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目的 探讨用细胞培养系统在体外培养新轮状病毒 (RV)。方法 含新RV的病人粪便标本经高浓度胰蛋白酶 (10 0 μg/ml) 37℃作用 1h ,接种于原代人胚肾 (PHEK)细胞 ,37℃吸附 1h后 ,补加无血清含胰蛋白酶 (10 0 μg/ml)的DMEM ,37℃转鼓培养 3d ,收获细胞培养液 ,冻融 1次 ,作为下一代培养的接种物 ,培养传代。用聚丙烯酰胺凝胶电泳、免疫荧光 (IF)、电镜 (EM)、免疫电镜 (IEM)等方法对细胞培养液进行病毒核酸、抗原及形态检查。结果 从 10份粪便标本中培养分离到 1株病毒 ,定名为J19。该病毒在PHEK细胞上稳定培养传代 (2 8代 ) ,其病毒核酸 (dsRNA)电泳图型与初始用于接种细胞的粪便标本中的dsRNA电泳图型完全一致 ,而与A、B、C组RVdsRNA电泳图型明显不同。J19株感染的细胞与病人恢复期血清呈IF阳性反应 ,与成人腹泻轮状病毒 (ADRV)腹泻病人恢复期血清、兔抗A组RV和兔抗ADRV血清均为IF阴性 ;EM和IEM在该病毒的细胞培养液中观察到 ,在形态上与初始接种物中病毒一样的轮状病毒颗粒 ,而且可被新RV腹泻病人恢复期血清凝集。结论 成功地从含新RV的成人腹泻粪便标本中分离培养出 1株轮状病毒———J19,并能在细胞稳定传代。J19株不属于A、B、C组RV ,而是一种新RV。关于新RV在细胞上培养传代 ,本文是第一次报?
Objective To investigate the in vitro culture of new rotavirus (RV) with cell culture system. Methods Stool samples from patients with new RV were inoculated into primary human embryonic kidney (PHEK) cells by high concentration trypsin (100 μg / ml) at 37 ℃ for 1 hour. After adsorption for 1 hour at 37 ℃, serum samples were collected and serum-free trypsin (10 0 μg / ml) in DMEM and cultured for 3 days at 37 ° C on a drum. The cell culture liquid was harvested and freeze-thawed once to serve as an inoculum of the next generation culture, and passaged for culture. The cell culture medium was tested for virus nucleic acid, antigen and morphology by polyacrylamide gel electrophoresis, immunofluorescence (IF), electron microscopy (EM) and immunoelectron microscopy (IEM). Results One strain of virus was isolated from 10 stool samples and named as J19. The virus was stably passaged on PHEK cells (passage 28). The virus nucleic acid (dsRNA) electrophoresis patterns were exactly the same as the dsRNA electrophoresis patterns in the stool specimens initially used for cell inoculation, but not in groups A, B and C RVdsRNA electrophoretic patterns are significantly different. Infected cells of J19 showed IF positive reaction with convalescent sera from patients, and IF convalescent sera from patients with adult diarrhea rotavirus (ADRV) diarrhea, rabbit anti-A and rabbit anti-ADRV sera were IF negative; EM and IEM were Virus rotavirus particles that are morphologically similar to those in the initial inoculum are observed in the cell culture fluid of the virus and may be convalesced by new RV diarrhea patients. Conclusion One strain of rotavirus, J19, was successfully isolated from stool samples of adult diarrhea patients with a new RV and was successfully passaged in cells. J19 strain does not belong to A, B, C RV, but a new RV. About the new RV in the cell culture passage, this article is the first newspaper?