SARS冠状病毒感染恒河猴的病毒、血清学检测研究

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。

Studies on virological and serological detection of SARS Animal Model of Rhesus monkeys

Successful development of anti-SARS drugs and vaccines depends greatly on a reliable and sensitive animal model. In April 2003, we firstly inoculated rats, mice, rabbits and guinea-pigs, but no animals were sensitive to SARS-CoV infection. Late in May, We tried to infect 3 kinds of primates, and then Rhesus monkey(Macaca mulata) and Cynomogus Macaques (Macaca fascicularis) were found to be sensitive. Here, we report that a SARS animal model has been successfully established by inoculating SARS coronavirus into Rhesus monkeys through the nasal cavity and bronchi. We have found that although all the 8 animals show light evident clinical signs except for a transient fever 2 to 3 days after inoculation of SARS SARS-CoV, the SARS virus RNA was detected for 10-15 days using nested RT-PCR in pharyngeal swab samples in 8 cases 5 days following inoculation, the SARS coronavirus-specific IgG was detcetbale in the serum of monkeys 11-15 days after inoculation. In addition, necropsy findings of lung tissues showed typical SARS changs. Taken together, the pathological changes, immune response and virus excretion in this SARS animal model may provide insight into the mechanism of SARS infection and may greatly facilitate the screening of anti-SARS drug and vaccine evaluation.