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標題: 假性狂犬病毒UL54蛋白單株抗體之特性分析
Characterization of Monoclonal Antibody against the pseudorabies virus UL54 Protein
作者: 鄧福慧
Teng, Hok-hui
關鍵字: pseudorabies virus;假性狂犬病毒;UL54;monoclonal antibody;nucleocytoplasmic shuttling;UL54;單株抗體;核質穿梭
出版社: 獸醫微生物學研究所
引用: 吳金英。2003。假性狂犬病毒早期調節基因UL54之選殖與表現及功能分析。國立中興大學獸醫微生物學研究所碩士論文。 陳麗芬。2006。假性狂犬病毒早期蛋白UL54之核停留及輸出訊號的定位。國立中興大學生命科學系學士論文。 黃雅如。2004。假性狂犬病毒早期蛋白UL54 之功能區分析。國立中興大學獸醫微生物學研究所碩士論文。 Ackermann, M., D. K. Braun, L. Pereira, and B. Roizman. 1984. Characterization of herpes simplex virus type 1 a proteins 0, 4, and 27 with monoclonal antibodies. J. Virol. 52:108–118. Alber G., U. M. Kent, and H. Metzger. 1992. Functional comparison of Fc epsilon RI, Fc gamma RII, and Fc gamma RIII in mast cells. J. Immunol. 149:2428–36. Baumeister, J., B. G. Klupp, and T. C. Mettenleiter. 1995. Pseudorabies virus and equine herpesvirus 1 share a nonessential gene which is absent in other herpesviruses and located adjacent to a highly conserved gene cluster. J. Virol. 69:5560–5567. Bogerd, H., R. Fridell, R. Benson, J. Hua, and B. Cullen. 1996. Protein sequence requirements for function of the human T–cell leukemia virus type 1 Rex nuclear export signal delineated by a novel in vivo randomization–selection assay. Mol. Cell. Biol. 16:4207–4214. Brewer, J. M., M. Conacher, A. Satoskar, H. Bluethmann, and J. Alexander. 1996. In interleukin–4–deficient mice, alum not only generates T helper 1 responses equivalent to Freund’s complete adjuvant, but continues to induce T helper 2 cytokine production. Eur. J. Immunol. 26:2062–2066. Brideau, A. D., B. W. Banfield, and L. W. Enquist. 1998. The Us9 gene product of pseudorabies virus, an alphaherpesvirus, is a phosphorylated, tail–anchored type II membrane protein. J. Virol. 72:4560–4570. Chang, Y. Y., H. W. Lin, M. L. Wong, and T. J. Chang. 2004. Regulation of the vhs gene promoter of pseudorabies virus by IE180 and EP0, and the requirement of a Sp1 Site for the promoter function. Virus Genes 28:247–258. Cheung, P., K. S. Ellison, R. Verity, and J. R. Smiley. 2000. Herpes simplex virus ICP27 induces cytoplasmic accumulation of unspliced polyadenylated alpha–globin pre–mRNA in infected HeLa cells. J. Virol. 74:2913–2919. Coffman, R. L., B. W. Seymour, D. A. Lebman, D. D. Hiraki, J. A. Christiansen, B. Shrader, H. M. Cherwinski, H. F. Salvelkoul, F. D. Finkelman, and M. W. Bond. 1988. The role of helper T cell products in mouse B cell differentiation and isotype regulation. Immunol. Rev. 102:5–28. de StGroth, S. F., and D. Scheidegger. 1980. Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods 35:1–21. Dimaano, C., and K. S. Ullman. 2004. Nucleocytoplasmic transport: integrating mRNA production and turnover with export through the nuclear pore. Mol. Cell. Biol. 24:3069–3076. Dingwall, C, J. Robbins, S. M. Dilworth, B. Roberts and W. D. Richardson. 1988. The nucleoplasmin nuclear location sequence is larger and more complex than that of SV–40 large T antigen. J. Cell Biol. 107:841–849. Dingwall, C. and R. A. Laskey. 1991. Nuclear targeting sequences––a consensus? Trends Biochem. Sci. 16:478–481. Enquist, L. W. 1999. Life beyond eradication: veterinary viruses in basic science. Arch. Virol. Suppl. 15:87–109. Faquim–Mauro, E. L., R. L. Coffman, I. A. Abrahamsohn, and M. S. Macedo. 1999. Cutting edge: mouse IgG1 antibodies comprise two functionally distinct types that are differentially regulated by IL–4 and IL–12. J. Immunol. 163:3572–3576. Faquim–Mauro, E. L., and M. S. Macedo. 