牛流行熱病毒( Bovine ephemeral fever virus，BEFV )於分類上屬於子彈型病毒科(Rhabdoviridae)，流行熱病毒屬(ephemerovirus)，牛流行熱病毒可經由庫蠓等節肢動物為媒介，經傳染之乳牛、黃牛及水牛有發燒、泌乳量驟降甚至停止的情形。BEFV具外套膜之負性單股RNA病毒，在外套膜中有一個貫膜性結構G醣蛋白(envelope glycoprotein G)，分子量為81KDa，具中和性抗原決定部位，可以刺激牛隻產生保護性免疫反應，但目前疫苗效力仍不佳。RNA干擾現象(RNA interference, RNAi)是指利用特定序列轉錄之小片段干擾性RNA(small interfering RNA, siRNA)，會形成帶有一段小片段環狀結構干擾性RNA (short harpin RNA)，經Dicer作用切成21-24 nt的小片段siRNAs，siRNAs會再和RISC (RNA-induced silencing complex)結合，降解同源的mRNAs，進而使mRNA無法轉譯，造成該特定基因沉默(gene silencing )。本研究探討病毒醣蛋白G基因的shRNA之病毒干擾效果，提供治療之參考。首先取西元2001年牛流行熱病毒分離株醣蛋白G基因的已知核酸序列，以該序列之前、中及後段設計三段shRNA序列，並將三段shRNA序列構築於pENTER/U6載體中(即pU6-shRNA345，pU6-shRNA701及pU6-shRNA1820)，利用RNA polymeraseIII U6 promoter驅動其表現。另外，將G的全長序列構築於pcDNA.3.1/V5-His-TOPO載體中(即pcDNA3.1/BEFV-G)，作為受siRNA作用之目標基因。並且將pcDNA3.1/BEFV-G載體轉染BHK-21細胞株(Baby Hamster Kidney cell - 21) ，分別於24 、48及72小時後，利用RT-PCR分析G 基因表現量，結果顯示G 基因表現量於轉染48小時後達到最大量。將pU6-shRNA345、pU6-shRNA701及pU6-shRNA1820轉染48小時後，分別以100及10 TCID50劑量病毒攻毒之，續培養48及72小時後，利用RT-PCR及免疫雜配法發現三組pU6-shRNAs不論病毒攻毒劑量多少皆可以明顯抑制G基因和G醣蛋白之表現，對於病毒本身之複製干擾比較無大的差異，100 TCID50劑量病毒攻毒24及48小時後，三組pU6-shRNAs感染細胞培養液，發現病毒效價(TCID50)分別為：pU6-shRNA345組，10-3.33及10-4.1；pU6-shRNA701組，10-4及10-4.2；pU6-shRNA1820組，10-3.8及10-4.64，對照組則為10-5及10-5.8，有些微干擾效果(大約10倍以上干擾), 細胞病變亦有緩和的趨勢。若改用共轉染pcDNA3.1/BEFV-G和三組pU6-shRNAs載體之測試結果顯示，於轉染後48小時，G基因表現量並無被抑制。由以上結果顯示，本研究針對BEFV G基因所設計之pU6-shRNAs的確可抑制G基因表現及干擾病毒複製，但必須在病毒感染前先轉染這些pU6-shRNAs，希望未來能進一步提昇其效力。

