cell culture. Significant increase of virus titer up to 107.1 TCID50 / 100 ml was found. This finding can be exploited to the primary virus isolation and the enrichment of virus cultivation.

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