2000. Induction of IL–4–dependent, anaphylactic–type and IL–4–independent, non–anaphylactic–type IgG1 antibodies is modulated by adjuvants. Int. Immunol. 12:1733–1740. Fischer, U., J. Huber, W. Boelens, I. Mattaj, and R. Luhrmann. 1995. The HIV–1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82:475–483. Fornerod, M., M. Ohno, M. Yoshida, and I. Mattaj. 1997. CRM1 is an export receptor for leucine–rich nuclear export signals. Cell 90:1051–1060. Fukuda, M., S. Asano, T. Nakamura, M. Adachi, M. Yoshida, M. Yanagida, and E. Nishida. 1997. CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390:308–311. Girard, J. P., H. Lehtonen, M. Caizergues–Ferrer, F. Amalric, D. Tollervey, and B. Lapeyre. 1992. GAR1 is an essential small nucleolar RNP protein required for pre–RNA processing in yeast. EMBO J. 11:673–682. Haasen, D., C. Kohler, G. Neuhaus, and T. Merkle. 1999. Nuclear export of proteins in plants: AtXPO1 is the export receptor for leucine–rich nuclear export signals in Arabidopsis thaliana. Plant J. 20:695–705. Hardwicke, M. A., and R. M. Sandri–Goldin. 1994. The herpes simplex virus regulatory protein ICP27 contributes to the decrease in cellular mRNA levels during infection. J. Virol. 68:4797–4810. Hardy, W. R., and R. M. Sandri–Goldin. 1994. Herpes simplex virus inhibits host cell splicing, and regulatory protein ICP27 is required for this effect. J. Virol. 68:7790–7799. Hazenbos, W. L., J. E. Gessner, F. M. Hofhuis, H. Kuipers, D. Meyer, I. A. Heijnen, R. E. Schmidt, M. Sandor, P. J. Capel, M. Daeron, J. G. van de Winkel, and J. S. Verbeek. 1996. Impaired IgG–dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice. Immunity 5:181–188. Henis,Y. I., Y. Herman–Barhom, B. Aroeti, and O. Gutman. 1989. Lateral mobility of both envelope proteins (F and HN) of Sendai virus in the cell membrane is essential for cell–cell fusion. J. Biol. Chem. 264:17119–17125. Hibbard, M. K., and R. M. Sandri–Goldin. 1995. Arginine–rich regions succeeding the nuclear localization region of the herpes simplex virus regulatory protein ICP27 are required for efficient nuclear localization and late gene expression. J. Virol. 69:4656–4667. Hope, T. J., N. P. Klein, M. E. Elder, and T. G. Parslow. 1992. trans–dominant inhibition of human immunodeficiency cirus type 1 Rev occurs through formation of inactive protein complexes. J. Virol. 66:1849–1855. Huang, C., and C. Y. Wu. 2004. Characterization and expression of the pseudorabies virus early gene UL54. J. Virol. Methods 119:129–136. Huang, Y. J., M. S. Chien, C. Y. Wu, and C. Huang. 2005. Mapping of functional regions conferring nuclear localization and RNA–binding activity of pseudorabies virus early protein UL54. J. Virol. Methods 130:102–107. Ihara, S., L. Feldman, S. Watanabe, and T. Ben–Porat. 1983. Characterization of the immediate–early fuctions of pseudorabies virus. Virology 131:437–454. Jean, S., K. M. LeVan, B. Song, M. Levine, and D. M. Knipe. 2001. Herpes simplex virus 1 ICP27 is required for transcription of two viral late (gamma 2) genes in infected cells. Virology 283:273–284. Kalderon, D., B. L. Roberts, W. D. Richardson, and A. E. Smith. 1984. A short amino acid sequence able to specify nuclear location. Cell 39:499–509. Kaplan, A. S. ,and A. E. Vatter. 1995. A comparison of herpes simplex and pseudorabies virus. Virology 7:394–407. Kiledjian, M., and G. Dreyfuss. 1992. Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box. EMBO J. 11:2655–2664. Klupp, B. G., C. J. Hengartner, T. C. Mettenleiter, and L. W. Enquist. 2004. Complete, annotated sequence of the pseudorabies virus genome. J. Virol. 78:424–440. Klupp, B. G., J. Alterschmidt, H. Granzow, W. Fuchs, and T. C. Mettenleiter. 