Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/93093
標題: Development of discriminating ELISA diagnostic reagents to classical swine fever virus (CSFV) and characterization of swine U6 promoter for short hairpin RNA expression
豬瘟病毒ELISA區別診斷試劑之開發與豬U6啟動子誘導表現shRNA之功能分析
作者: 吳經緯
Ching-Wei Wu
關鍵字: 豬瘟病毒
酵素連結免疫分析法
短髮夾型核醣核酸
classical swine fever virus
enzyme-linked immunosorbent assay
short hairpin RNA
引用: 李淑慧、鐘明華、林有良、丁履紉、陽喜金,1997。豬實驗感染野外豬瘟病毒之組織病理變化。台灣省家畜衛生試驗所研究報。33:13-28。 劉振軒、張文發、邱慧英。2002。甲類動物傳染病之簡介。財團法人台灣養豬科學研究所。苗栗。臺灣。 蕭雅如、王群、徐華山、鐘明華,2006。比較豬瘟中和抗體與ELISA抗體力價之關係。行政院農業委員會家畜衛生試驗所研討會報告。臺北。臺灣。 許岳蒼,2002。酵素結合免疫吸附法對豬瘟抗體、抗原之檢測及應用。碩士論文。國立中興大學獸醫病理學研究所。臺中。臺灣。 陳俊維,2003。應用酵母菌Pichia pastoris表現系統生產豬瘟病毒醣蛋白E0並作為ELISA診斷試劑。碩士論文。國立中興大學獸醫微生物學研究所。臺中。臺灣。 劉庭妤,2007。豬瘟病毒醣蛋白Erns之RNase活性功能區分析。碩士論文。國立中興大學獸醫微生物學研究所。臺中。臺灣。 游家進,2009。結合小鼠鈣網蛋白基因與豬瘟病毒結構性醣蛋白E2 DNA疫苗於BALB/c小鼠之效力評估。碩士論文。國立中興大學獸醫微生物學研究所。臺中。臺灣。 黃靜如,2012。豬瘟病毒醣蛋白Erns之單株抗體的製備及其應用於間接三明治ELISA抗體檢測方法之建立。碩士論文。國立中興大學微生物暨公共衛生學研究所。臺中。臺灣。 林光展,2012。酵母菌表現豬瘟病毒醣蛋白E2次單位疫苗之效力分析。博士論文。國立中興大學微生物學暨公共衛生學研究所。臺中。臺灣。 Classical swine fever (hog cholera). OIE Terrestrial Manual CHAPTER 2.8.3. 1092-1106 Horzinek, M.C. 1981. Non-arthropod-borne Togaviruses. London: Academic Press. 65-75. Lin, T.C. and Lee, C.T. 1981. An overall report on the development of a highly safe and potent lapinized hog cholera virus strain for hog cholera control in Taiwan. NSC Spec. Publ. 5, 1-44. Lindenbach, B.D. and Rice, C. 2001. Flaviviridae: the viruses and their replication. Fields Virology 1, 991-1041. MacLachlan, N.J. and Dubovi, E.J. 2011. Flaviviridae. In: Barthold, S.W., Bowen, R.A., Hedrick, R.P., Knowles, D.P., Lairmore, M.D., Parrish, C.R., Saif, L.J., Swayne, D.E. (eds) Fenner's Veterinary Virology (4th ed.). London: Elsevier. van Oirschot, J.T. 1988. Vaccinology of classical swine fever and related viral. Liess, B. (eds) Boston: Martinus Nijhoff publishing Wei, P. and Qin, A.J. 2008. Molecular Biology of Important Animal Viruses. China: Science Press Agapov, E.V., Murry, C.L., Frolov, I., Qu, L., Myers, T.M. and Rice, C.M. 2004. Uncleaved NS2-3 is required for production of infectious bovine viral diarrhea virus. J. Virol. 78, 2414-2425. Andrew, M., Morris, K., Coupar, B., Sproat, K., Oke, P., Bruce, M., Broadway, M., Morrissy, C., and Strom, D. 2006. Porcine interleukin-3 enhances DNA vaccination against classical swine fever. Vaccine 24, 3241-3247. Baker, J.A. 1946. Serial passage of hog cholera virus in rabbits. Proc. Soc. Exp. Biol. Med. 63, 183-187. Baretl, D.P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-297. Bartal, A.H. and Hirshaut, Y. 1987. Methods of hybridoma formation, vol 7. Humana Pr Inc. Bauhofer, O., Summerfield, A., McCullough, K.C., and Ruggli, N. 2005. Role of double-stranded RNA and Npro of classical swine fever virus in the activation of monocyte-derived dendritic cells. Virology 343, 93-105. Becher, P., Avalos Ramirez, R., Orlich, M., Cedillo Rosales, S., Konig, M., Schweizer, M., Stalder, H., Schirrmeier, H., and Thiel, H.J. 2003. Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification. Virology 311, 96-104. Becher, P., Orlich, M., Kosmidou, A., Konig, M., Baroth, M., and Thiel, H.J. 1999. Genetic diversity of pestiviruses: identification of novel groups and implications for classification. Virology 262, 64-71. Beer, M., Reimann, I., Hoffmann, B., and Depner, K. 2007. Novel marker vaccines against classical swine fever. Vaccine 25, 5665-5670. Behrense, S.E., Tomei, L, and De Francesco, R. 1996. Identification and properties of the RNA-dependent RNA polymerase of hepatitis C virus. EMBO J. 15. 12-22. Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366. Bjorklund, H., Lowings, P., Stadejek, T., Vilcek, S., Greiser-Wilke I., Paton, D., and Belak, S. 1999. Virus Genes 19, 189-195. Blome, S., Meindl-Bohmer, A., Loeffen, W., Thuer, B., and Moenning, V. 2006. Assessment of classical swine fever diagnostics and vaccine performance. Rev. Sci. Tech. 25, 1025-1038. Bouma, A., deSmit, A.J., de Jong, M.C.M., de Kluijver, E.P., and Moormann, R.J.M. 2000. Determination of the onset of the herd-immunity induced by the E2 subunit vaccine against classical swine fever virus. Vaccine 18, 1374-1381. Brea, D., Meurens, F., Dubois, A.V., Gaillard, J., Chevaleyre, C., Jourdan, M.L., Winter, N., Arbeille, B., Si-Tahar, M., Gauthier, F., and Attucci, S. 2012. The pig as a model for investigating the role of neutrophil serine proteases in human inflammatory lung disease. Biochem. J. 447, 363-370. Brummelkamp, T.R., Bernards, R., and Agami, R. 2002. A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-553. Bruschke, C., Hulst, M.M., Moormann, R.J., van Rijn, P.A., and van Oirschot, J.T., 1997. Glycoprotein Erns of pestiviruses induces apoptosis in lymphocytes of several species. J. Virol. 71, 6692-6696. Carbrey, E.A., Stewart, W.C., Kresse, J.L., and Snyder, M.L. 1976. Natural infection of pigs with bovine diarrhea virus and its deferential diagnosis from hog cholera. J. Am. Vet. Med. Assoc. 