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研究生:宋心愉
研究生(外文):Hsin-Yu Sung
論文名稱:使用重組病毒偵測G1和G9輪狀病毒中和抗體與螺旋藻抽取液Apogen在動物體抗輪狀病毒活性之研究
論文名稱(外文):Detection of G1 and G9 rotavirus neutralizing antibodies using reassortant viruses and Study of in vivo anti-rotavirus activity of Apogen, an ex-tract of the microalga Spirulina platensis
指導教授:李君男李君男引用關係
指導教授(外文):Chun-Nan Lee
口試委員:高全良張淑媛
口試日期:2013-07-15
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學檢驗暨生物技術學研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:65
中文關鍵詞:重組輪狀病毒G1P[12]中和試驗螺旋藻抽取液ApogenR小鼠實驗抑制輪狀病毒之活性
外文關鍵詞:G1P[12] reassortant virustype-specific neutralizing antibodyneutralization assayspirulina extract ApogenRanti-rotavirus activity
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輪狀病毒是引起全球幼童腹瀉的主要致病原,可藉由兩個能引起中和抗體產生的外層結構性蛋白質VP7和VP4來分型,分別為G 以及P type。感染人類常見的型別為G1~G4及P[4]、P[6]、P[8],除此之外,G9輪狀病毒株已經成為世界上第五大流行的輪狀病毒。從2000~2005年台大醫院輪狀病毒陽性檢體中,G9輪狀病毒所佔比例最多,再加上現行施打的輪狀病毒疫苗並未包括G9輪狀病毒,因此了解G9輪狀病毒在台灣的流行情況有其重要性並對於未來疫苗發展有幫助。透過測定血清特異抗體可了解過去是否感染過特定型別的輪狀病毒,過去本實驗室發現相同血清檢體之G1、G3、G9輪狀病毒中和抗體效價之結果差異不大,因此欲藉由製作重組輪狀病毒的方法排除掉因為帶有相似的VP4而造成中和試驗的交叉反應。在長滿MA104細胞的玻璃管斜面上同時感染輪狀病毒Wa ( G1P[8] )以及FI14 ( G3P[12] ),並在繼代培養過程中加入抗血清anti-YO ( G3P[8] ) 進行篩選,透過RT-PCR分型確認是否篩選出G1P[12] 病毒。重複進行九次溶斑純化試驗 ( plaque purification ) 得到純化的G1P[12]輪狀病毒。最後將G1P[12]以及G9P[12]重組病毒,分別置換過去中和試驗裡使用的Wa ( G1P[8] ) 以及VR010591 ( G9P[8] ),針對1999和2009年6-7歲學童血清檢體再次進行中和試驗。結果顯示,使用G1P[12]以及G9P[12]重組輪狀病毒能夠去除VP4之干擾,進而可區分G1以及G9之中和抗體效價,並顯示G9輪狀病毒中和抗體效價在2009年比1999年來的高。
螺旋藻 ( Spirulina spp .) 又稱為藍藻,因形體為螺旋狀而得名,目前已經有文獻證明螺旋藻萃取成份在細胞或動物實驗中都具有抗病毒的活性。過去本實驗室觀察到螺旋藻抽取液ApogenR在細胞實驗中具有抑制輪狀病毒之活性,因此欲進一步在動物實驗中觀察是否同樣具有抑制輪狀病毒之效果。首先,在6天大BALB/c新生鼠給予cell-adapted EDIM輪狀病毒,透過活體增殖wild-type EDIM輪狀病毒 ( wtEDIM )。接著將wtEDIM輪狀病毒序列稀釋後給予6週大BALB/c成鼠,並在感染後每天蒐集兩顆小鼠糞便以ELISA方式偵測糞便中排出的輪狀病毒抗原,了解能夠造成50% 6週大BALB/c成鼠,從糞便中偵測到輪狀病毒抗原的wtEDIM輪狀病毒劑量 ( 50% shedding dose, SD50 ), SD50的實驗結果約為原wtEDIM培養液的250倍稀釋液。之後以25 SD50的wtEDIM輪狀病毒感染6週大BALB/c成鼠,並從感染前1天到感染後4天每天給予不同濃度的螺旋藻抽取液ApogenR,並同樣以ELISA方式偵測糞便中排出的輪狀病毒抗原,了解能夠讓50% 6週大BALB/c成鼠的糞便病毒排出量有效減少的ApogenR劑量 ( 50% effective concentration, EC50 ),結果顯示EC50約為95 mg/kg/day ApogenR。比較ApogenR實驗組與病毒控制組在感染後糞便輪狀病毒排出量之差異程度,發現給予200、100、50 mg/kg/day ApogenR實驗組相較於病毒控制組, p值之結果在感染後的第4到第6天都有明顯差異,並且隨著ApogenR的濃度越高,病毒的排出量隨之降低,可觀察到藥效與劑量成正相關。本研究發現螺旋藻抽取液ApogenR在成鼠體內對於輪狀病毒之感染具有抑制的現象,未來仍需更多研究來證明其對於幼鼠症狀緩解之效果以及其有效成分或抗病毒之作用機轉,才能確定是否可發展為抗輪狀病毒藥物。

Rotavirus is a major pathogen associated with severe gastroenteritis in infants and young children worldwide, and there are neutralization epitopes on both outer capsid proteins, VP7 and VP4, which can be respectively defined into G and P types. G1 ~ G4 and P [4], P [6], P [8] types are most prevalent in human, and recently G9 rotavirus has become the fifth popular rotavirus in the world. From 2000 to 2005 rotavirus-positive specimens in National Taiwan University Hospital, G9 rotavirus was noticed with high proportion. Since current rotavirus vaccines do not include G9 strain, to understand the G9 rotavirus epidemic situation in Taiwan is helpful for vaccine development in the fu-ture. Patients infected with specific rotavirus serotype in the past can be monitored by serotype-specific antibody, which is usually determined by neutralization assay. Previ-ously our laboratory found that the neutralizing antibodies detected with G1, G3, and G9 rotavirus were similar in titers, it was possibled due to the cross-reactivity induced by VP4 epitopes.