2005. Identification and characterization of the pseudorabies virus UL43 protein. Virology 334:224–233. Koffa, M. D., J. B. Clements, E. Izaurralde, S. Wadd, S. A. Wilson, I. W. Mattaj, and S. Kuersten. 2001. Herpes simplex virus ICP27 protein provides viral mRNAs with access to the cellular mRNA export pathway. EMBO J. 20:5769–5778. la Cour, T., R. Gupta, K. Rapacki, K. Skriver, F. M. Poulsen, and S. Brunak. 2003. NESbase version 1.0: a database of nuclear export signals. Nucleic Acids Res. 31:393–396. LaCasse, E. C. and Y. A. Lefebvre. 1995. The SWISS–PROT protein sequence database and its supplement TrEMBL in 2000. Nucleic Acids Res. 23:1647–1656. Lanford, R. E., and J. S. Butel. 1984. Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell 37:801–813. Ledford, H. 2008. Monoclonal antibodies come of age. Nature 455:437–437. Lei, E. P., and P. A. Silver. 2002. Protein and RNA export from the nucleus. Dev. Cell 2:261–272. Lengyel, J., C. Guy, V. Leong, S. Borge, and S. A. Rice. 2002. Mapping of functional regions in the amino–terminal portion of the herpes simplex virus ICP27 regulatory protein: importance of the leucine–rich nuclear export signal and RGG box RNA–binding domain. J. Virol. 76:11866–11879. Lengyel, J., A. K. Strain, K. D. Perkins, and S. A. Rice. 2006. ICP27–dependent resistance of herpes simplex virus type 1 to leptomycin B is associated with enhanced nuclear localization of ICP4 and ICP0. Virology 352:368–379. Lindberg, A., and J. P. Kreivi. 2002. Splicing inhibition at the level of spliceosome assembly in the presence of herpes simplex virus protein ICP27. Virology 294:189–198. Martin, K. J., J. W. Lillie, and M. R. Green. 1990. Transcriptional activation by the pseudorabies virus immediate early protein. Genes Dev. 4:2376–2382. Mattaj, I. W. 1993. RNA recognition: a family matter? Cell 73:837–840. McCarthy, A. M., L. McMahan, and P. A. Schaffer. 1989. Herpes simplex virus type 1 ICP27 deletion mutants exhibit altered patterns of transcription and are DNA deficient. J. Virol. 63:18–27. McGregor, F., A. Phelan, J. Dunlop, and J. B. Clements. 1996. Regulation of herpes simplex virus poly(A) site usage and the action of immediate–early protein IE63 in the early–late switch. J. Virol. 70:1931–1940. McMahan, L., and P. A. Schaffer. 1990. The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to Cterminal regions and are required to modulate viral gene expression very early in infection. J. Virol. 64:3471–3485. Mears, W. E., V. Lam, and S. A. Rice. 1995. Identification of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27. J. Virol. 69:935–947. Mears, W. E., and S. A. Rice. 1996. The RGG box motif of the herpes simplex virus ICP27 protein mediates an RNA–binding activity and determines in vivo methylation. J. Virol. 70:7445–7453. Mettenleiter, T. C., L. Zsak, F. Zuckermann, N. Sugg, H. Kern, and T. Ben–Porat. 1990. Interaction of glycoprotein gIII with a cellular heparinlike substance mediates adsorption of pseudorabies virus. J. Virol. 64:278–286. Mettenleiter, T. C. 2000. Aujeszky’s disease (pseudorabies) virus: the virus and molecular pathogenesis–sates of the art, June 1999. Vet. Res. 31:99–115. Mettenleiter, T. C. 2006. Intriguing interplay between viral proteins during herpesvirus assembly or not: The herpesvirus assembly puzzle. Vet. Microbiol. 113:163–169. Meyer, B. E., and M. H. Malim. 1994. The HIV–1 Rev trans–activator shuttles between the nucleus and the cytoplasm. Genes Dev. 8:1538–1547. Minson, A. C., A. J. Davison, R. C. Desrosiers, B. Fleckenstein, D. J. Mc–Geoch, P. E. Pellett, B. Roizman, and D. M. J. Studdert. 