169, 1217-1219. Cereghino, J.L. and Cregg, J.M. 2000. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24, 45-66. Chang, C.Y., Huang, C.C., Lin, Y.J., Deng, M.C., Chen, H.C., Tsai, C.H., Chang, W.M., and Wang, F.I. 2010. Antigenic domains analysis of classical swine fever virus E2 glycoprotein by mutagenesis and conformation-dependent monoclonal antibodies. Virus Res. 149, 183-189. Chang, C.Y., Huang, C.C., Deng, M.C., Huang, Y.L., Lin, Y.J., Liu, H.M., Lin, Y.L. and Wang, F.I. 2012. Identification of conformational epitopes and antigen-specific residues at the D/A domains and the extramembrane C-terminal region of E2 glycoprotein of classical swine fever virus. Virus Res. 168, 56-63. Chen, N., Tong, C., Li, D., Wan, J., Yuan, X., Li, X., Peng, J., and Fang, W. 2010. Antigenic analysis of classical swine fever virus E2 glycoprotein using pig antibodies identifies residues contributing to antigenic variation of the vaccine C-strain and group 2 strains circulating in China. Virol. J. 7, 378. Cinar, P. and Tempero, M.A. 2012. Monoclonal antibodies and other targeted therapies for pancreatic cancer. Cancer J. 18. 653-664. Clavijo, A., Lin, M., Riva, J., Mallory, M., Lin, F., and Zhou, E.M. 2001. Development of competitive ELISA using a truncated E2 recombinant protein as antigen for detection of antibodies to classical swine fever virus. Res. Vet. Sci. 70, 1-7. Cole, C.G., Henley, R.R., Dale, C.N., Mott, L.O., Torrey, J.P., and Zinober, M.R. 1962. History of hog cholera research in the US. Department of Agriculture 1884-1960. Cregg, J.M., Cereghino, J.L., Shi, J., and Higgins, D.R. 2000. Recombinant protein expression in Pichia pastoris. Mol. Biotechnol. 16, 23-52. Cregg, J., Tschopp, J., Stillman, C., Siegel, R., Akong, m., Craig, W., Buckholz, R., Madden, K., Kellaris, P., and Davis, G. 1987. High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Nature Biotechnology 5, 479-485. Daniel-Carlier, N., Sawafta, A., Passet, B., Thepot, D., Leroux-Coyau, M., Lefevre, F., Houdebine, L.M., and Jolivet, G. 2012. Viral infection resistance conferred on mice by siRNA transgenesis. Transgenic Res. 22, 489-500. de las Mulas, T.M., Ruiz-Villamor, E., Donoso, S., Quezada, M.,Lecocq, C., and Sierra, M.A. 1997. Immunohistochemical detection of hog cholera viral glycoprotein 55 in paraffin-embedded tissues. J. Vet. Diagn. Invest. 9, 10-16. de Smit, A.J., Bouma, A., de Kluijver, E.P., Terpstra, C., and Moormann, R.J. 2001. Duration of the protection of an E2 subunit marker vaccine against classical swine fever after a single vaccination. Vet. Microbiol. 78, 307-317. Deng, M.C., Huang, C.C., Huang, T.S., Chang, C.Y., Lin, Y.J., Chien, M.S., and Jong, M.H. 2005. Phylogenetic analysis of classical swine fever virus isolated from Taiwan. Vet. Microbiol. 106, 187-193. Depner, K., Gruber, A., and Leiss, B. 1994. Experimental infection of wiener pigs with a field isolate of hog cholera/classical swine fever virus derived from a recent outbreak in Lower Saxony. I: Clinical, virological and serological findings. Weiner Tierarztliche Monatsschrift 81, 370-373. Depner, K., Paton, D.J., Cruciere, C., de Mia, G.M., Muller, A., Koenen, F., Stark, R., Liess, B. 1995. Evaluation fo the enzyme-linked immunosorbent assay for the rapid screening and detection of classical swine fever virus antigens in the blood of pigs. Rev. Sci. Tech. 14, 677-689. Dong, X.N. and Chen, Y.H. 2007. Marker vaccine strategies and candidate CSFV marker vaccines. Vaccine 25, 205-230. Dong, X.N., Wei, K.,Liu, Z.Q., and Chen, Y.H. 2002. Candidate peptide vaccine induced protection against classical swine fever virus. Vaccine 21, 167-173. Dunne, H.W., Benbrood, S.C. Smith, E.M., and Runnells, R.A. 1957. Bone structure changes in pigs infected with hog cholera. Am. J. Vet. Med. Assoc. 130, 260-265. Edwards, S., Fukusho, A., Lefevre, P., Lipowski, A., Pejsak, Z., Roehe, P., and Westergaard, J. 2000. Classical swine fever the global situation. Vet. Microbiol. 73, 103-119. Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschi, T. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498. Fernandez-Sainz, I., Holinka, L.G., Gavrilov, B.K., Prarat, M.V., Gladue, D., Lu, Z., Jia, W., Risatti, G.R., and Borca, M.V. 2009. Alteration of the N-linked glycosylation condition in E1 glycoprotein of classical swine fever virus strain Brescia alters virulence in swine. Virology 386, 210-216. Fernandez-Sainz, I., Holinka, L.G., Gladue, D., O'Donnell, V., Lu, Z., Gavrilov, B.K., Risatti, G.R., and Borca, M.V. 2011. Substitution of specific cysteine residues in the E1 glycoprotein of classical swine fever virus strain Brescia affects formation of E1-E2 heterodimers and alters virulence in swine. J. Virol. 85, 7264-7272. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811. Fletcher, S.P., Ali, I.K., Kaminski, A., Digard, P., and Jackson, R.J. 2002. The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes. RNA, 8, 1558-1571. Floegel-Niesmann, G. 2001. Classical swine fever (CSF) marker vaccine Trial III. Evaluation of discriminatory ELISAs. Vet. Microbiol. 