In order to generate G1P[12], Wa ( G1P[8] ) and FI14 ( G3P[12] ) were conjugated in the same glass tube with MA104 cell, and anti-YO ( G3P[8] ) serum was added in the maintenance medium. The G and P types of reassortant rotavirus were analyzed by RT-PCR. After plaque purification method nine times , pure progeny of G1P[12] reas-sortant virus were generated by adding anti-YO (G3P[8]) serum in the agarose overlay. For the same serum specimens in 1999 and 2009, use G1[12] and G9P[12] reassortant rotaviruses to do neutralization assay. The results show significant differences compare with the neutralization titers which were detected with G9P[12] reassortant rotavirus in 1999 and 2009, and the titers in 2009 are higher than in 1999.
Spirulina platensis is also known as blue-green algae. Early as the thirteenth century, there had been record on human consumption of Spirulina platensis as a food source. Previously, studies have shown that the extrac of Spirulina platensis have multiple antiviral functions in cell or animal experiments. The spirulina extract ApogenR used in this study was developed by FEBICO. Our laboratory had previously observed that spirulina extract Apogen R had inhibition activities in cell experiments of rotavirus in-fection. In this study, we want to observe whether the effects of inhibiting rotavirus will also be observed in animal experiments.
Cell-adapted EDIM rotavirus was orally inoculated to 6-day-old newborn mice to enrich wild-type EDIM rotaivurs ( wtEDIM ) . Ten-fold serially diluted wtEDIM was inoculated to 6-week-old mice in order to understand the 50% shedding dose ( SD50 ) of wtEDIM. The SD50 of wtEDIM was the 250-fold diluted viral suspension. In oder to understand the 50% effective dose ( EC50 ) of ApogenR, 25 SD50 wtEDIM was orally inoculated to 6-week-old mice, and also feeding different concentrations of ApogenR were also fed to 3 groups of mice from day -1 to day 4 post infection. The EC50 of Ap-ogenR was calculated as 95 mg/kg/day.
Comparing the amounts of rotavirus antigen detected in the feces of ApogenR test groups and virus control, we found that groups inoculated with 200, 100, and 50 mg/kg/day ApogenR showed significant shedding decrease in feces from day 4 to day 6 post infection. The concentration of ApogenR was positively correlated with the inhibi-tion activities of rotavirus infection.
In this study, we found that the spirulina extract ApogenR had anti-rotavirus activi-ties in adult mice. More research is still needed in the future to prove the anti-rotavirus activities of ApogenR in neonatal mice, the main effective component of ApogenR and its mechanism, then it would be able to confirm if ApogenR could be developed into an-ti-rotavirus drugs.

