臺灣博碩士論文加值系統

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.

中文摘要----------------------------------------------------I
英文摘要---------------------------------------------------II
誌謝-------------------------------------------------------IV
目錄--------------------------------------------------------V
表目錄------------------------------------------------------VIII
圖目錄-------------------------------------------------------IX
縮寫及符號---------------------------------------------------XI
緒論---------------------------------------------------------1
一、牛冠狀病毒簡介-------------------------------------------1
(一)牛冠狀病毒感染症-----------------------------------------1
(二)病毒之分類構造及特殊生物活性-----------------------------2
二、病毒親緣關係分析-----------------------------------------5
三、病毒之分離及確認-----------------------------------------6
四、牛冠狀病毒疫苗現況---------------------------------------6
五、研究目的-------------------------------------------------8
貳、材料與方法-----------------------------------------------9
一、材料-----------------------------------------------------9
(一)細胞株---------------------------------------------------9
(二)檢體病毒株-----------------------------------------------9
(三)實驗動物-------------------------------------------------9
(四)引子----------------------------------------------------10
(五)藥品----------------------------------------------------11
(六)器材----------------------------------------------------13
(七)儀器----------------------------------------------------14
二、方法----------------------------------------------------16
(一)細胞培養及保存------------------------------------------16
(二)病牛檢體病毒株之初步偵測(RT-PCR) -----------------------16
(三)檢體病毒株之分離----------------------------------------17
(四)病毒株純化----------------------------------------------17
(五)病毒濃縮純化及蛋白組成分析------------------------------18
(六)病毒感染力(TCID50)測定----------------------------------19
(七)曾發病牛場血清抗體調查及病毒排放檢測--------------------19
(八)電子顯微鏡鏡檢------------------------------------------19
(九)免疫螢光抗體檢測----------------------------------------20
(十)破壞紅血球受器酵素(receptor destroy enzyme)活性測定-----21
(十一)病毒株對濕黴素(hygromycin B)之感受性試驗--------------21
(十二)病毒株基因定序----------------------------------------21
(1)病毒RNA之抽取--------------------------------------------21
(2)RT-PCR增幅目標基因---------------------------------------22
(3)DNA電泳及純化--------------------------------------------23
(4)核酸定序-------------------------------------------------23
(十三)基因比對與親緣樹狀圖分析------------------------------23
(十四)疫苗及各種免疫佐劑製備--------------------------------24
(十五)疫苗安全試驗------------------------------------------24
(十六)疫苗接種及抗體反應分析--------------------------------25
(1)疫苗接種-------------------------------------------------25
(2)血球凝集抑制抗體力價測定---------------------------------25
(3)病毒細胞培養中和抗體效價測定-----------------------------25
(十七)病毒培養方法之改良------------------------------------26
參、結果----------------------------------------------------27
一、檢體採集及病理解剖結果----------------------------------27
二、以RT-PCR檢測檢體BCoV病毒結果----------------------------27
三、檢體BCoV病毒之分離及效價測定結果------------------------27
四、分離病毒株破壞紅血球受器酵素(RDE)活性測定結果-----------28
五、分離病毒株對濕黴素(hygromycin B)感受性試驗結果----------28
六、分離病毒株電子顯微鏡鏡檢結果----------------------------29
七、病毒細胞培養方法之改進結果------------------------------29
八、分離病毒株細胞培養免疫螢光抗體試驗及檢體
組織免疫檢查結果--------------------------------------------29
九、曾發病牛場血清抗體調查及牛隻排病毒檢測結果--------------30
十、病毒濃縮純化及蛋白組成分析結果--------------------------30
十一、各分離病毒株P、N、HE及S基因檢測(RT-PCR)結果-----------31
十二、台灣分離株P、N、HE及S基因及定序及與美國參考
病毒株Mebus比對結果-----------------------------------------31
十三、台灣分離病毒株與其它冠狀病毒P基因比對與親緣樹狀圖
分析結果----------------------------------------------------34
十四、台灣病毒分離株疫苗試製及評估--------------------------35
肆、討論----------------------------------------------------38
伍、結論----------------------------------------------------44
陸、參考文獻------------------------------------------------45
柒、表------------------------------------------------------55
捌、圖------------------------------------------------------68
玖、附錄----------------------------------------------------98
拾、自述-------------------------------------------------- 107

陸、參考文獻
呂榮修, 三浦康男, 蘇建榮, 王建元, 李永林, 林地發, 林本欽. (1983).由牛冠狀病毒引起乳牛呼吸及下痢症. 台灣省畜牧獸醫會會報 41:57-63.

