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  • 學位論文

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

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

摘要


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。

並列摘要


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.

參考文獻


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