83, 121-136. Floegel-Niesmann, G., Bunzenthal, C., Fischer, S., Moennig, V., and Kaaden, O.R. 2003. Virulence of recent and former classical swine fever virus isolates evaluated by their clinical and pathological signs. J. Vet. Med. B. Infect. Dis. Vet. Public Health 50, 214-220. Frey, C.F., Bauhofer, O., Ruggli, N., Summerfield, A., Hofmann, M.A., and Tratschin, J.D. 2006. Classical swine fever virus replicon particles lacking the Erns gene: a potential marker vaccine for intradermal application. Vet. Res. 37, 655-670. Gabriel, C., Blome, S., Urniza, A., Juanola, S., Koenen, F., and Beer, M. 2012. Towards licensing of CP7_E2alf as marker vaccine against classical swine fever-Duration of immunity. Vaccine 30, 2928-2936. Gallei, A., Orlich, M., Thiel, H.J. 2005. Noncytopathogenic pestivirus strains generated by nonhomologous RNA recombination: alterations in the NS4A/NS4B coding region. J. Virol. 79, 14261-14270. Ganges, L., Barrera, M., Nunez, J.I., Blanco, I., Frias, M.T., Rodriguez, F., and Sobrino, F. 2005. A DNA vaccine expressing the E2 protein of classical swine fever virus elicits T cell responses that can prime for rapid antibody production and confer total protection upon viral challenge. Vaccine 23, 3741-3752. Gavrilov, B.K., Rogers, K., Fernandez-Sainz, I.J., Holinka, L.G., Borca, M.V., and Risatti, G.R. 2011. Effects of glycosylation on antigenicity and immunogenicity of classical swine fever virus envelope proteins. Virology 420, 135-145. Gladue, D.P., Holinka, L.G., Fernandez Sainz, I., Prarat, M.V., O'Donnell, V., Vepkhvadze, N., Lu, Z., Rogers, K., Risatti, G.R., and Borca, M.V. 2010. Effects of the interactions of classical swine fever virus Core protein with proteins of the SUMOylation pathway on virulence in swine. Virology 407, 129-136. Gladue, D.P., Holinka, L.G., Largo, E., Fernandez Sainz, I., Carrillo, C., O'Donnell, V., Baker-Branstetter, R., Lu, Z., Ambroggio, X., Risatti, G.R., Nieva, J.L., and Borca, M.V. 2012. Classical swine fever virus p7 protein is a viroporin involved in virulence in swine. J. Virol. 86, 6778-6791. Greiser-Wilke, I., Blome, S., and Moennig, V. 2007. Diagnostic methods for detection of Classical swine fever virus – Status quo and new developments. Vaccine 25, 5524-5530. Greiser-Wilke, I., Depner, K., Fritzemeier, J., Haas, L., Moennig, V. 1998. Application of a computer program for genetic typing of classical swine fever virus isolated from Germany. J. Virol. Methods 75, 141-150. 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 asymmetrically distributed. Cell 81, 611-620. Hamasaki, K., Nakao, K., Matsumoto, K., Ichikawa, T., Ichikawa, H., and Eguchi, K. 2003. Short interfering RNA-directed inhibition of hepatitis B virus replication. FEBS let. 543, 51-54. Hammond, J.M., Jansen, E.S., Morrissy, C.J., Williamson, M.M., Hodgson, A.L., and Johnson, M.A. 2001. Oral and sub-cutaneous vaccination of commercial pigs with a recombinant porcine adenovirus expressing the classical swine fever virus gp55 gene. Arch. Virol. 146, 1787-1793. Harada, T., Tautz, N., and Thiel, H.J. 2000. E2-p7 region of the bovine viral diarrhea virus polyprotein: processing and functional studies. J. Virol. 74, 9498-9506. Hericourt, F., Blanc, S., Redeker, V., and Jupin, I. 2000. Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus-insect cell system. Biochem. J. 349, 417-425. Hofmann, M.A., Brechtbuehl, K., and Staeuber, N. 1994. Rapid characterization of new pestivirus strains by directsequencing of PCR-amplified cDNA from the 50 noncoding region. Arch. Virol. 139, 217-229. Huang, C., Chien, M.S., Hu, C.M., Chen, C.W., and Hsieh, P.C. 2006. Secreted expression of the classical swine fever virus glycoprotein Erns in yeast and application to a sandwich blocking ELISA. J. Virol. Methods 132, 40-47. Hulst, M.M. and Moormann, R.J.M. 1997. Inhibition of pestivirus infection in cell culture by envelope proteins Erns and E2 of classical swine fever virus: Erns and E2 interact with different receptors. J. Gen. Virol. 78, 2779-2787. Hulst, M.M. and Morrmann, R.J.M. 2001. Erns protein of pestiviruses. Methods Enzymol. 342, 431-440. Hulst, M.M., Westra, D.F., Wensvoort, G., and Moormann, R.J.M. 1993. Glycoprotein E1 of hog cholera virus expressed in insect cells protects swine from hog cholera. J. Virol. 67, 5435-5442. Isken, O., Grassmann, C.W., Yu, H., and Behrens, S.E. 2004. Complex signals in the genomic 3' nontranslated region of bovine viral diarrhea virus coordinate translation and replication of the viral RNA. RNA 10, 1637-1652. James, D.C., Freedman, R.B., Hoare, M., Oqonah, O.W., Rooney, B.C., Larionov, O.A., Dobrovolsky, V.N., Laqutin, O.V., and Jenkins, N. 1995. N-glycosylation of recombinant human interferon-g produced in different animal expression systems. Nat. Biotechnol. 