李儼峰, 趙典樹, 李淑惠, 黎南榮. (1996). 仔牛輪狀病毒與冠狀病毒混合感染
症. 中華民國獸醫病理學會八十五年組織病理研討會專輯, 台北, 台灣, pp. 39-41.

Abraham,S., Kienzle,T.E., Lapps,W., and Brian,D.A. (1990). Deduced sequence of the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site. Virology 176: 296-301.

Air,G.M., Els,M.C., Brown,L.E., Laver,W.G., and Webster,R.G. (1985). Location of antigenic sites on the three-dimensional structure of the influenza N2 virus neuraminidase. Virology 145: 237-248.

Air,G.M., and Laver,W.G. (1995). Red cells bound to influenza virus N9 neuraminidase are not released by the N9 neuraminidase activity. Virology 211: 278-284.

Baric,R.S., Nelson,G.W., Fleming,J.O., Deans,R.J., Keck,J.G., Casteel,N., and Stohlman,S.A. (1988). Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription. Journal of Virology 62: 4280-4287.

Benfield,D.A., and Saif,L.J. (1990). Cell culture propagation of a coronavirus isolated from cows with winter dysentery. Journal of Clinical Microbiology 28: 1454-1457.

Brian,D.A., Hogue,B.G., and Kienzle,T.E. (1995). The coronavirus hemagglutinin esterase glycoprotein. In: S.G. Siddell (ed.), The Coronaviridae, Plenum Press, Inc., New York, N.Y. , pp.165-179.

Cavanagh,D. (1995). The coronavirus surface glycoprotein, In: S.G. Siddell (ed.), The Coronaviridae, Plenum Press, Inc., New York, N.Y., pp. 73-173.

Cavanagh,D., Lai,M.M. (1997). The molecular biology of coronaviruses. Advances in Virus Research 48: 1-100.

Cho,K.O., Hasoksuz,M., Nielsen,P.R., Chang,K.O., Lathrop,S., and Saif,L.J. (2001). Cross-protection studies between respiratory and calf diarrhea and winter dysentery coronavirus strains in calves and RT-PCR and nested PCR for their detection. Archives of Virology 146: 2401-2419.

Clark,M.A. (1993). Bovine coronavirus. British Veterinary Journal 149: 51-70.

Crouch,C.F., Oliver,S., Hearle,D.C., Buckley,A., Chapman,A.J., and Francis,M.J. (2000). Lactogenic immunity following vaccination of cattle with bovine coronavirus. Vaccine 19: 189-196.

Cruciere,C., and Laporte,J. (1988). Sequence and analysis of bovine enteritic coronavirus (F15) genome. I. Sequence of the gene coding for the nucleocapsid protein; analysis of the predicted protein. Annales de L'Institut Pasteur Virology 139: 123-138.

Deregt,D., Gifford,G.A., Ijaz,M.K., Watts,T.C., Gilchrist,J.E., Haines,D.M., and Babiuk,L.A. (1989). Monoclonal antibodies to bovine coronavirus glycoproteins E2 and E3: demonstration of in vivo virus-neutralizing activity. Journal of General Virology 70: 993-998.

Deregt,D., Sabara,M., and Babiuk,L.A. (1987). Structural proteins of bovine coronavirus and their intracellular processing. Journal of General Virology 68: 2863-2877.

Drosten,C., Gunther,S., Preiser,W., van der Werf,S., Brodt,H.R., Becker,S., Rabenau,H., Panning,M., Kolesnikova,L., Fouchier,R.A., Berger,A., Burguiere,A.M., Cinatl,J., Eickmann,M., Escriou,N., Grywna,K., Kramme,S., Manuguerra,J.C., Muller,S., Rickerts,V., Sturmer,M., Vieth,S., Klenk,H.D., Osterhaus,A.D., Schmitz,H., and Doerr,H.W. (2003). Identification of a novel coronavirus in patients with severe acute respiratory syndrome. The New England Journal of Medicine 348: 1967-1976.

El-Ghorr,A.A., Snodgrass,D.R., Scott,F.M., and Campbell,I. (1989). A serological comparison of bovine coronavirus strains. Archives of Virology 104: 241-248.

El-Kanawati,Z.R., Tsunemitsu,H., Smith,D.R., and Saif,L.J. (1996). Infection and cross-protection studies of winter dysentery and calf diarrhea bovine coronavirus strains in colostrum-deprived and gnotobiotic calves. American Journal of Veterinary Research 57: 48-53.

