Objective: To investigate the methodology of cultivation of the new rotavirus that causes adult diarrhea.
Methods: 10% suspension of new rotavirus positive stool specimens in DMEM with 100 microgram/ml trypsin was made and centrifuged at the speed of 3 000 rpm for 10 minutes. The supernatant was filtered with 0.45 micrometer-pore-sized membrane filter, and the filtrate was incubated at 37 degrees C for 60 minutes. Primary human embryo kidney (PHEK) cells were prepared and seeded into roller tubes at the concentration of 1 ml odf cell suspension per tube. At 1 hour prior to inoculation, the PHEK cells were rinsed and refed with serum-free DMEM. Immediately before inoculation, the DMEM was decanted, and 200 microliter of prepared filtrate was inoculated into each tube. The tubes were incubated at 37 degrees C for 1 hour. At the end of 1 hour 800 microliter of DMEM containing trypsin (100 microgram per ml) were added to each tube. The tubes were placed in a roller tube apparatus at 37 degrees C for 3 days, removed and frozen-thawed once. 200 microliter 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.