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

• Main Authors: Hsin-Yu Sung, 宋心愉
• Other Authors: Chun-Nan Lee
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/25545124371376540316

碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 101 === 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.