第一章 緒論 1
1.1. 輪狀病毒 1
1.1.1. 病毒結構 1
1.1.2. 疫苗發展 2
1.1.3. G9輪狀病毒 3
1.2. 螺旋藻Splirulina Plantensis 4
1.2.1. 螺旋藻Splirulina Plantensis抗病毒活性 4
1.2.2. 遠東生技ApogenR 4
1.2.3. 小鼠輪狀病毒EDIM 5
1.3. 研究目的及實驗設計 5
第二章 材料與方法 7
2.1. 重組輪狀病毒 7
2.1.1. MA104細胞繼代培養 7
2.1.2. 輪狀病毒株 7
2.1.3. 病毒培養 7
2.1.4. 螢光抗體染色 ( fluorescent focus assay ) 8
2.1.5. 輪狀病毒共同感染( co-infection )與抗體篩選 8
2.1.6. 溶斑純化試驗 ( plaque purification ) 9
2.1.7. 萃取病毒核酸 10
2.1.8. VP7以及VP4基因RT-PCR 10
2.1.9. 以PCR區分VP7以及VP4基因型 11
2.1.10. 血清檢體 12
2.1.11. 中和抗體測定 ( NELISA , ELSIA-based antigen reduction neutralization assay ) 12
2.2. 螺旋藻抽取液ApogenR抑制輪狀病毒活性之動物實驗 13
2.2.1. 小鼠品系 13
2.2.2. 增殖cell-adapted EDIM 病毒 13
2.2.3. 增殖wild-type ( wt ) EDIM 病毒 14
2.2.4. 螺旋藻萃取物ApogenR 15
2.2.5. 純化cell-adapted EDIM 病毒 15
2.2.6. 50% shedding dose與初步測試ApogenR抑制輪狀病毒之活性 17
2.2.7. 測定ApogenR之50% effective concentration 18
2.2.8. 偵測糞便中輪狀病毒抗原- ELISA 18
3.1. G1P[12]重組輪狀病毒之製備 20
3.1.1. Wa與FI14輪狀病毒株以螢光抗體染色定量 20
3.1.2. Wa與FI14輪狀病毒株共同感染 ( co-infection ) 20
3.1.3. 重組病毒溶斑純化試驗 ( Plaque purification ) 20
3.2. 1999年以及2009年6-7歲學童血清之NELISA結果 21
3.3. 螺旋藻抽取液ApogenR在動物體抑制輪狀病毒之活性 22
3.3.1. 增殖cell-adapted EDIM輪狀病毒 22
3.3.2. 在新生幼鼠增殖wtEDIM輪狀病毒 22
3.3.3. 濃縮cell-adapted EDIM輪狀病毒 23
3.3.4. 測定wtEDIM輪狀病毒的50% shedding dose ( SD50 ) 23
3.3.5. 測定ApogenR的50% effective concentration ( EC50 ) 24
第四章 討論 29
4.1. 使用重組輪狀病毒偵測G1和G9輪狀病毒中和抗體 29
4.2. ApogenR在動物體內抑制輪狀病毒之活性 33
參考文獻 62

1.Kawai, K., et al., Burden of rotavirus gastroenteritis and distribution of rotavirus strains in Asia: a systematic review. Vaccine, 2012. 30(7): p. 1244-54.
2.Parashar, U.D., et al., Global illness and deaths caused by rotavirus disease in children. Emerg Infect Dis, 2003. 9(5): p. 565-72.
3.Molbak, K., T.K. Fischer, and C.S. Mikkelsen, The estimation of mortality due to rotavirus infections in sub-Saharan Africa. Vaccine, 2000. 19(4-5): p. 393-5.
4.Lu, C.Y., et al., Disease burden and related medical costs of rotavirus infections in Taiwan. BMC Infect Dis, 2006. 6: p. 176.
5.Vesikari, T., et al., Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine. N Engl J Med, 2006. 354(1): p. 23-33.
6.Angel, J., M.A. Franco, and H.B. Greenberg, Rotavirus vaccines: recent developments and future considerations. Nat Rev Microbiol, 2007. 5(7): p. 529-39.
7.Greenberg, H.B. and M.K. Estes, Rotaviruses: from pathogenesis to vaccination. Gastroenterology, 2009. 136(6): p. 1939-51.