2000. Herpesviridae, p. 203–255. In van Regenmortel, M. H., C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle, and R. B. Wickner (ed.), Virus taxonomy. Academic Press, New York, N.Y. Moon, H. B., E. Severinson, C. Heusser, S. G. Johansson, G. Moller, and U. Persson. 1989. Regulation of IgG1 and IgE synthesis by interleukin 4 in mouse B cells. Scand. J. Immunol. 30:355–61. Nair, R., P. Cater, and B. Rost. 2003. NLSdb: database of nuclear localization signals. Nucleic Acids Res. 31:397–399. Nauwynck, H., S. Glorieux, H. Favoreel, and M. Pensaert. 2007. Cell biological and molecular characteristics of pseudorabies virus infections in cell cultures and in pigs with emphasis on the respiratory tract. Vet. Res. 38:229–241. Neville, M., F. Stutz, L. Lee, L. I. Davis, and M. Rosbash. 1997. The importin–beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export. Curr. Biol. 7:767–775. Okazaki, K., T. Matsuzaki, Y. Sugahara, J. Okada, M. Hasebe, Y. Iwamura, M. Ohnishi, T. Kanno, M. Shimizu, and E. Honda. 1991. BHV–1 adsorption is mediated by the interaction of glycoprotein gIII with heparin–like moiety on the cell surface. Virology 181:666–670. Ossareh–Nazari, B., F. Bachelerie, and C. Dargemont. 1997. Evidence for a role of CRM1 in signal–mediated nuclear protein export. Science 278:141–144. Ossareh–Nazari, B., C. Gwizdek, and C. Dargemont. 2001. Protein export from the nucleus. Traffic 2:684–689. Pensaert, M. B., and J. P. Kluge. 1989. “Pseudorabies virus (Aujesky’s disease)” virus infections of porcine. Elsevier Science Publishers, B. V. Amsterdam. Phelan, A., and J. B. Clements. 1997. Herpes simplex virus type 1 immediate early protein IE63 shuttles between nuclear compartments and the cytoplasm. J. Gen. Virol. 78:3327–3331. Pinol–Roma, S., and G. Dreyfuss. 1991. Transcription–dependent and transcription–independent nuclear transport of hnRNP proteins. Science 253:312–314. Pinol–Roma, S., and G. Dreyfuss. 1992. Shuttling of pre–mRNA binding proteins between nucleus and cytoplasm. Nature 355:730–732. Pomeranz, L. E., A. E. Reynolds, and C. J. Hengartner. 2005. Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol. Mol. Biol. Rev. 69:462–500. Reichert, J. M., and V. E. Valge–Archer. 2007. Development trends for monoclonal antibody cancer therapeutics. Nature Rev. Drug Discov. 6:349–356. Rice, S. A., and D. M. Knipe. 1990. Genetic evidence for two distinct transactivation functions of the herpes simplex virus a protein ICP27. J. Virol. 64:1704–1715. Rice, S. A., V. Lam, and D. M. Knipe. 1993. The acidic amino–terminal region of herpes simplex virus type I alpha protein ICP27 is required for an essential lytic function. J. Virol. 67:1778–1787. Rice, S. A., and V. Lam. 1994. Amino acid substitution mutations in the herpes simplex virus ICP27 protein define an essential gene regulation function. J. Virol. 68:823–833. Robbins, J., S. M. Dilworth, R. A. Laskey and C. Dingwall. 1991. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64:615–623. Sacks, W. R., C. C. Greene, D. P. Aschman, and P. A. Schaffer. 1985. Herpes simplex virus type 1 ICP27 is an essential regulatory protein. J. Virol. 55:796–805. Sandri–Goldin, R. M. 1998. ICP27 mediates HSV RNA export by shuttling through a leucine–rich nuclear export signal and binding viral intronless RNAs through an RGG motif. Genes Dev. 12:868–879. Schwartz, J. A., E. E. Brittle, A. E. Reynolds, L. W. Enquist, and S. J. Silverstein. 2006. UL54–null pseudorabies virus is attenuated in mice but productively infects cells in culture. J. Virol. 