Fitz,W., Rosenthal,P.B., and Wong,C.H. (1996). Synthesis and inhibitory properties of a thiomethylmercuric sialic acid with application to the X-ray structure determination of 9-O-acetylsialic acid esterase from influenza C virus. Bioorganic & Medicinal Chemistry 4: 1349-1353.

Fukutomi,T., Tsunemitsu,H.,and Akashi,H. (1999). Detection of bovine coronaviruses from adult cows with epizootic diarrhea and their antigenic and biological diversities.
Archives of Virology 144: 997-1006.

Gelinas,A.M., Boutin,M., Sasseville,A.M., and Dea,S. (2001). Bovine coronaviruses associated with enteric and respiratory diseases in Canadian dairy cattle display different reactivities to anti-HE monoclonal antibodies and distinct amino acid changes in their HE, S and ns4.9 protein. Virus Research 76: 43-57.

Hasoksuz,M., Lathrop,S., Al-dubaib,M.A., Lewis,P., and Saif,L.J. (1999). Antigenic variation among bovine enteric coronaviruses (BECV) and bovine respiratory coronaviruses (BRCV) detected using monoclonal antibodies. Archives of Virology 144: 2441-2447.

Hasoksuz,M., Sreevatsan,S., Cho,K.O., Hoet,A.E., and Saif,L.J. (2002). Molecular analysis of the S1 subunit of the spike glycoprotein of respiratory and enteric bovine coronavirus isolates. Virus Research 84: 101-109.

Hirano,N., Sada,Y., Tuchiya,K., Ono,K., and Murakami,T. (1985). Plaque assay of bovine coronavirus in BEK-1 cells. Nippon. Juigaku. Zasshi. 47: 679-681.

Hofling,K., Brossmer,R., Klenk,H., and Herrler,G. (1996). Transfer of an esterase-resistant receptor analog to the surface of influenza C virions results in reduced infectivity due to aggregate formation. Virology 218: 127-133.

Hofmann,M., and Wyler,R. (1988). Propagation of the virus of porcine epidemic diarrhea in cell culture. Journal of Clinical Microbiology 26: 2235-2239.

Hogue,B.G., Kienzle,T.E., and Brian,D.A. (1989). Synthesis and processing of the bovine enteric coronavirus haemagglutinin protein. Journal of General Virology 70: 345-352.

Ismail,M.M., Cho,K.O., Ward,L.A., Saif,L.J., and Saif,Y.M. (2001). Experimental bovine coronavirus in turkey poults and young chickens. Avian Disease 45: 157-163.

Kapil,S., Richardson,K.L., Maag,T.R., and Goyal,S.M. (1999). Characterization of bovine coronavirus isolates from eight different states in the USA. Veterinary Microbiology 67: 221-230.

Kapil,S., Richardson,K.L., Radi,C., and Chard-Bergstrom,C. (1996). Factors affecting isolation and propagation of bovine coronavirus in human rectal tumor-18 cell line. Journal of Veterinary Diagnostic Investigation 8: 96-99.

Kapil,S., Trent,A.M., and Goyal,S.M. (1990). Excretion and persistence of bovine coronavirus in neonatal calves. Archives of Virology 115: 127-132.

Kapke,P.A., and Brian,D.A. (1986). Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene. Virology 151: 41-49.

Kienzle,T.E., Abraham,S., Hogue,B.G., and Brian,D.A. (1990). Structure and orientation of expressed bovine coronavirus hemagglutinin-esterase protein. Journal of Virology 64: 1834-1838.

Klausegger,A., Strobl,B., Regl,G., Kaser,A., Luytjes,W., and Vlasak,R. (1999). Identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza C virus and bovine coronavirus. Journal of Virology 73: 3737-3743.

Kristiansen,M., Froystad,M.K., Rishovd,A.L., and Gjoen,T. (2002). Characterization of the receptor-destroying enzyme activity from infectious salmon anaemia virus. Journal of General Virology 83: 2693-2697.

Ksiazek,T.G., Erdman,D., Goldsmith,C.S., Zaki,S.R., Peret,T., Emery,S., Tong,S., Urbani,C., Comer,J.A., Lim,W., Rollin,P.E., Dowell,S.F., Ling,A.E., Humphrey,C.D., Shieh,W.J., Guarner,J., Paddock,C.D., Rota,P., Fields,B., DeRisi,J., Yang,J.Y., Cox,N., Hughes,J.M., LeDuc,J.W., Bellini,W.J., Anderson,L.J. and SARS Working Group. (2003). A novel coronavirus associated with severe acute respiratory syndrome. New England Journal of Medicine 348: 1953-1966.