8.Khin Maung, U., et al., Comparison of glucose/electrolyte and maltodextrin/glycine/glycyl-glycine/electrolyte oral rehydration solutions in acute diarrhea in children. J Pediatr Gastroenterol Nutr, 1991. 13(4): p. 397-401.
9.Mukherjee, A. and M. Chawla-Sarkar, Rotavirus infection: a perspective on epidemiology, genomic diversity and vaccine strategies. Indian J Virol, 2011. 22(1): p. 11-23.
10.Pesavento, J.B., et al., Rotavirus proteins: structure and assembly. Curr Top Microbiol Immunol, 2006. 309: p. 189-219.
11.Feng, N., et al., Role of interferon in homologous and heterologous rotavirus infection in the intestines and extraintestinal organs of suckling mice. J Virol, 2008. 82(15): p. 7578-90.
12.Estes, M.K. and A.Z. Kapikian, Rotaviruses in Fields Virology, 2007: p. 1917-1974.
13.Marthaler, D., et al., Detection of substantial porcine group B rotavirus genetic diversity in the United States, resulting in a modified classification proposal for G genotypes. Virology, 2012. 433(1): p. 85-96.
14.Papp, H., et al., Review of group A rotavirus strains reported in swine and cattle. Vet Microbiol, 2013.
15.Gentsch, J.R., et al., Serotype diversity and reassortment between human and animal rotavirus strains: implications for rotavirus vaccine programs. J Infect Dis, 2005. 192 Suppl 1: p. S146-59.
16.Patton, J.T., Rotavirus diversity and evolution in the post-vaccine world. Discov Med, 2012. 13(68): p. 85-97.
17.Gentsch, J.R., et al., Identification of group A rotavirus gene 4 types by polymerase chain reaction. J Clin Microbiol, 1992. 30(6): p. 1365-73.
18.Gouvea, V., et al., Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol, 1990. 28(2): p. 276-82.
19.Ianiro, G., et al., Genetic diversity of G9P[8] rotavirus strains circulating in Italy in 2007 and 2010 as determined by whole genome sequencing. Infect Genet Evol, 2013. 16C: p. 426-432.
20.Matthijnssens, J., et al., Uniformity of rotavirus strain nomenclature proposed by the Rotavirus Classification Working Group (RCWG). Arch Virol, 2011. 156(8): p. 1397-413.
21.Payne, D.C., et al., Secular variation in United States rotavirus disease rates and serotypes: implications for assessing the rotavirus vaccination program. Pediatr Infect Dis J, 2009. 28(11): p. 948-53.
22.Taniguchi, K. and S. Urasawa, Diversity in rotavirus genomes. Seminars in Virology, 1995. 6(2): p. 123-131.
23.Postmarketing monitoring of intussusception after RotaTeq vaccination--United States, February 1, 2006-February 15, 2007. MMWR Morb Mortal Wkly Rep, 2007. 56(10): p. 218-22.
24.Gentsch, J.R., et al., Review of G and P typing results from a global collection of rotavirus strains: implications for vaccine development. J Infect Dis, 1996. 174 Suppl 1: p. S30-6.
25.Santos, N. and Y. Hoshino, Global distribution of rotavirus serotypes/genotypes and its implication for the development and implementation of an effective rotavirus vaccine. Rev Med Virol, 2005. 15(1): p. 29-56.
26.Rotavirus surveillance--worldwide, 2001-2008. MMWR Morb Mortal Wkly Rep, 2008. 57(46): p. 1255-7.
27.Lu, Y.M., W.Z. Xiang, and Y.H. Wen, Spirulina (Arthrospira) industry in Inner Mongolia of China: current status and prospects. J Appl Phycol, 2011. 23(2): p. 265-269.
28.Ciferri, O., Spirulina, the edible microorganism. Microbiol Rev, 1983. 47(4): p. 551-78.
29.Deng, R. and T.J. Chow, Hypolipidemic, antioxidant, and antiinflammatory activities of microalgae Spirulina. Cardiovasc Ther, 2010. 28(4): p. e33-45.
30.Skjanes, K., C. Rebours, and P. Lindblad, Potential for green microalgae to produce hydrogen, pharmaceuticals and other high value products in a combined process. Crit Rev Biotechnol, 2013. 33(2): p. 172-215.