13, 592-596. Jensen, R.C., Wang, Y., Hardin, S.B., and Stumph, W.E. 1998. The proximal sequence element (PSE) plays a major role in establishing the RNA polymerase specificity of Drosophila U-snRNA genes. Nucleic Acids Res. 26, 616-622. Jones, D.M., Patel, A.H., Targett-Adams, P., and McLauchlan, J. 2009. The hepatitis C virus NS4B protein can trans-complement viral RNA replication and modulates production of infectious virus. J. Virol. 83, 2163-2177. Kaden, V., Lange, E., Polster, U., Klopfleisch, R., and Teifke, J.P. 2004. Studies on the virulence of two field isolates of the classical swine fever virus genotype 2.3 Rostock in wild boars of different age groups. J. Vet. Med. B Infect. Dis. Vet. Public Health 51, 202-208. Kaden, V., Ziegler, U., Lange, E., and Dedek, J. 2000. Classical swine fever virus: clinical, virological, serological and hematological findings after infection of domestic pigs and wild boars with the field isolate 'Spante' originating from wild boar. Berl. Munch. Tierarztl. Wochenschr. 113, 412-416. Kalliampakou, K.I., Kalamvoki, M., and Mavromara, P. 2005. Hepatitis C virus (HCV) NS5A protein downregulates HCV IRES-dependent translation. J. Gen. Virol 86, 1015-1025. Kang, T.H., Chung, J.Y., Monie, A., Pai, S.I., Hung, C.F., and Wu, T.C. 2010. Enhancing DNA vaccine potency by co-administration of xenogenic MHC class-I DNA. Gene Ther. 17, 531-540. Katz, J.B., Ridpath, J.F., and Bolin, S.R. 1993. Presumptive diagnostic differentiation of hog cholera virus from bovine viral diarrhea, and border disease viruses by using a cDNA nested-amplification approach. J. Clin. Microbiol. 3, 565-568. Kim, S.M., Lee, K.N., Lee, S.J., Ko, Y.J., Lee, H.S., Kweon, C.H., Kim, H.S., and Park, J.H. 2010. Multiple shRNAs driven by U6 and CMV promoter enhances efficiency of antiviral effects against foot-and –mouth disease virus. Antiviral Res. 87, 307-317. Kohler, G. and Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497. Konig, M., Lengsfeld, T., Pauly, T., Stark, R., and Thiel, H.J. 1995. Classical swine fever virus: independent induction of protective immunity by two structural glycoproteins. J. Virol. 69, 6479-6486. Kolupaeva, V.G., Pestova, T.V. and Hellen,C.U. 2000. Ribosomal binding to the internal ribosomal entry site of classical swine fever virus. RNA 6, 1791-1807. Koprowski, H., James, T.R., and Cox, H.R. 1946. Propagation of hog cholera virus in rabbits. Proc. Soc. Exp. Biol. Med. 63, 178-183. La Rocca, S.A., Herbert, R.J., Crooke, H., Drew, T.W., Wileman, T.E., and Powell, P.P. 2005. Loss of interferon regulatory factor 3 in cells infected with classical swine fever virus involves the N-terminal protease, Npro. J. Virol. 79, 7239-7247. Lackner, T., Muller, A., Pankraz, A., Becher, P., Thiel, H.J., Gorbalenya, A.E., and Tautz, N. 2004. Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus. J. Virol. 78. 10765-19775. Langedijk, J., vanVeelen, P., Schaaper, W., de Ru, A., Meloen, R., and Hulst, M. 2002. A structural model of pestivirus Erns based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide. J. Virol. 76, 10383-10392. Lazer, C., Zitzmann, N., Dwek, R.A., and Branza-Nichita, N. 2003. The pestivirus Erns glycoprotein interacts with E2 in both infected cells and mature virions. Virology 314, 696-705. Leifer, I., Everett, H., Hoffmann, B., Sosan, O., Crooke, H., Beer, M., and Blome, S., 2010. Escape of classical swine fever C-strain vaccine virus from detection by C-strain specific real-time RT-PCR caused by a point mutation in the primer-binding site. J. Virol. Methods 166, 98-100. Li, C., Candotti, D., and Allain, J.P. 2001. Production and characterization of monoclonal antibodies specific for a conserved epitope within hepatitis C virus hypervariable region 1. J. Virol. 75. 12412-12420. Liess, B. and Parger, D. 1976. Detection of neutralizing antibodies (NIF test): use of new technical equipment (CCSC system) for laboratory swine fever diagnosis. CEC report on diagnosis and epizootiology of classical swine fever and African swine fever, 5486EUR, 187-197. Lin, G.J., Deng, M.C., Chen, Z.W., Liu, T.Y., Wu, C.W., Cheng, C.Y., Chien, M.S., and Huang, C. 2012. Yeast expressed classical swine fever E2 subunit vaccine candidate provides complete protection against lethal challenge infection and prevents horizontal virus transmission. Vaccine 30, 2336-2341. Lin, G.J., Liu, T.Y., Tseng, Y.Y., Chen, Z.W., You, C.C., Hsuan, S.L., Chien, M.S., and Huang, C. 2009. Yeast-expressed classical swine fever virus glycoprotein E2 induces a protective immune response. Vet. Microbiol. 139, 369-374. Lin, M., Lin, F., Mallory, M., and Clavijo, A. 2000. Deletions of structural glycoprotein E2 of classical swine fever virus strain alfort/187 resolve a linear epitope of monoclonal antibody WH303 and the minimal N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum. J. Virol. 