CVB3病毒RNA拯救方法的建立、评价与应用

发布时间：2018-03-04 02:16

本文选题：病毒拯救　切入点：CVB3　出处：《中国疾病预防控制中心》2017年硕士论文　论文类型：学位论文

【摘要】：CVB3病毒属于小RNA病毒科肠道病毒属,是引起病毒性心肌炎的重要病原体。其基因组为单股正链RNA。基因组含有5'和3'非编码区(UTRs),1个开放阅读框。开放阅读框编码一条约250kDa的多聚蛋白。该多聚蛋白进一步被水解为4个结构蛋白(VP1-VP4)和7个非结构蛋白(2A、2B、2C,3A、3B、3C和3D)。单股正链RNA病毒的RNA具有自我复制特点,因此,通过单链RNA拯救病毒可能是一种病毒分离培养方法的重要补充。我们以CVB3病毒为模式病毒,成功建立了 RNA拯救病毒的方法,摸索出RNA拯救CVB3病毒的最佳条件:确定小RNA病毒核酸的最适提取试剂、最适转染试剂及最适转染剂量。利用建立的拯救病毒条件,开展了小RNA病毒科病毒的CVB3、EV71、CA16、ECH03、HRV16、HRV86和EMCV等7种病毒的RNA拯救实验,并成功拯救出7种病毒,再次验证了建立的小RNA病毒的RNA拯救病毒的方法可行性。为了提高RNA拯救病毒的效率,我们使用7.15×105PFU/ml滴度的CVB3病毒提取RNA时,增加RNA纯化洗脱次数,通过3次洗脱,每次拯救的病毒噬斑数分别为150±15、11.67±2.08、1.67±0.58,结果提示通过多次洗脱可提高拯救病毒量。为了进一步提高病毒的拯救效率,我们选择7.15×105PFU,7.15×104PFU和7.15×103PFU3种滴度的CVB3病毒,提取RNA,分别转染HeLa细胞、293T细胞和Vero细胞等3种细胞,比较RNA拯救CVB3病毒效率。转染7.15× 105 PFU CVB3病毒的RNA 24h后,293T细胞发生100%病变,而HeLa细胞和Vero细胞未出现明显病变;转染48h后,HeLa细胞和Vero细胞出现少许病变(20%CPE);转染72h,HeLa细胞和Vero细胞均全部病变。转染7.15×104PFUCVB3病毒的RNA 48h后,293T细胞100%出现病变,而HeLa细胞和Vero细胞未出现明显的病变;转染72h后,HeLa细胞100%出现病变,Vero细胞50%出现病变。转染7.15×103PFU CVB3病毒的RNA 48h后,293T细胞100%出现病变,而转染72h后,HeLa细胞25%发生病变,Vero细胞未出现病变。使用293T细胞进行RNA转染拯救病毒,拯救的病毒再接种HeLa细胞产生的毒力最高。结果表明病毒的RNA在293T细胞内具有更好的复制能力。RNA通过293T细胞拯救CVB3病毒的效率较HeLa细胞、Vero细胞高,同时,分离病毒时间最短。在此基础上,为了克服不同病毒需要特定的敏感细胞的限制,我们建立了基于293T细胞的一种通用小RNA病毒拯救方法,成功实现CVB3、EV71、CA16、ECHO3、HRV16、HRV86 和 EMCV 等 7 种病毒的 RNA 拯救。为了克服心脏、胰腺组织在病毒分离过程中产生的细胞毒性,进一步了解组织样本对CVB3病毒拯救方法的影响,同时,评价RNA拯救方法在CVB3病毒感染的心肌炎模型中病毒的复制规律,我们将7.15×103 PFU CVB3病毒通过腹腔接种Balb/c小鼠建立CVB3感染心肌炎模型(6只/组)。在感染的第1天、第3天、第5天、第7天、第9天、第11天、第13天和第15天,取血,分离血清;采集心脏和胰腺组织,一半固定,制备切片进行HE染色,评价病毒引起的心肌和胰腺的病理变化,一半匀浆,提取病毒核酸,进行RNA拯救,评价病毒在小鼠心肌和胰腺的复制规律。HE染色发现CVB3病毒感染诱导了典型的心肌炎变化:在感染的第5天可观察到心肌出现明显的炎症细胞浸润,心肌细胞出现玻璃样变和纤维化;感染的第9天,病理变化达到高峰,随后炎症反应逐步下降,至感染的13天接近正常水平。胰腺组织随着感染时间的延长,病理损伤程度加重,感染第7天达高峰,持续至感染15天。镜下可见间质水肿、腺泡水肿,炎性细胞浸润,随病程的进展导管出现不同程度的扩张。通过RNA拯救方法开展了CVB3病毒在感染的小鼠心肌炎模型中的动态分布特点研究,发现CVB3病毒在感染的早期(感染的3-5天)在心脏、胰腺和血清中的拷贝数急速升高,达到高峰,高峰维持2-3天,随后病毒的复制拷贝数快速下降。病毒在胰腺、心脏和血清中的复制能力各不相同。感染的第5天,小鼠胰腺组织中的病毒滴度达到高峰,为826.6±28.0PFU/mg;感染的第3天,心脏和血清中的病毒滴度达到高峰,分别为24.4±1.9PFU/mg和10953.6±807 PFU/mL。我们的结果提示感染早期的心脏和胰腺组织损伤来自于CVB3病毒的直接损伤,感染后期则为炎症反应导致的损伤。综上所述,我们通过对实验条件优化,建立了 CVB3病毒RNA拯救的方法,并成功应用于7种小RNA病毒。摸索建立了以293T细胞为转染细胞的通用小RNA病毒拯救技术。利用该方法评价了 CVB3病毒感染小鼠心肌炎模型中病毒在心脏、胰腺和血清中的复制规律。
[Abstract]:CVB3 virus belongs to the small RNA virus, enterovirus, is an important pathogen of causing viral myocarditis. Its genome is a single strand RNA. genome containing 5'and 3' non encoding region (UTRs), 1 open reading frames. The open reading frame encoding a 250kDa poly protein. The protein was further poly hydrolysis of 4 structural proteins (VP1-VP4) and 7 non structural proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). Single strand RNA virus RNA with self replication characteristics, therefore, the single stranded RNA virus rescue may be an important supplement of virus isolation and culture method. We the CVB3 virus as a model virus was successfully established RNA method to save the virus, we find out the best condition of RNA CVB3 virus rescue: to determine the optimum RNA virus nucleic acid extraction reagent, the optimum transfection reagent and the optimum transfection dose. The rescued virus condition, carry out small RNA virus A virus in CVB3, EV71, CA16, ECH03, HRV16, HRV86 and EMCV 7 RNA virus rescue experiments, and successfully rescued 7 viruses, again to verify the feasibility of the established method of small RNA virus RNA virus rescue. In order to improve the efficiency of RNA to save the virus, we use 7.15 x 105PFU/ml titer CVB3 virus RNA extraction, RNA increased by 3 times of elution, elution, virus plaque number every rescue were 150 + 15,11.67 + 2.08,1.67 + 0.58. The results suggest that through multiple elution can improve the rescued virus. In order to further improve the virus rescue efficiency, we choose 7.15 * 105PFU, 7.15 * 104PFU and 7.15 103PFU3 titer of CVB3 virus, RNA extraction, HeLa cells were transfected 293T 3 cells and Vero cells, RNA CVB3 virus rescue efficiency. With 7.15 * 105 PFU CVB3 RNA 24h virus, 293T cells occurred in 100% lesions, and He La and Vero cells showed no obvious