31.Hayashi, K., T. Hayashi, and I. Kojima, A natural sulfated polysaccharide, calcium spirulan, isolated from Spirulina platensis: in vitro and ex vivo evaluation of anti-herpes simplex virus and anti-human immunodeficiency virus activities. AIDS Res Hum Retroviruses, 1996. 12(15): p. 1463-71.
32.Hayakawa, Y., et al., Calcium spirulan as an inducer of tissue-type plasminogen activator in human fetal lung fibroblasts. Biochim Biophys Acta, 1997. 1355(3): p. 241-7.
33.Lee, J.B., et al., Effects of structural modification of calcium spirulan, a sulfated polysaccharide from Spirulina platensis, on antiviral activity. Chem Pharm Bull (Tokyo), 2001. 49(1): p. 108-10.
34.Shih, S.R., et al., Inhibition of enterovirus 71-induced apoptosis by allophycocyanin isolated from a blue-green alga Spirulina platensis. J Med Virol, 2003. 70(1): p. 119-25.
35.Kraft, L.M., Observations on the control and natural history of epidemic diarrhea of infant mice (EDIM). Yale J Biol Med, 1958. 31(3): p. 121-37.
36.Sheridan, J.F., et al., Virus-specific immunity in neonatal and adult mouse rotavirus infection. Infect Immun, 1983. 39(2): p. 917-27.
37.Kraft, L.M., Studies on the etiology and transmission of epidemic diarrhea of infant mice. J Exp Med, 1957. 106(5): p. 743-55.
38.Wilsnack, R.E., J.H. Blackwell, and J.C. Parker, Identification of an agent of epizootic diarrhea of infant mice by immunofluorescent and complement-fixation tests. Am J Vet Res, 1969. 30(7): p. 1195-204.
39.Menchaca, G., et al., Serotype specificity of the neutralizing-antibody response induced by the individual surface proteins of rotavirus in natural infections of young children. Clin Diagn Lab Immunol, 1998. 5(3): p. 328-34.
40.Lai, H.C., et al., Phylogenetic analyses of human rotavirus in central Taiwan in 1996, 2001 and 2002. J Clin Virol, 2005. 32(3): p. 199-217.
41.Sung, Y.L., et al., Emergence of G9 serotype rotavirus as a major cause of infectious gastroenteritis in southern Taiwan. J Microbiol Immunol Infect, 2004. 37(6): p. 322-6.
42.Larralde, G., et al., Serotype-specific epitope(s) present on the VP8 subunit of rotavirus VP4 protein. J Virol, 1991. 65(6): p. 3213-8.
43.Gorrell, R.J. and R.F. Bishop, Production of reassortant viruses containing human rotavirus VP4 and SA11 VP7 for measuring neutralizing antibody following natural infection. Clin Diagn Lab Immunol, 1997. 4(5): p. 509-14.
44.Troupin, C., et al., Rearranged genomic RNA segments offer a new approach to the reverse genetics of rotaviruses. J Virol, 2010. 84(13): p. 6711-9.
45.Komoto, S., et al., Generation of recombinant rotavirus with an antigenic mosaic of cross-reactive neutralization epitopes on VP4. J Virol, 2008. 82(13): p. 6753-7.
46.Ward, R.L., M.M. McNeal, and J.F. Sheridan, Development of an adult mouse model for studies on protection against rotavirus. J Virol, 1990. 64(10): p. 5070-5.
47.Reimerink, J.H., et al., Systemic immune response after rotavirus inoculation of neonatal mice depends on source and level of purification of the virus: implications for the use of heterologous vaccine candidates. J Gen Virol, 2007. 88(Pt 2): p. 604-12.
48.Dector, M.A., et al., Rotavirus gene silencing by small interfering RNAs. EMBO Rep, 2002. 3(12): p. 1175-80.
49.Tam, K.I. and M.R. Roner, Characterization of in vivo anti-rotavirus activities of saponin extracts from Quillaja saponaria Molina. Antiviral Res, 2011. 90(3): p. 231-41.
50.Takahashi, K., et al., Protective efficacy of a sulfated sialyl lipid (NMSO3) against human rotavirus-induced diarrhea in a mouse model. Antimicrob Agents Chemother, 2002. 46(2): p. 420-4.
51.Wu, C.R., et al., Effects of QWBZP on T-cell subsets and their cytokines in intestinal mucosa of HRV infection suckling mice. J Ethnopharmacol, 2010. 131(1): p. 130-4.

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