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[目的]对感染SARS-CoV的恒河猴进行病毒、血清学等指标检测及研究,确定模型动物成功感染,并为SARS发病机制,疫苗评价,药物筛选确定参考指标。[方法]SARSCo-V经鼻腔接种8只恒河猴,在感染的第1天开始到5、7、10、15、20、30和60天分别安乐死时,不同时间取咽拭子、血液和脏器,进行病毒分离,RT-PCR检测和抗体测定。[结果]用巢式RT-PCR在感染后每天提取的咽拭子标本中检测SARS-CoV的RNA,以细胞培养冠状病毒为阳性对照,以正常恒河猴咽拭子为阴性对照,在8只动物病毒接种第5天开始可检测到大小为797bp的目的条带,阳性检出最长可持续到第15天。进一步用病毒分离实验对PCR结果进行确证,8只动物中的5只恒河猴接种5天的咽拭子标本中,经Vero细胞培养,细胞产生了典型细胞病变(CPE),提示SARS冠状病毒能感染恒河猴并有病毒的复制和排毒。IFA方法证实为SRAS-CoV抗原存在。SARS-CoV感染恒河猴后,可以检测出免疫反应。在SARS冠状病毒接种前和接种后第5、8、11、15、19、23、26、30、34、每隔4-7天以及安乐死时采血,制备血清测定抗体,8只恒河猴接种病毒前均血清中SARS冠状病毒特异性抗体IgG为阴性,10天后安乐处死的5只感染猴在11-15天开始,至安乐死时,均为阳性。IgG阳性的5只恒河猴均有一定的中和抗体产生,且对SARS病毒感染细胞有一定的保护性。感染SARS病毒猴后与正常猴比较,其细胞杀伤效应明显增强。感染SARS-CoV的恒河猴不仅出现与SARS患者类似的临床和病理学改变,也在一定时期内排毒,出现特异免疫反应,这些指标均可作为药物筛选、疫苗评价等方面的重要参数。
[Objective] To detect and study virus, serology and other indicators of rhesus monkeys infected with SARS-CoV to confirm the successful infection of model animals and to establish the reference index for pathogenesis, vaccine evaluation and drug screening of SARS. [Methods] Eight chickens were inoculated intranasally with SARSCo-V and were euthanized at day 5, 7, 10, 15, 20, 30, and 60, respectively. Organ, virus isolation, RT-PCR detection and antibody determination. [Results] The RNA of SARS-CoV was detected by nested reverse transcription-polymerase chain reaction (RT-PCR) in throat swab samples extracted daily. The positive control of cell culture coronavirus was used as the positive control, The target band of 797 bp in size was detectable only on the fifth day after the animal virus was inoculated, and the positive detection could last up to the fifteenth day. PCR was further confirmed by virus isolation experiments. Five of 8 animals were inoculated with throat swabs for 5 days and cultured in Vero cells. The cells developed typical cytopathic effect (CPE), suggesting that SARS coronavirus Can infect rhesus monkeys and has viral replication and detoxification. The IFA method confirmed the presence of SRAS-CoV antigen. After the infection of rhesus monkeys with SARS-CoV, the immune response can be detected. Serum assayed antibodies were prepared before and 5, 8, 11, 15, 19, 23, 26, 30, 34 and every 4-7 days after inoculation of the SARS coronavirus and euthanasia, and 8 rhesus monkeys Pre-virus serum SARS coronavirus-specific IgG was negative, 10 days after the euthanasia killed 5 infected monkeys in 11-15 days, until euthanasia, were positive. The five IgG positive Rhesus macaques all had some neutralizing antibodies, and had some protection against SARS virus infected cells. Infection with SARS virus monkey compared with the normal monkey, the cell killing effect was significantly enhanced. Rhesus monkeys infected with SARS-CoV not only show clinical and pathological changes similar to SARS patients, but also detoxify and specific immune responses within a certain period of time. These indicators can be used as an important parameter in drug screening and vaccine evaluation.