lesions; 48h after transfection of HeLa cells and Vero cells appeared a few lesions (20%CPE); transfection of 72h, HeLa and Vero cells were all lesions. With 7.15 * 104PFUCVB3 virus RNA 48h, 293T and HeLa cells in 100% lesions, and Vero cells did not appear obvious lesion; 72h after transfection of HeLa cells 100% lesions, Vero cells appeared 50% lesions. With 7.15 * 103PFU CVB3 RNA 48h virus, 293T cells appeared 100% lesions, and 25% 72h after transfection, HeLa cell lesions, Vero cells did not appear lesions. RNA transfected 293T cells using the rescued virus, virus rescue inoculated HeLa cells produced the highest toxicity. The results show that RNA has better efficiency of the virus in 293T cells by.RNA 293T cells replicating CVB3 virus rescue than HeLa cells, Vero cells, at the same time, the time of virus isolation Short. On this basis, in order to overcome the different virus need to be sensitive to cell specific restrictions, we established a general small RNA virus 293T cell rescue method based on the successful implementation of CVB3, EV71, CA16, ECHO3, HRV16, HRV86 and EMCV 7 kinds of viruses to save RNA. In order to overcome the heart, cell toxicity in the process of virus isolation in pancreatic tissue, to further understand the effects of tissue samples of the CVB3 virus rescue method, copy law evaluation of RNA rescue virus in viral myocarditis model of CVB3 virus infection in, we will be 7.15 * 103 PFU CVB3 virus by intraperitoneal inoculation of Balb/c mice myocarditis model of CVB3 infection (6 rats / group). On the first day of infection for third days, fifth days, seventh days, Ninth days, eleventh days, thirteenth days and fifteenth days, blood, serum separation; collecting the heart and pancreas tissue, half fixed sections were prepared for HE staining to evaluate disease Half of the pathological changes, drug induced myocardial and pancreas homogenate extraction, viral nucleic acid, RNA rescue, evaluation of virus.HE replication of staining in the myocardium and CVB3 infected mice pancreas induced myocarditis: typical changes in infected fifth days can be observed in myocardium appeared obvious inflammatory cell infiltration, myocardial cells of glass degeneration and fibrosis; ninth days of infection, pathological changes reached the peak, then gradually decreased to inflammation, infection 13 days close to normal pancreatic tissue. With prolonged infection, pathological injury degree aggravating, infection in seventh Tianda peak, until the infection 15 days. Under the microscope, interstitial edema, alveolar edema and the infiltration of inflammatory cells, with the progress of the course of catheter with varying degrees of expansion. The RNA method was carried out to save the CVB3 virus in mice model of viral myocarditis infection in the dynamic distribution characteristics research The early detection of CVB3 virus infection (infection in 3-5 days) in heart, pancreas and copy number in the serum increased rapidly, and reached the peak, the peak for 2-3 days, then the replication of the virus copy number decreased rapidly. The virus replication in the pancreas, heart and serum in each. The fifth day of infection the virus titer, mouse pancreatic tissue peaked at 826.6 + 28.0PFU/mg; third days of infection, the virus titer in the serum and heart peak were 24.4 + 1.9PFU/mg and 10953.6 + 807 PFU/mL. our results suggest that the infection of direct injury of heart and pancreas tissue injury from CVB3 virus infection, late is the inflammation caused by injury. In summary, we optimized the experimental conditions, to establish a method of CVB3 RNA virus rescue, and successfully applied to 7 kinds of small RNA virus has been established in 293T cells transfected with fine The universal small RNA virus rescue technology is used. The method of virus replication in heart, pancreas and serum of CVB3 virus infected myocarditis model is evaluated by this method.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373

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