80:769–784. Sedlackova, L., and S. A. Rice. 2008. Herpes simplex nirus type 1 immediate–early protein ICP27 is required for efficient incorporation of ICP0 and ICP4 into virions. J. Virol. 82:268–277. Shieh, M. T., D. WuDunn, R. I. Montgomery, J. D. Esko, and P. G. Spear. 1992. Cell surface receptors for herpes simplex virus are heparin sulfate proteoglycans. J. Cell Biol. 116:1273–1281. Siomi, H., M. C. Siomi, R. L. Nussbaum, and G. Dreyfuss. 1993. The proteinproduct of the fragile X gene, FMR1, has characteristics of an RNA–binding protein. Cell 74:291–298. Smith, G. A. and L. W. Enquist. 2002. Break ins and break outs: viral interactions with the cytoskeleton of Mammalian cells. Annu. Rev. Cell. Dev. Biol. 18:135–161. Snapper, C. M., F. D. Finkelman, and W. E. Paul. 1988. Differential regulation of IgG1 and IgE synthesis by interleukin 4. J. Exp. Med. 167:183–96. Sobell, H. M. 1985. Actinomycin and DNA transcription. Proc. Natd. Acad. Sci. 82: 5328–5331. Soliman, T. M., R. M. Sandri–Goldin, and S. J. Silversteinb. 1997. Shuttling of the herpes simplex virus type 1 regulatory protein ICP27 between the nucleus and cytoplasm mediates the expression of late proteins. J. Virol. 71:9188–9197. Soliman, T. M., and S. J. Silverstein. 2000. Identification of an export control sequence and a requirement for the KH domain in ICP27 from herpes simplex virus type 1. J. Virol. 74:7600–7609. Spear, P. G., R. J. Eisenberg, and G. H. Cohen. 2000. Three Classes of Cell Surface Receptors for Alphaherpesvirus Entry. Virology 275:1–8. Spencer, C. A., M. E. Dahmus, and S. A. Rice. 1997. Repression of host RNA polymerase II transcription by herpes simplex virus type 1. J. Virol. 71:2031–2040. Stade, K., C. Ford, C. Guthrie, and K. Weis. 1997. Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90:1041–1050. Stevens, T. L., A. Bossie, V. M. Sanders, R. Fernandez–Botran, R. L. Coffman, T. R. Mosmann, and E. S. Vitetta. 1988. Regulation of antibody isotype secretion by subsets of antigen–specific helper T cells. Nature 334:255–258. Taharaguchi, S., E. Ono, S. Yamada, Y. Shimizu, and H. Kida. 1995. Mapping of a functional region conferring nuclear localization of pseudorabies virus immediate early protein. Arch. Virol. 140:1737–1746. Tinland, B., Z. Koukolikova–Nicola, M. N. Hall, and B. Hohn. 1992. The TDNA–linked VirD2 protein contains two distinct functional nuclear localization signals. Proc. Natl Acad. Sci. USA 89:7442–7446. Van Oers, M. H. J., A. A. P. A. M. Van der Heyden, and L. A. Aarden. 1988. Interleukin 6 (IL–6) in serum and urine of renal transplant recipients. Clin. Exp. Immunol. 71:314–319. Venkatesh, L. K., and G. Chinnadurai. 1990. Mutants in a conserved region near the carboxyl–terminus of HIV–1 Rev identify functionally important residues and exhibit a dominant negative phenotype. Virology 178:327–330. Wen,W., J. Meinkoth, R. Tsien, and S. Taylor. 1995. Identification of a signal for rapid export of proteins from the nucleus. Cell 82:463–473. Wittmann, G., and H. J. Rziha. 1989. Aujeszky’s disease (pseudorabies) in pigs, p. 230–325. In G. Wittmann (ed.), Herpesvirus diseases of cattle, horses and pigs. Kluwer Academic Publishers, Boston, Mass. WuDunn, D., and P. G. Spear. 1989. Initial interaction of herpes simplex virus with cells is binding to heparan sulfate. J. Virol. 63:52–58. Yamada, S., and M. Shimizu. 1994. Pseudorabies virus immediate–early regulatory protein IE180 expressed by recombinant baculovirus is functional. Virology 202:491–495. Yu, F. L. 1980. Selective inhibition of rat liver nuclear RNA polymerase II by actinomycin D in vivo. Carcinogenesis 1:577-581.
