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《National Medical Journal of China》 2002-01
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Cultivation and serial propagation of a new rotavirus causing adult diarrhea in primary human embryo kidnay cells

JI Shaozhong, BI Ye, YANG Hongyan, YANG Fengrong, SONG Junqi, TAO Xiaoxia, CUI Xiaoying Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Beijing 102206, China  
Objective To investigate the mothodology of cultivation of the new rotavirus that causes adult diarrhea. Methods 10% suspension of new rotavitrus positive stool specimens in DMEM with 100 μg/ml trypsin was made and centrifuged at the speed of 3 000 rpm for 10 minutes. The supernatant was iiltered with 0.45 μm pore sized membrane filter, and the filtrate was incubated at 37℃ for 60 inutes. Primary human embryo kidnay (PHEK) cells were prepared and seeded into roler thubes at the concentration of 1 ml odf cell suspension per tube. At 1 hour prior to inoculation, the PHEK cells were rinsed and reefed with serum free DMEM Immediately before inoculation, the DMEM was decanted, and 200 μl of prepared filtrate was inoculated into each tube The tubes were incubated at 37℃ for 1 hour At the end of 1 hour 800 μl of DMEM containing trypsin (100 μg per ml) were added to each tube The tubes were placed in a roller tube apparatus at 37℃ for 3 days, removed and frozen thawed once 200 μl of the cell culture fluids (CCF) were used for inoculation of each tube for next virus passage Detection of new rotavirus from CCF was carried out by PAGE, IF, EM and IEM Results New rotavirus was successfully isolated in cultured cells from 1 of 10 specimens The isolated virus, designated J19, was propagated in PHEK cells for 28 passages RNA patterns of J19 strain were identical to that of original new rotavirus inoculum and different from that of group A,B,C rotavirus PHEK cells infected with the J19 strain were detected by IF Infected cells reacted with convalescent antisera to the new rotavirus, but did not react with convalescent antisera to ADRV and hyperimmune antisera against group A,B rotavirus Rotavirus particles were detected by EM, IEM in infected CCF of J19 strain Other viral particles were not observed The particles were aggregated with convalescent antisera to new rotavirus Conclusion We were successful in adapting a new rotavirus to serial propagation in PHEK cells The J19 strain is a kind of new rotavirus different from group A,B,C rotavirus This is the first report on the cultivation and propagation of the new rotavirus in cultured cells.
【Fund】: 国家自然科学基金资助项目 (3 9870 679)
【CateGory Index】: R373.2
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