Kunkel,F., and Herrler,G. (1993). Structural and functional analysis of the surface protein of human coronavirus OC43. Virology 195: 195-202.

Lapps,W., Hogue,B.G., and Brian,D.A. (1987). Sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes. Virology 157: 47-57.

Lin,X.Q., O eilly,K.L., Storz,J., Purdy,C.W., and Loan,R.W. (2000). Antibody responses to respiratory coronavirus infections of cattle during shipping fever pathogenesis. Archives of Virology 145: 2335-2349.

Luytjes,W., Bredenbeek,P.J., Noten,A.F., Horzinek,M.C., and Spaan,W.J. (1988). Sequence of mouse hepatitis virus A59 mRNA 2: indications for RNA recombination between coronaviruses and influenza C virus. Virology 166: 415-422.

Luytjes,W., van Marle,G., van der Most,R.G., van der Straaten,T., and Spaan,W.J. (1995). Regulation of transcription of coronaviruses. Advances in Experimental Medicine & Biology 380: 507-510.

Macintyre,G., Curry,B., Wong,F., and Anderson,R. (1991). hygromycin B therapy of a murine coronaviral hepatitis. Antimicrobial Agents and Chemotherapy 35: 2125-2127.

Mebus,C.A., Stair,E.L., Rhodes,M.B., and Twiehaus,M.J. (1973). Neonatal calf diarrhea: propagation, attenuation and characteristics of a coronavirus-like agent. American Journal of Veterinary Research 34: 145-150.

Milane,G., Kourtesis,A.B., and Dea,S. (1997). Characterization of monoclonal antibodies to the hemagglutinin-esterase glycoprotein of a bovine coronavirus associated with winter dysentery and cross-reactivity to field isolates. Journal of Clinical Microbiology 35: 33-40.

Naslund,K., Traven,M., Larsson,B., Silvan,A., and Linde,N. (2000). Capture ELISA systems for the detection of bovine coronavirus-specific IgA and IgM antibodies in milk and serum. Veterinary Microbiology 72: 183-206.

Parker,M.D., Cox,G.J., Deregt,D., Fitzpatrick,D.R., and Babiuk,L.A. (1989). Cloning and in vitro expression of the gene for the E3 haemagglutinin glycoprotein of bovine coronavirus. Journal of General Virology 70: 155-164.

Parker,M.D., Cox,G.J., Yoo,D.W., Fitzpatrick,D.R., and Babiuk,L.A. (1990). The haemagglutinin of bovine coronavirus exhibits significant similarity to the haemagglutinin of type C influenza virus. Advances in Experimental Medicine & Biology 276: 103-108.

Parker,M.D., Yoo,D., and Babiuk,L.A. (1990). Expression and secretion of the bovine coronavirus hemagglutinin-esterase glycoprotein by insect cells infected with recombinant baculoviruses. Journal of Virology 64: 1625-1629.

Payne,H.R., and Storz,J. (1988). Analysis of cell fusion induced by bovine coronavirus infection. Archives of Virology 103: 27-33.

Pfleiderer,M., Routledge,E., and Siddell,S.G. (1990). Functional analysis of the coronavirus MHV-JHM surface glycoproteins in vaccinia virus recombinants. Advances in Experimental Medicine & Biology 276: 21-31.

Poutanen,S.M., Low,D.E., Henry,B., Finkelstein,S., Rose,D., Green,K., Tellier,R., Draker,R., Adachi,D., Ayers,M., Chan,A.K., Skowronski,D.M., Salit,I., Simor,A.E., Slutsky,A.S., Doyle,P.W., Krajden,M., Petric,M., Brunham,R.C., McGeer,A.J.; National Microbiology Laboratory, Canada; Canadian Severe Acute Respiratory Syndrome Study Team. (2003). Identification of severe acute respiratory syndrome in Canada. The New England Journal of Medicine 348: 1995-2005.

Reddy,P.S., Idamakanti,N., Zakhartchouk,L.N., Babiuk,L.A., Mehtali,M., and Tikoo,S.K. (2000). Optimization of bovine coronavirus hemagglutinin-estrase glycoprotein expression in E3 deleted bovine adenovirus-3. Virus Research 70: 65-73.