假性狂犬病毒 (pseudorabies virus, PRV) 是一種引起猪隻重要傳染病的alphaherpesvirus,除了高等靈長類以外大部份的哺乳類動物皆可被感染。PRV早期蛋白UL54是一種核質穿梭蛋白由363個胺基酸所組成且分子量約40 kDa,其蛋白序列上帶有核輸入 (nuclear localization signal, NLS) 以及核輸出訊號 (nuclear export signal, NES) 的功能區。本研究之目的為製備特異性抗PRV UL54單株抗體並進一步利用此單株抗體以監測UL54蛋白在PRV感染期間於感染細胞內分佈與表現的情形。首先利用E. coli來大量表現UL54 重組蛋白並經純化後做為免疫BLAB/c小鼠之抗原以進行融合瘤細胞實驗。本實驗成功製備出一株可特異性辨認PRV UL54 蛋白之單株抗體 (8-8G),進一步針對一系列UL54缺損重組蛋白進行epitope mapping後定出其所辨識抗原決定位 (epitope) 的相關位置乃位於UL54蛋白N端第84到141個胺基酸的區域。此外,利用單株抗體8-8G針對PRV感染的細胞進行間接免疫螢光法 (indirect immunofluorescence assay, IFA) 之結果顯示,隨著感染時間的增加UL54蛋白在細胞質表現後逐漸往細胞核移動並大量累積在細胞核內。但添加了轉錄抑制劑actinomycin D後因使UL54蛋白的核輸入功能受阻導致UL54蛋白逐漸由細胞核移動到細胞質,進一步證實PRV UL54蛋白具有核質穿梭功能。

Pseudorabies virus (PRV) is a porcine alphaherpesvirus that can infect most mammals except for higher-order primates. The PRV UL54 gene encodes a 40 kD protein of 363 amino acids. The early protein UL54 is a nucleo-cytoplasmic shuttling protein with nuclear localization signal (NLS) and nuclear export signal (NES). The purpose of this study was to prepare and characterize the monoclonal antibody against PRV UL54 protein and then to examine the UL54 expression and distribution during PRV infection. The UL54 protein was expressed in E. coli in a large amount and used
as an antigen to immunize BALB/c mice for further hybridoma experiment. One monoclonal antibody (8-8G) was obtained which could specifically recognize the PRV UL54 protein and the epitope recognized by this monoclonal antibody was mapped to the N-terminal amino acid residues 84-141. Furthermore, the transcriptional inhibitor actinomycin D was treated to monitor the intracellular distribution of UL54 protein during PRV infection by indirect immunofluorescence assay (IFA). As infection progressed, UL54 protein localized from the cytoplasm to the nucleus and accumulated abundantly in the nucleus. After the addition of actinomycin D, the UL54 protein moved to the cytoplasm and finally demonstrated more diffuse staining throughout the nucleus and cytoplasm, confirming its shuttling activity.
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