　Bovine ephemeral fever virus (BEFV) is caused by an arthropod-borne rhabdovirus, classified as the genus Ephemerovirus, and can cause an acute febrile diease in cattle and water buffalo. The BEFV virion contains a single-stranded, negative-sense RNA genome and a lipid envelope. The 81 KDa BEFV envelope G glycoprotein contains type-specific and neutralizing antigenic sites and can induce protective immunity in cattle. RNA interference (RNAi) is a process of sequence-specific post-transcriptional gene silencing induced mediated by double stranded RNA and is a powerful genetic approach to analyze gene function in animals and plants. Three short hairpin RNAs (shRNAs) are constructed into pENTER/U6 vector containing RNA polymerase Ⅲ U6 promoter and siRNA sequence. These three shRNAs vectors are designated as pU6-shRNA345, pU6-shRNA701 and pU6-shRNA1820. The full length G gene was constructed into the pcDNA.3.1/V5-His-TOPO vector and designated as pcDNA3.1/BEFV-G. Then transfection of pcDNA3.1/BEFV-G into BHK-21 cell, after 24, 48, and 72 hrs, total cellular RNA was extracted and analyzed by RT-PCR. G gene was found to maximally expressed after 48 hrs culture. BHK-21 cells were pre-transfected with these three shRNA constructrs and incubated for 48, and 72 hrs, BEFV virus was then inoculated (10 TCID50 and 100 TCID50 dose). All pU6-shRNAs could silence the expression of G gene and G glycoprotein of BEFV by RT-PCR and Western blot in all viral inoculated cells. The BHK-21 cells transfected with three shRNAs and challenged with 100 TCID50 dose for 24 hrs and 48 hrs, obtained viral TCID50 titers as followes : pU6-shRNA345, was 10-3.33 and 10-4.1; pU6-shRNA701，was 10-4 and 10-4.2; pU6-shRNA1820，was 10-3.8 and 10-4.64 , respectively. TCID50 titers of the control cells were calculated to be 10-5 and 10-5.8. We suggested that three pU6-shRNAs did not silence the virus replication very effectively in our case. When pU6-shRNAs and glycoprotein G were cotransfected, G gene expression was not inhibited. Our results showed that by transfecting the pU6-shRNAs could silence the G gene expression and interfere with the virus replication by transfecting these pU6-shRNAs before viral infection. The fact revealed that a more high antivirus capability should be increased in our feature study.

丁履紉. (2000). 牛流行熱疫情監控. 農政與農情 102 : 75-78.

呂榮修、李永林、黃士則、蔡向榮、廖永剛、林地發、曾俊憲、宋華聰.(1992). 1989年發生在台灣的牛流行熱病毒學研究. 台灣畜牧獸醫學會會報，60：51-56.

李太矜. (1988). 本省牛流行熱緊急防疫. 台灣歷年家畜防疫記述. 臺灣省政府農林廳編印，pp.60-65.

林朝舜. (1973). 牛流行感冒. 家畜衛生. 台灣省政府農林廳暨中國農村復興聯合委員會編印，pp.54-61.

馮翰鵬. (2002). 牛流行熱. 中化藥訊 52 : 13-20.

鄺懋勁, 曾俊憲, 黎南榮, 楊喜金, 林士鈺. (1998). 應用反轉錄聚合酶鏈反應檢測1996年臺灣流行之牛流行熱.臺灣省家畜衛生試驗所研究報告. 34 : 1-7.

Anderson, J., Banerjea ,A., Planelles, V., Akkina, R.（2003）Potent suppression of HIV type 1 infection by a short hairpin anti-CXCR4 siRNA. AIDS Research and Human Retroviruses 19:699-706.

Angela, R., Devin, L., Queta. B., and Stephen, S. (2004). Rational siRNA design for RNA interference. Nature Biotechnology 22:326-330.

Barik, S. (2004). Control of non segmented negative-strand RNA virus replication by siRNA. Virus Research 102:27-35.

Bass,B.L.2001. Tge short answer. Nature 411. 428-429.

Brummelkamp, T. R., Bernards, R. and Agami, R. (2002a). A system for stable expression of short interfering RNAs in mammalian cells. Science 295:550-553.

Brummelkamp, T. R., Bernards, R. and Agami, R. (2002b). Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2:243-247

Caplen, N.J., Parrish, S., Imani, F., Fire, A., Morgan, R.A. (2001). Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proceedings of the national academy of sciences of the United States of America 98:9742-9747.

Capodici, J., Kariko, K. and Weissman, D. (2002). Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference.　Journal of Immunology 169:5196-5203.

Chen,　W., Yan,　W., Du,　Q., Fei,　L., Liu,　M., Ni,　Z., Sheng,　Z., and Zheng, Z. (2005).　RNA Interference Targeting VP1 Inhibits Foot-and-Mouth Disease Virus Replication in BHK-21 Cells and Suckling Mice. Journal of Virology 78: 6900–6907.

Chen, Y., Du, D., Wu. J., Chen, C.P., Tan, Y., Kung, H-F., and He, M.L. (2003). Inhibition of hepatitis B virus replication by stably expressed shRNA. Biochemica and Biophysical Research Communications 311:398-404.