Reynolds,D.J., Debney,T.G., Hall,G.A., Thomas,L.H., and Parsons,K.R. (1985). Studies on the relationship between coronaviruses from the intestinal and respiratory tracts of calves. Archives of Virology 85: 71-83.

Scheid,A., Caliguiri,L.A., Compans,R.W., and Choppin,P.W. (1972). Isolation of paramyxovirus glycoproteins. Association of both hemagglutinating and neuraminidase activities with the larger SV5 glycoprotein. Virology 50: 640-652.

Schultze,B., Gross,H.J., Brossmer,R., Klenk,H.D., and Herrler,G. (1990). Hemagglutinating encephalomyelitis virus attaches to N-acetyl-9-O-acetylneuraminic acid-containing receptors on erythrocytes: comparison with bovine coronavirus and influenza C virus. Virus Research 16: 185-194.

Schultze,B., Wahn,K., Klenk,H.D., and Herrler,G. (1991). Isolated HE-protein from hemagglutinating encephalomyelitis virus and bovine coronavirus has receptor - destroying and receptor-binding activity. Virology 180: 221-228.

Senanayake,S.D., and Brian,D.A. (1997). Bovine coronavirus I protein synthesis follows ribosomal scanning on the bicistronic N mRNA. Virus Research 48: 101-105.

Shieh,C.K., Lee,H.J., Yokomori,K., La Monica,N., Makino,S., and Lai MM. (1989). Identification of a new transcriptional initiation site and the corresponding functional gene 2b in the murine coronavirus RNA genome. Journal of Virology 63: 3729-3736.

Shi,S.T., Schiller,J.J., Kanjanahaluethai,A., Baker,S.C., Oh,J.W., and Lai,M.M. (1999). Colocalization and membrane association of murine hepatitis virus gene 1 products and De novo-synthesized viral RNA in infected cells. Journal of Virology 73: 5957-5969.

Siddell,S.G., Pohl-Koppe,A., Raabe,T, and ter Meulen,V. (1995). Detection of human coronavirus 229E-specific antibodies using recombinant fusion proteins. Journal of Virological Methods 55: 175-183.

Snijder,E.J., Bredenbeek,P.J., Dobbe,J.C., Thiel,V., Ziebuhr,J., Poon,L.L., Guan,Y., Rozanov,M., Spaan,W.J.M., and Gorbalenya,A.E. (2003). Unique and Conserved eatures of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage Journal of Molecular Biology 331: 991-1004.

Snijder,E.J., Bredenbeek,P.J., Noten,F.H., den Boon,J.A., Schaaper,W.M., Horzinek,M.C., and Spaan,W.J. (1990). The polymerase gene of corona- and toroviruses: evidence for an evolutionary relationship. Advances in Experimental Medicine & Biology 276: 307-316.

Snijder,E.J., den Boon,J.A., Horzinek,M.C., and Spaan,W.J. (1991). Comparison of the genome organization of toro- and coronaviruses: evidence for two nonhomologous RNA recombination events during Berne virus evolution. Virology 180: 448-452.

Snijder,E.J., Wassenaar,A.L., Den Boon,J.A., and Spaan,W.J. (1995). Proteolytic processing of the arterivirus replicase. Advances in Experimental Medicine & Biology 380: 443-451.

Spaan,W., Cavanagh,D., and Horzinek,M.C. (1988). Coronaviruses: structure and genome expression. Journal of General Virology 69: 2939-2952.

Stephensen,C.B., Casebolt,D.B., and Gangopadhyay,N.N. (1999). Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay. Virus Research 60: 181-189.

Storz,J., Herrler,G., Snodgrass,D.R., Hussain,K.A., Zhang,X.M., Clark,M.A., and Rott,R. (1991). Monoclonal antibodies differentiate between the haemagglutinating and the receptor-destroying activities of bovine coronavirus. Journal of General Virology 72: 2817-2820.

Storz,J., Purdy,W., Lin,X., Burrell,M., Truax,R.E., Briggs,R.E., Frank,G.H., and
Loan,R.W. (2000). Isolation of respiratory bovine coronavirus, other cytocidal viruses, and Pasteurella spp. from cattle involved in two natural outbreaks of shipping fever.
Journal of the American Veterinary Medical Association 216: 1599-1604.

Storz,J., and Rott,R. (1981). Reactivity of antibodies in human serum with antigens of
an enteropathogenic bovine coronavirus. Medical. Microbiology ＆ Immunology
169: 169-178.