74, 11619-11625. Lin, T.C., Shieh, C.M. and Su, J.F. 1974. Virus multiplication in pigs inoculated with lapinized hog cholera live vaccine. Zhonghua Minguo wei sheng wu xue za zhi 7, 13-19. Liu, J.J., Wong, M.L., and Chang, T.J. 1998. The recombinant nucleocapsid protein of classical swine fever virus can act as a transcriptional regulator. Virus Res. 53, 75-80. Liu, L., Hoffmann, B., Baule, C., Beer, M., Belak, S., Widen, F. 2009. Two real-time RT-PCR assays of classical swine fever virus, developed for the genetic differentiation of naturally infected from vaccinated wild boars. J. Virol. Methods 159, 131-133. Liu, L., Xia, H., Everett, H., Sosan, O., Crooke, H., Meindl-Bohmer, A., Qiu, H.J., Moennig, V., Belak, S., and Widen, F. 2011. A generic real-time TaqMan assay for specific detection of lapinized Chinese vaccines against classical swine fever. J. Virol. Methods 175, 170-174. Loeffen, W. 2005. Evaluation of live commercially available CSF-ELISA kits. Report on the annual meeting of National Swine Fever Laboratories (Brussels) 143. Lowings, P., Ibata, G., Needham, J., Paton, D. 1996. Classical swine fever virus diversity and evolution. J. Gen. Virol. 77, 1311-1321. Mayer, D.M., Hofmann, A., and Tratschin, J.D. 2004. Attenuation of classical swine fever virus by deletion of the viral Npro gene. Vaccine 22, 317-328. McGoldrick, A., lowings, J.P., Ibata, G., Sands, J.J., Belak, S., and Paton, D.J. 1998. A novel apporoach to the detection of classical swine fever virus by RT-PCR with a fluorogenic probe (TagMan). J. Virol. Methods. 72, 125-135. Meyers, G. and Thiel, H.J. 1995. Cytopathogenicity of classical swine fever virus caused by defective interfering particles. J. Virol. 69, 3683-3689. Meyers, G., Enaph, T.R., and Thiel, H.J. 1989. Molecular cloning and nucleotide sequence of the genome of the hog cholera virus. Virol. 171, 125-135. Moennig, V. and Plagemann, P.G.W. 1992. The pestivituses. Adv. Virus Res. 41, 53-98. Moennig, V. 2000. Introduction to classical swine fever: virus, disease, and control policy. Vet Microbiol. 73, 93-102. Moormann, R.J.M., Bouma, A., Kramps, J.A., Terpstra, C., and de Smit, H.J. 2000. Development of a classical swine fever subunit marker vaccine and companion diagnostic test. Vet. Microbiol. 73, 209-219. Mori, Y. Okabayashi, T., Yamashita, T., Zhao, Z., Wakita, T., Yasui, K., Hasebe, F., Tadano, M., Konishi, E., Moriishi, K., and Matsuura, Y. 2005. Nuclear localization of Japanese encephalitis virus core protein enhances viral replication. J. Virol. 79, 3448-4358. Moser, C., Ruggli, N., Tratschin, J.D., and Hofmann, M.A. 1996. Detection of antibodies against classical swine fever virus in swine sera by indirect ELISA using recombinant envelope glycoprotein E2. Vet. Microbiol. 51, 41-53. Moser, C., Stettler, P., Tratschin, J.D., and Hofmann, M.A. 1999. Cytopathogenic and noncytopathogenic RNA replicons of classical swine fever virus. J. Virol. 73, 7787-7794. Nadar, M., Chan, M.Y., Huang, S.W., Tseng, J.T., and Tsai, C.H. 2011. HuR binding to AU-rich elements present in the 3' untranslated region of classical swine fever virus. Virol. J. 8: 340. Napoli, C., Lemieux, C., and Jorgensen, R. 1990. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. The Plant Cell. 2, 279-289. Narita, M., Kawasaima, K., Kimura, K., Mikami, O., Shibahara, T., Yamada, S., and Sakoda, Y. 2000. Comparative immunohistopathology in pigs infected with highly virulent or less virulent strains of hog cholera virus. Vet. Pathol. 37, 402-408. Omari, K.E., Iourin, O., Harlos, K., Grimes, J.M., and Stuart, D.I. 2013. Structure of a pestivirus envelope glycoprotein E2 clarifies its role in cell entry. Cell Rep. 3, 30-35. Paton, D.J., Lowings, J.P., and Barrett, A.D.T. 1992. Epitope mapping of the gp53 envelopment protein of bovine viral diarrhea virus. Virology 190, 763-772. Paton, D.J., McGoldrick, A., Belak, S., Mittelholzer, C., Koenen, F., Vanderhallen, H., Biagetti, M., de Mia, G.M., Stadejek, T., Hofmann, M.A., and Thuer, B. 2000. Classical swine fever virus: a ring test to evaluate RT-PCR detection methods. Vet. Microbiol. 73, 159-174. Poton, D.J., McGoldrick, A., Greiser-Wilke, I., Parchariyanon, S., Song, J.Y., Liou, P.P. Stadejek, T., Lowings, J.P., Bjorklund, H., and Belak, S. 2000. Genetic typing of classical swine fever virus. Vet. Microbiol. 73, 137-157. Rangelova, D., Nielsen, J., Strandbygaard, B., Koenen, F., Blome, S., and Uttenthal, A. 2012. Efficacy of marker vaccine candidate CP7_E2alf in piglets with maternally derived C-strain antibodies. Vaccine 30, 6376-6381. Reimann, I., Depner, K., Trapp, S., and Beer, M. 2004. An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus. Virology 322, 143-157. Reusken, C.B., Dalebout, T.J., Eerligh, P., Bredenbeek, P.J., and Spaan, W.J. 2003. Analysis of hepatitis C virus /classical swine fever virus chimeric 5' NTRs: sequences within the hepatitis C virus IRES are required for viral RNA replication. J. Gen. Virol. 84, 1761-1769. Riedel, C., Lamp, B., Heimann, M., and Rumenapf, T. 2010. Characterization of essential domains and plasticity of the classical swine fever virus Core protein. J. Virol. 84, 11523-11531. Risatti, G.R., Holinka, L.G., Fernandez Sainz, I., Carrillo, C., Lu, Z., and Borca, M.V. 2007. N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine. J. Virol. 