Cheng, C.H., Lee, Y.F., Hsieh, Y.C., Yin,C.C., Huang, C.Y., and Chen, S.H. (2003). Study of bovine ephemeral fever virus nonstructural glycoprotein GNS in Taiwan. The 37th Annual Meeting of the Chinese Society of Microbiology, December 7, Taichung, Taiwan.

Chiu, S. Y. (1986). The isolation of bovine ephemeral fever virus in Taiwan in 1984. Journal Chinese Society of Veterinary Science 12：275-288.
Chiu, S. Y. and Lu, Y. S. (1987). The epidemiology of bovine ephemeral fever in Taiwan in 1984. Journal Chinese Society of Veterinary Science 13：1-9.

Clemens, J., Worby, C., Simonson-Leff, N., Muda, M., Maehama, T., Hemminmgs, B.A., and Dixon, J.(2000). Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways. Proceedings of the national academy of sciences of the United States of America 97:6499-6503.

Cybinski, D. H. and George, St T. D. (1993). Antigenic variation in the bovine ephemeral fever virus glycoprotein. In “Bovine Ephemeral Fever and Related Rhabdoviruses ACIAR Proceedings”,Edited by T. D. St George, M. F. Uren, P. L. Young and D. Hoffman, No.44, pp.131-137, ACIAR, Canberra.

Cybinski, D. H., Davis, S. S., and Zakrzewski, H. (1992). Antigenic variation of the bovine ephemeral fever virus glycoprotein. Archives of Virology 124：211-224.

Cybinski, D.H., and Muller,M.J.(1990).Isolation of Arboviruses from cattle and insects at two sentinel sites in Queensland Australia 1979-1985. Australian Journal of Zoology 38：25-32.

de los S. ,T., Wu, Q., de Avila B. S, Grubman , M.J. (2005). Short hairpin RNA targeted to the highly conserved 2B nonstructural protein coding region inhibits replication of multiple serotypes of foot-and-mouth disease virus. Virology 10:222-31.

Dykxhoorn, D.M., Novina, C.D., Sharp, P.A.( 2003). Killing the messenger: short RNAs that silence gene expression. Nature Reviews Molecular Cell Biology 4:457-67.

Elbashir, S. M. Harborth, J., Weber, K., and Tuschl, T. (2002). Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 20.199-213.

Elbashir, S. M., Lendeckel, and W., Tuschl, T. (2001). RNA interference is mediated by 21-and 22-nucleotide RNAs. Genes Development 15,188-200.

Fields, B.N., Knipe, D.M., Howley, P.M. and Griffin, D.E. (2001). Rhabdoviridae: the viruses and their replication. In: Fields virology, 4th ed. Lippincott Williams & Wilkins, pp.1221-1244.

Fire, A. (1999). RNA-triggered gen silencing. Trends in Genetics 15,358-363.

Fire, A., Xu, s., Montgomery, M.K., Kostas, S.A., Driver, S.E., Mello, C.C. (1998). Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature 391:806-811.

Gil, J., and Esteban, M. (2004). Vaccinia virus recombinants as a model system to analyze interferon-induced pathways. Journal of Interferon and Cytokine Research 24(11):637-46.

Guo, S., and Kemphues, K. J. (1995). Par-1, a gene required for establishing polarity in C.elegans embryos, encodes a putative Ser/Thr kinase that is asynnetrically distributed . Cell 81:611-620.

Gupta Sunita, R. A., Schoer, J. E., Egan. G. J., Hannon, and Mittal, V. (2004). Inducible , reversible , and stable RNA interference in mammalian cells. Proceedings of National Academy Science of the United States of America 101:1927-1932.

Hannon, G. J. (2002). RNA interference. Nature 418 : 244-51.

Hammond S.M., Bernstein E., Beach D., and Hannon G.J. 2000. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404:293-296.

Hertig, C., Pye, A.D., Hyatt, A.D., David, S.S., McWillian, S.M., Heine, H.G., Walker, P.J. and Boyle, D.B. (1995). Vaccinia virus-expressed bovine ephemeral fever virus G but not GNS glycoprotein induces neutralizing antibodies and protects against experimental infection. Journal of General Virology 77:631-40.