Storz,J., Zhang,X.M., and Rott,R. (1992). Comparison of hemagglutinating, receptor-destroying, and acetylesterase activities of avirulent and virulent bovine coronavirus strains. Archives of Virology 125: 193-204.

Sturman,L.S., and Holmes,K.V. (1983). The molecular biology of coronaviruses.
Advance in Virus Research 28: 35-112.

Takamura,K., Matsumoto,Y., and Shimizu,Y. (2002). Field study of bovine coronavirus vaccine enriched with hemagglutinating antigen for winter dysentery in dairy cows. Canada Journal of Veterinary Research 66: 278-281.

Takamura,K., Okada,N., Ui,S., Hirahara,T., and Shimizu,Y. (2000). Protection studies on winter dysentery caused by bovine coronavirus in cattle using antigens prepared from infected cell lysates. Canada Journal of Veterinary Research 64: 138-140.

Thomas,L.H., Gourlay,R.N., Stott,E.J., Howard,C.J., and Bridger,J.C. (1982). A search for new microorganisms in calf pneumonia by the inoculation of gnotobiotic calves. Research of Veterinary Science 33: 170-82.

Traven,M., Naslund,K., Linde,N., Linde,B., Silvan,A., Fossum,C., Hedlund,K.O., and Larsson,B. (2001). Experimental reproduction of winter dysentery in lactating cows using BCV -- comparison with BCV infection in milk-fed calves. Veterinary Microbiology 81: 127-151.

Tsunemitsu,H., and Saif,L.J. (1995). Antigenic and biological comparisons of bovine coronaviruses derived from neonatal calf diarrhea and winter dysentery of adult cattle. Archives of Virology 140: 1303-1311.

Tsunemitsu,H., Smith,D.R., and Saif,L.J. (1999). Experimental inoculation of adult dairy cows with bovine coronavirus and detection of coronavirus in feces by RT-PCR.
Archives of Virology 144: 167-175.

Van Kruiningen,H.J., Khairallah,L.H., Sasseville,V.G., Wyand,M.S., and Post,J.E. (1987). Calfhood coronavirus enterocolitis: a clue to the etiology of winter dysentery. Veterinary Pathology 24: 564-567.

Vlasak,R., Luytjes,W., Leider,J., Spaan,W., and Palese,P. (1988). The E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity. Journal of Virology 62: 4686-4690.

Vlasak,R., Luytjes,W., Spaan,W., and Palese,P. (1988). Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses. Proceedings of the national academy of sciences of the United States of America 85: 4526-4529.

Vlasak,R., Muster,T., Lauro,A.M., Powers,J.C., and Palese,P. (1989). Influenza C virus esterase: analysis of catalytic site, inhibition, and possible function. Journal of Virology 63: 2056-2062.

Yoo,D., and Deregt,D. (2001). A single amino acid change within antigenic domain II of the spike protein of bovine coronavirus confers resistance to virus neutralization.
Clinical and Diagnostic Laboratory Immunology 8: 297-302.

Yoo,D., Graham,F.L., Prevec,L., Parker,M.D., Benko,M., Zamb,T., and Babiuk,L.A. (1992). Synthesis and processing of the haemagglutinin-esterase glycoprotein of bovine coronavirus encoded in the E3 region of adenovirus. Journal of General Virology 73: 2591-2600.

Yoo,D.W., Parker,M.D., and Babiuk,L.A. (1991). The S2 subunit of the spike glycoprotein of bovine coronavirus mediates membrane fusion in insect cells. Virology 180: 395-399.

Yoo,D.W., Parker,M.D., Song,J., Cox,G.J., Deregt,D., and Babiuk,L.A. (1991). Structural analysis of the conformational domains involved in neutralization of bovine coronavirus using deletion mutants of the spike glycoprotein S1 subunit expressed by recombinant baculoviruses. Virology 183: 91-98.

Yokomori,K., La Monica,N., Makino,S., Shieh,C.K., and Lai,M.M. (1989). Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus. Virology 173: 683-691.

Zhang,X., Herbst,W., Kousoulas,K.G., and Storz,J., (1994). Comparison of the genes and the biological properties of respiratory and the biological properties of respiratory and enteropathogenic bovine coronaviruses. Archives of Virology 134: 421-426.

Zhou,J., Crawford,L., Sun,X.Y., and Frazer,I.H. (1991). The hygromycin –resistance -encoding gene as a selection marker for vaccinia virus recombinants. Gene 107: 307-312.