81, 924-933. Ritchie, A. and Fernelius, A. 1968. Direct immune-electron microscopy and some morphological features of hog cholera virus. Arch Gesamte Virusforsch 23, 292-298. Roelz, R., Pilz, I.H., Mutschler, M., and Pahl, H.L., 2010. Of mice and men: human RNA polymerase III promoter U6 is more efficient than its murine homologue for shRNA expression from a lentiviral vector in both human and murine progenitor cells. Exp. Hematol. 38, 792-797. Romano, N. and Macino, G. 1992. Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with in homologous sequences. Mol. Microbiol. 6, 3343-3353. Ruggli, N., Bird, B.H., Liu, L., Bauhofer, O., Tratschin, J.D., and Hofmann, M.A. 2005. Npro of classical swine fever virus is an antagonist of double-stranded RNA mediated apoptosis and IFN-alpha/beta induction. Virology 340, 265-276. Rumenapf, T., Stark, R., Heiman, M., and Thiel, H.J. 1998. N-terminal protease of pestiviruses: identification of putative catalytic residues by site-directed mutagenesis. J. Virol. 72, 2544-2547. Rumenapf, T., Stark, R., Meyers, G., and Thiel, H.J. 1991. Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity. J. Virol. 65, 589-597. Rumenapf, T., Unger, G., Strauss, J.H., and Thiel, H.J. 1993. Processing of the envelope glycoproteins of pestiviruses. J. Virol. 67, 3288-3294. Sainz, I.F., Holinka, L.G., Lu, Z., Risatti, G.R., and Borca, M.V. 2008. Removal of a N-linked glycosylation site of classical swine fever virus strain Brescia Erns glycoprotein affects virulence in swine. Virology 370, 122-129. Sanchez-Cordon P.J. Nunez, A., Salguero, F.J., Pedrera, M., Fernandez de Marco, M., and Gomez-Villamandos, J.C. 2005. Lymphocyte apoptosis and thrombocytopenia in spleen during classical swine fever: role of macrophages and cytokines. Vet. Pathol, 42, 477-488. Sato, M., Mikami, O., Kobayashi, M., Nakajima, Y. 2000. Apoptosis in the lymphatic organs of piglets inoculated with classical swine fever virus. Vet. Microbiol. 75, 1-9. Schneider, R., Unger, G., Stark, R., Schneider-Scherzer, E., and Thiel, H.J. 1993. Identification of a structural glycoprotein of a RNA virus as a ribonuclease. Science 261, 1169. Seago, J., Hilton, L., Reid, E., Doceul, V., Jeyatheesan, J., Moganeradj, K., McCauley, J., Charlenston, B., and Goodbourn, S., 2007. The Npro product of classical swine fever virus and bovine viral diarrhea virus uses a conserved mechanism to target interferon regulatory factor-3. J. Gen. Virol. 88, 3002-3006. Sui, G., Soohoo, C., Affar, E.B., Gay, F., Shi, Y., Forrester, W.C. and Shi, Y. 2002. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. U S A. 99, 5515-5520. Summerfield, A., Alves, M., Ruggli, N., de Bruin, M.G., and McCullough, K.C. 2006. High IFN-alpha responses associated with depletion of lymphocytes and natural IFN-producing cells during classical swine fever. J. Interferon Cytokine Res. 26, 248-255. Summerfield, A., Knotig, S.M., and Mccullough, K.C. 1998. Lymphocyte apoptosis during classical swine fever: implication of activation-induced cell death. J. Virol. 72, 1853-1861. Suradhat, S. and Damrongwatanapokin, S. 2003. The influence of maternal immunity on the efficacy of a classical swine fever vaccine against classical swine fever virus, genogroup 2.2, infection. Vet. Microbiol. 92, 187-194. Suzich, J.A., Tamura, J.K., Palmer-Hill, F., Warrener, P., Grakoui, A., Rice, C.M., Feinstone, S.M., and Collett, M.S. 1993. Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and Flavivirus enzymes. J. Virol. 67, 6152-6158. Tang, Q., Zhang, Y., Fan, L., Tong, G., He, L., and Dai, C. 2010. Classic swine fever virus NS2 protein leads to the induction of cell cycle arrest at S-phase and endoplasmic reticulum stress. Virol. J. 7, 4. Tautz, N., Elbers, K., Stoll, D., Meyers, G., and Thiel, H. 1997. Serine protease of pestiviruses: determination of cleavage sites. J. Virol. 71, 5415-5422. Tellinghuisen, T.L., Paulson, M.S., and Rice, C.M. 2006. The NS5A protein of bovine viral diarrhea virus contains an essential zinc-binding site similar to that of the hepatitis C virus NS5A protein. J. Virol. 80, 7450-7458. Terpstra, C., Bloemraad, M., and Gielkens, A.L.J. 1984. The neutralizing peroxidase-linked assay for detection of antibody against swine fever virus. Vet. Microbiol. 9, 113-120. Terpstra, C. and Wensvoort, G. 1988. The protective value of vaccine-induced neutralizing antibody titres in swine fever. Vet. MIcrobiol. 16, 123-128. Thiel, H., Stark, R., Weiland, E., Rumenapf, T., and Meyers, G. 1991. Hog cholera virus: molecular composition of virions from a pestivirus. J. Virol. 65, 4705-4712. Tighe, H., Corr, M., Roman, M., and Raz, E. 1998. Gene vaccination: plasmid DNA is more than just a blueprint. Immunology Today 19, 89-97. Tschopp, J.F., Brust, P.F., Cregg, J.M., Stillman, C.A., and Gingeras, T.R. 1987. Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acid Res. 15, 3859-3876. Tratschin J.D., Moser, C., Ruggli, N. and Hofmann, M.A. 1998. Classical swine fever virus leader proteinase Npro is not required for viral replication in cell culture. J. Virol. 