Hsieh, Y.C., Chen, S.H., Chou, C.C., Ting, L.J., Itakura, C., and Wang, F.I. (2005). Bovine Ephemeral Fever in Taiwan (2001-2002) . Journal of Veterinary Medical Science 67: 411-416.

Hsieh, Y.C., Lee, Y.F., Chen, C.W., and Chen, S.H. (2002). Study of glycoprotein gene variation of bovine ephemeral fever virus isolated in southern Taiwan. The 36th Annual Meeting of the Chinese Society of Microbiology, December 15, Taipei, Taiwan.

Hung, S.L., Lee, P.L., Chen, H.WQ., Chen, L.K., Kao,C.L., King, C.C.(1999). Analysis of the steps involved in dengue virus entry into host cells. Virology. 257:156-167.

Inaba, Y., Kurogi, H., Takahashi, A., Sato, K. and Matumoto, M. (1974). Vaccination of cattle against bovine ephemeral fever with live attenuated virus followed by killed virus. Archiv fur die Gesamte Virusforschung 44：121-132.

Inaba, Y., Kurogi, H., Sato, K., Goto, Y., Omori, T. and Matumoto, M. (1973). Formalin-inactivated, aluminum phosphate gel-adsorbed vaccine of bovine ephemeral fever virus. Archiv fur die Gesamte Virusforschung 42：42-53.

Jacque, J. M., Triques, K. and Stevenson, M. (2002). Modulation of HIV-1 replication by RNA interference. Nature 18:435-438.

Jayakar, H.R., Jeetendra, E., and Whitt, M.A. (2004). Rhabdovirus assembly and budding. Virus Research 106:1147-132.

Jorgensen, R. (1990). Altered gene expression in plants due to trans interactions between homologous genes. Trends in Biotechnology 8:340-344.

Kongsuwan, K., Cybinski, D.H., Cooper, J. and Walker, P.J. (1998). Location of neutralizing epitopes on the G protion of bovine ephemeral fever rhabdovirus. Journal of General Virology 79:2573-81.

Kurenova, E.,Champion, L., Biessmann, H., and Mason, JM.　(1998). Directional gene silencing induced by a complex subtelomeric satellite from Drosophila. Chromosoma 107:311-20.

Lee, Y., Ahn, C., Han, J., Choi, . Kim, J., Yim, J., Lee, J., Provoast, P., Radmark, O., Kim, S., Kim, VN. (2003). The nuclear RNAase III Drosha initiates microRNA processing. Nature 425:415-419.

Lin, X., Yang, J., Chen, J., Gunasekera, A., Fesik, S.W., Shen, Y. (2004). Development of a tightly regulated U6 promoter for shRNA expression. FEBS Letters 577:376-380.

Lindbo,J.A., Silva-Rosales, L., Proebsting, W.M., and Dougherty, W.G.(1993). Induction of a highly specific antiviral state in transgenic plants: Implications for gene regulation and virus-resistance. Plant Cell 5:1749-1759.

Matzke, M.A. and Matzke, A.J.M. (1995). How and why do plants inactivate homologous transgene ? Plant physiology 107:679-685.

McCaffrey,AP., Nakai,H., Pandey, K., Huang, Z., Salazar, FH., Xu, H., Wieland, SF., Marion, PL., and Kay, MA. (2003). Inhibition of hepatitis B virus in mice by RNA interference. Nature Biotechnology 6 :639-44.

Mc William, S. M., Kongsuwan, K., Cowley, J. A., Byrne, K. A. and Walker, P. J. (1997). Genome organization and transcription strategy in the complex GNS-L intergenic region of bovine ephemeral fever rhabdovirus. Journal of General Viroogy 78：1309-1317.

Meins, J.F., and Kunz, C. (1994). Silencing of chitinase expression in transgenic plants : An autoregulatory model. In Homologous recombination and gene silencing in plants (ed. J. Paszkowski).pp.335-348.

Miyagishi, M. and Taira, K. (2002). U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnoogy 20:497-500.

Mol, J.,Van, B.R., and Kooter, J. (1991). More about cosuppression. Trends in Biotechnoogy 9:182-183.

Nandi, S., and Negi, B.S. (1999). Bovine ephemeral fever: a review. Comparative Immunology , Microbiology and Infectious Diseases 22:81-91.

Napoli, C., Lemieux, C. and Jorgensen, R. (1990). Introduction of a chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2(4):279-289.

Qing, G.., Mcmanus, M. T., Nguyen, T., Shen, C. H., Sharp, P. A., Eisen, H. N. and Chen, J. (2003). RNA interfence of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proceedings of National Academy Science of the United States of America 100:2718-2723.

Randall, G., Grakoui, A. and Rice, C. M. (2003). Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs .　Proceedings of National Academy Science of the United States of America 100:235-240.

Salen, B. (2004). Control of nonsegmented negative-strand RNA virus replication by siRNA. Virus Research 102:27-35.

Saporito-Irwin, S.M., Geist, R.T. and Gutmann, D.H. (1997). Ammonium acetate protocol for the preparation of plasmid DNA suitable for mammalian cell transfections. Biotechniques 23:424-7.

Shen, C., and Reske, S.N. (2000). Adenovirus-Delivered siRNA. Methods in Molecular Biology 252:523-32.

Singh, J., Goel, V. and Klar, A. J. (1998). A novel function of the DNA repair gene rhp6 in mating-type silencing by chromatin remodeling in fission yeast. Molecular and Cellular Biology 18:5511-22.

Sioud, M. (2004). Therapeutic siRNAs. Trends in Pharmacological Science 25:22-28.

Stephanie, P., and Richard, D. I.( 2004). Determinants of interferon-stimulated gene induction by RNAi vector. Differentiation 72:103-111.
St. George, T. D. and Standfast, H. A. (1988). Bovine ephemeral fever in the Arboviruses. Epidemiology and Ecology 2:71-86.

Theodoridis, A., Boshoff, S. E. T. and Botha, M. J. (1973). Syudies on the development of a vaccine against bovine ephemeral fever. Onderstepoort of Journal Veterinary Research 40：77-82.

Theodoridis, A., Nevill, E. M., Els, H. J. and Boshoff, S. T. (1979). Viruses isolated from Culicoides midges in South Africa during unsuccessful attempts to isolate bovine ephemeral fever virus. Onderstepoort of Journal Veterinary Research 46：191-198.

Timoney, J. F., Gillespie, J.H., Scott, F. W. and Barlough, J. E. (1988). Ephemeral fever. In 〞Hagan and Bruner´s Microbiology and Infectious Disease of Domestic Animals〝, 8th ed, pp 851-855,Cornell University Press, Ithaca.

Tzipori, S. and Spradbrow, P.B. (1978). A cell culture vaccine against bovine ephemeral fever. Australian Veterinary Journal 54：323-328.

Tzipori, S. and Spradbrow, P. B. (1974). Development and behaviour of a strain of bovine ephemeral fever virus with unusual host range. Journal of Comparative Pathoogy 84：1-8.

Tzipori, S. and Spradbrow, P. B. (1973). Studies on vaccines against bovine ephemeral fever. Australian Veterinary Journal 49：183-187.

Ui-Tei, K., Zenno, S., Miyata, Y. and Schultz, R.M. (2000). Sensitive assay of RNA interference in Drosophila and Chinese hamster cultured cells using firefly luciferase gene as target. FEBS Letters 479:79-82.

Uren, M. F., Walker, P. J., Zakrzewski, H., St George, T. D. and Byrne, K.A. (1994). Effective vaccination of cattle using the virion G protein of bovine ephemeral fever virus as an antigen. Vaccine 12：845-850.

Van der Vlugtt R.A.A., Ruiter, R.K., and Stuitje, A.R. (1990). Flavonoid genes in petnia: Addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2:631-639.

Vanselow, B. A., Walthall, J. C. and Abetz, I. (1995). Field trials of ephemeral fever vaccines. Veterary Microbiolgy 46：117-130.

Vanselow, B.A., Abetz, I., and Trenfield, K. (1985). A bovine ephemeral fever vaccine incorporating adjuvant Quil A : a comparative study using adjuvants Quil A, aluminium hydroxide gel and dextran sulhate. Veterary Records 117:37-43.