72, 7681-7684. van Oirschot, J.T. 1999. Diva vaccine that reduce virus transmission. J. Biotechnol. 73, 195-205. van Oirschot, J.T. 2003. Vaccinology of classical swine fever: from lab to field. Vet. Microbiol. 96, 367-384. van Oirschot, J.T. and Terpstra, C.A. 1977. A congenital persistent swine fever infection. I. Clinical and virological observations. Vet. Microbiol. 2, 121-132. van Rijn, P.A., Bossers, A., Wensvoort, G., and Moormann, R.J. 1996. Classical swine fever virus (CSFV) envelope glycoprotein E2 containing one structural antigenic unit protects pigs from lethal challenge. J. Gen. Virol. 77, 2737-2745. van Rijn, P.A., Miedema, G., Wensvoort, G., van Gennip, H., and Moormann, R. 1994. Antigenic structure of envelope glycoprotein E1 of hog cholera virus. J. Virol. 68, 3934-3942. van Rijn, P.A., van Gennip, R.G., de Meijer, E.J., and Moormann,, R.J. 1992. A preliminary map of epitopes on envelope glycoprotein E1 of HCV strain Brescia. Vet. Microbiol. 33, 221-230. van Zijl, M., Wensvoort, G., de Kluyver, E., Hulst, M., van der Gulden, H., and Gielkens, A. 1991. Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera. J. Virol. 65, 2761-2765. Warrener, P. and Collett, M.S. 1995. Pestivirus NS3 (p80) protein possesses RNA helicase activity. J. Virol. 69, 1720-1726. Waterham, H.R., Digan, M.E., Koutz, P.J., Lair, S.V., and Cregg, J.M. 1997. Isolation of the Pichia pastoris glyceraldehydes-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186, 37-44. Weesendorp, E., Stegeman, A., and Loeffen, W.L. 2009. Quantification of classical swine fever virus in aerosols originating from pigs infected with strains of high, moderate or low virulence. Vet. Microbiol. 17, 129-140. Weiland, E., Ahl, R., Stark, R., Weiland, F., and Thiel, H.J. 1992. A second envelope glycoprotein mediates neutralization of a pestivirus, hog cholera virus. J. Virol. 66, 3677-3682. Weiland, E., Stark, R., Haas, B., Rumenapf, T., Meyers, G., Thiel, H.J. 1990. Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer. J. Virol. 64, 3563-3569. Wensvoort, G., Bloemarrd, M., and Terpstra, C. 1988. An enzyme immunoassay employing monoclonal antibodies and detection specifically antibodies to classical swine fever virus. Vet. Microbiol. 17, 129-140. Windisch, J.M., Schneider, R., Stark, R., Weiland, E., Meyers, G., and Thiel, H.J. 1996. RNase of classical swine fever virus : biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies. J. Virol. 70, 352-358. Wirz, B., Tratschin, J.D., Muller, H.K., and Mitchell, D.B. 1993. Detection of hog cholera virus and differentiation from other pestiviruses by polymerase chain reaction. J. Clin. Microbiol. 31, 1148-1158. Wiskerchen, M. and Collett, M.S. 1991. Pestivirus gene expression: protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing. Virology 184, 341-350. Xia, H., Mao, Q., Paulson, H.L., and Davidson, B.L. 2002. siRNA-mediated gene silencing in vitro and in vivo. Nat. Biotechnol. 20, 1006-1010. Xia, X.G., Zhou, H., Ding, H., Affar, E.B., Shi, Y., and Xu, Z. 2003. An enhanced U6 promoter for synthesis of short hairpin RNA. Nucleic Acid Res. 31. e100. Xiao, M., Chen, J., Wang, Y., Zhen, Y.,Lu, W., Chen, J., and Li, B. 2004. Sequence, necessary for initiating RNA synthesis, in the 3'-noncoding region of the classical swine fever virus genome. Mol. Biol. 38, 343-351. Xiao, M., Zhang, C.Y., Pan, Z.S., Wu,H.X., and Guo, J.Q. 2002. Classical swine fever virus NS5B-GFP fusion protein possesses a RNA-dependent RNA polymerase activity. Arch. Virol. 147, 1779-1787. Xu, X., Guo, H., Xiao, C., Zha, Y., Shi, Z., Xia, X., and Tu, C. 2008. In vitro inhibition of classical swine fever virus replication by siRNAs targeting Npro and NS5B genes. Antiviral Res. 78, 188-193. Yin, C.H., Qin, L.T., Sun, M.Y., Gao, Y.L., Qi, X.L., Gao, H.L., Wang, Y.Q., and Wang, X.M. 2013. Antigenic analysis of monoclonal antibodies against different epitopes of σB protein of avian reovirus. PLoS One 8: e81533. Yin, S., Shang, Y., Zhou, G., Tian, H., Liu, Y., Cai, X., and Liu, X. 2010. Development and evaluation of rapid detection of classical swine fever virus by reverse transcription loop-mediated isothermal amplification (RT-LAMP). J. Biotechnol. 146, 147-150. Yoon, J.B., Murphy, S., Bai, L., Wang, Z., and Roeder, R.G., 1995. Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes. Mol. Cell. Biol. 15, 2019-2027. Yu, H., Isken, O., Grassmann, C.W., and Behrens, S.E. 2000. A stem-loop motif formed by the immediate 5' terminus of the bovine viral diarrhea virus genome modulates translation as well as replication of the viral RNA. J. Virol. 74, 5825-5835. Yue, T., Goldstein, I.J., Hollingsworth, M.A., Kaul, K., Brand, R.E. and Haab, B.B. 2009. The prevalence and nature of glycan alterations on specific proteins in pancreatic cancer patients revealed using antibody-lectin sandwich arrays. Mol. Cell Proteomics 8, 1697-1707.
摘要: Classical swine fever virus (CSFV) belongs to the genus Pestivirus of the Flaviviridae family. CSFV infection causes a highly contagious and severe disease resulting in large economic losses in the pig industry worldwide. Systematic vaccination and non-vaccination stamping-out policy are the two main strategies to control CSF. Recently, CSF E2 subunit marker vaccines and companion enzyme-linked immunosorbent assays (ELISAs) detecting the antibody to Erns have been considered a discriminating strategy for differentiating infected from vaccinated animals (DIVA). The purpose of this study was to produce specific monoclonal antibodies (MAbs) to Erns and E2 for further developing as diagnostic ELISA kits. The MAb CW813 was shown to specifically recognize Pichia pastoris yeast-expressed Erns (yErns), and the MAb CW813 and yErns based indirect sandwich ELISA showed the sensitivity of 96.6% (28/29) and specificity of 100% (25/25) that may provide an alternative method for developing a discriminating diagnostic kit. In addition, the MAb 1B6 was specific to yeast-expressed E2 (yE2), and panels of swine sera were tested by peroxidase-conjugated MAb 1B6-based blocking ELISA using yE2 N330 subunit as coated antigen. The assay demonstrated the sensitivity of 100% (35/35) and specificity of 100% (17/17) that may provide the routine serodiagnosis to swine antibody against CSFV E2. Further comparison of the MAb-1B6-based blocking ELISA with other commercial ELISA kits using a great amounts of field swine sera showed the sensitivity of 89.1% (49/55) and specificity of 93.9% (31/33) for the IDEXX E2 blocking ELISA identified sera, and the sensitivity of 84.9% (140/165) and specificity of 95.1% (117/123) for the BioChek E2 indirect ELISA determined samples. Moreover, expression of short hairpin RNAs (shRNAs) by the RNA polymerase type III U6 promoter is an effective and widely used strategy for RNA interference (RNAi) which has become a powerful biological tool for studying gene function and fighting viral disease by targeting the mRNA of viral genes. In this study, the swine U6 promoter driven shRNA expression system was constructed, and the transcription efficiency of swine U6 was analyzed to compare with its human or mouse homologue. The swine U6 promoter is stronger than the human or mouse homologue and can efficiently mediate RNA interference in these three different species of mammalian cell lines. The study result also revealed that the shRNA expressed by the swine U6 promoter could mediate efficient RNAi in reducing CSFV replication by 90.7%. The shRNA expression vector driven by the swine U6 promoter might be widely used for vector-based RNAi-induced gene silencing, especially to inhibit replication of other important swine virus. Taken together, the monoclonal antibodies specific to CSFV glycoproteins and developed ELISA methods for discriminating serodiagnosis as well as the anti-CSFV shRNA expression driven by swine U6 promoter may provide useful diagnostic reagents and strategies for studies and controls of CSFV with easy manipulation and low coat.
豬瘟病毒(classical swine fever virus, CSFV)屬於黃病毒科(Flaviviridae)之瘟疫病毒屬(Pestivirus),豬隻感染豬瘟病毒可引發高度傳染性及嚴重疾病導致養豬業極大的經濟損失。而目前對於豬瘟病毒之防治策略包括施打豬瘟減毒疫苗或全面撲殺受病毒感染豬隻。現今亦已發展使用豬瘟病毒醣蛋白E2標識次單位疫苗(E2 subunit marker vaccine)並利用偵測病毒醣蛋白Erns抗體的酵素連結免疫吸附分析法(enzyme-linked immunosorbent assay, ELISA)而建立一個能區別診斷受野外病毒感染與疫苗接種之動物(differentiate infected from vaccinated animals, DIVA),可針對受野外病毒感染的豬隻進行清除的防治策略。本研究之目的為製備出能辨識豬瘟病毒醣蛋白Erns與E2之特異性單株抗體並將其應用於ELISA之開發。單株抗體CW813能夠辨識以酵母菌表現之Erns抗原且以此抗體所建立之間接三明治型ELISA檢測豬隻血清顯示良好的敏感性(96.6%)與特異性(100%),可發展做為抗體區別診斷套組。而單株抗體1B6可特異性辨識酵母菌表現之E2醣蛋白,且以此抗體連結過氧化酵素所建立之阻斷型ELISA檢測豬隻血清顯示其敏感性與特異性皆為100%,可做為例行豬瘟E2抗體檢測之血清學診斷方法。進一步以此E2 blocking ELISA與市售商業套組進行野外豬隻血清之測定比較,依照市售阻斷型ELISA (IDEXX)測定之結果於本方法所測得之敏感性為89.1%且特異性為93.9%;而以市售間接型ELISA (BioChek)測定之結果於本方法所測得之敏感性為84.9%且特異性為95.1%。此外,利用RNA polymerase III的U6啟動子所誘導shRNA (short hairpin RNA)之表現系統已成為有效且廣泛用於表現干擾性核醣核酸(RNA interference, RNAi)的方式,並可做為基因功能或抗病毒的研究工具。本研究另外建立了豬U6啟動子誘導shRNA之表現系統並進行特性分析與抗病毒研究。結果顯示,以豬U6啟動子誘導shRNA表現之能力優於人或小鼠U6啟動子且將其應用於抑制豬瘟病毒複製之實驗結果亦顯示良好的抑制效果。本研究不僅製備了豬瘟病毒醣蛋白Erns與E2之特異性單株抗體進而建立了ELISA抗體區別檢測方法,亦建立了豬U6啟動子誘導shRNA之表現系統,這些研究成果將可提供對於豬瘟病毒之良好研究工具與診斷試劑,可望有助於豬瘟防疫。
URI: http://hdl.handle.net/11455/93093
文章公開時間: 2017-01-29
Appears in Collections:微生物暨公共衛生學研究所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.