Venter, G.J., Hamblin, C. and Paweska, J.T. (2003). Determination of the oral susceptibility of South African livestock-associated biting midges, Culicoides species, to bovine ephemeral fever virus. Medical and Veterinary Entomology 17:133-7.

Voinnet O. (2001).RNA silencing as a plant immune system against viruses. Trends in Genetics 17:449-459.

Walker,P. J., and Kritaya, K.(1999).Deduced structural model for animal rhabdovirus glycoproteins. Journal of General Virology 80:1211-1220.

Walker, P. J., Wang, Y., Cowley, J. A., Mc William, S. M. and Prehaud, C. J. (1994). Structural and antigenic analysis of the nucleoprotein of bovine ephemeral fever rhabdovirus. Journal of General Virology 75：1889-1899.

Walker, P. J., Byrne, K. A., Riding, G. A., Cowley, J. A., Wang, Y. and Mc William, S. (1992). The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes. Virology 191：49-61.

Walker, P. J., Byrne, K. A., Cybinski, D. H., Doolan, D. L. and Wang, Y. H. (1991). Proteins of bovine ephemeral fever virus. Journal of General Virology 72：67-74.

Wagner, R., Heuer, D., Wolff, T., Herwig, A., and Klenk, H.D. (2002). N-Glycans attached to the stem domain of haemagglutinin efficiently regulate influenza Avirus replication. Journal of General Virology 83:601-609.

Wang, Y., Mc William, S. M., Cowley, J. A., and Walker, P. J. (1994). Complex genome organization in the GNS-L intergenic region of Adelaide River rhabdoviruses. Virology 203:63-72.

Wang, F. I., Hsu, A. M., and Huang, K. J. (2001). Bovine ephemeral fever in Taiwan . Veterary Diagnosis and Invesigationt 13(6): 462-7.

Wang, Y. and Walker, P. J. (1993). Adelaide River rhabdovirus expresses consecutive glycoprotein genes as polycistronic mRNAs: new evidence of gene duplication as an evolutionary process. Virology 195:719-731.

Waterouse, P.M., Wang, M.B., and Finnegan, E.J. (2001). Role of short RNAs in gene silencing. Trends in Plant Science 7:297-301.

Wilson, J. A., Jayasena, S., Khvorova, A., Sabations, S., Rodrigues-Gervais, I. G., Arya, S., Sarangi, F., Harris-brandts, M., Beaulieu, S. and Richardson, C. D. (2003). RNA interference blocks gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells . Proceedings of National Academy Science of the United States of America 100:2783-2788.

Yokota, T., Sakamoto, N., Enomoto, N., Tanabe, Y., Miyagishi, M., Maekawa, S., Yi, L., Kurosaki, M., Taira, K., Watanabe, M., and Mizusawa, H. (2003). Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs. EMBO Reports 4：602-608.

Young, P. L. and Spradbrow, P. B. (1990). Clinical response of cattle to experimental infection with bovine ephemeral fever virus. Veterinary Record 126：86-88.

Yu, J.Y., Deruiter, S.L., and Turner, L. (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proceedings of National Academy Science of the United States of America 99:6047-6052.

Yuan, J., Cheung, P. K. M., Zhang, H. M., Chau, D. and Yang, D. (2005). Inhibition of Coxsackievirus B3 replication by small interfering RNAs requires perfect sequence match in the central region of the viral positive strand. Journal of Virolog 79 (4): 2151-2159.

Zakrzewski, H., Cybinski, D.H. and Walker, P.J. (1992). A blocking ELISA for the detection of specific antibodies to bovine ephemeral fever virus. Journal of Immunological Methods 151:289-97.

Zhang, X. N., Xiong, W., Wang, J. D., Hu, Y. W., Xiang, L. and Yuan, Z. H. (2004a). siRNA-mediated inhibition of HBV replication and expression. World Journal of Gastroenterogy 10:2967-71.

Zhang, Y., Li, T., Fu, L., Yu, C., Li, Y., Xu, X., Wang, Y., Ning, H., Zhang, S., Chen, W., Babiuk, L.A., and Chang, Z. (2004b). Silencing SARS-CoV Spike protein expression in cultured cells by RNA interference. FEBS Letters 560:141-6.