本文選題：病毒拯救　切入點(diǎn)：CVB3　出處：《中國(guó)疾病預(yù)防控制中心》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：CVB3病毒屬于小RNA病毒科腸道病毒屬,是引起病毒性心肌炎的重要病原體。其基因組為單股正鏈RNA�；蚪M含有5'和3'非編碼區(qū)(UTRs),1個(gè)開放閱讀框。開放閱讀框編碼一條約250kDa的多聚蛋白。該多聚蛋白進(jìn)一步被水解為4個(gè)結(jié)構(gòu)蛋白(VP1-VP4)和7個(gè)非結(jié)構(gòu)蛋白(2A、2B、2C,3A、3B、3C和3D)。單股正鏈RNA病毒的RNA具有自我復(fù)制特點(diǎn),因此,通過單鏈RNA拯救病毒可能是一種病毒分離培養(yǎng)方法的重要補(bǔ)充。我們以CVB3病毒為模式病毒,成功建立了 RNA拯救病毒的方法,摸索出RNA拯救CVB3病毒的最佳條件:確定小RNA病毒核酸的最適提取試劑、最適轉(zhuǎn)染試劑及最適轉(zhuǎn)染劑量。利用建立的拯救病毒條件,開展了小RNA病毒科病毒的CVB3、EV71、CA16、ECH03、HRV16、HRV86和EMCV等7種病毒的RNA拯救實(shí)驗(yàn),并成功拯救出7種病毒,再次驗(yàn)證了建立的小RNA病毒的RNA拯救病毒的方法可行性。為了提高RNA拯救病毒的效率,我們使用7.15×105PFU/ml滴度的CVB3病毒提取RNA時(shí),增加RNA純化洗脫次數(shù),通過3次洗脫,每次拯救的病毒噬斑數(shù)分別為150±15、11.67±2.08、1.67±0.58,結(jié)果提示通過多次洗脫可提高拯救病毒量。為了進(jìn)一步提高病毒的拯救效率,我們選擇7.15×105PFU,7.15×104PFU和7.15×103PFU3種滴度的CVB3病毒,提取RNA,分別轉(zhuǎn)染HeLa細(xì)胞、293T細(xì)胞和Vero細(xì)胞等3種細(xì)胞,比較RNA拯救CVB3病毒效率。轉(zhuǎn)染7.15× 105 PFU CVB3病毒的RNA 24h后,293T細(xì)胞發(fā)生100%病變,而HeLa細(xì)胞和Vero細(xì)胞未出現(xiàn)明顯病變;轉(zhuǎn)染48h后,HeLa細(xì)胞和Vero細(xì)胞出現(xiàn)少許病變(20%CPE);轉(zhuǎn)染72h,HeLa細(xì)胞和Vero細(xì)胞均全部病變。轉(zhuǎn)染7.15×104PFUCVB3病毒的RNA 48h后,293T細(xì)胞100%出現(xiàn)病變,而HeLa細(xì)胞和Vero細(xì)胞未出現(xiàn)明顯的病變;轉(zhuǎn)染72h后,HeLa細(xì)胞100%出現(xiàn)病變,Vero細(xì)胞50%出現(xiàn)病變。轉(zhuǎn)染7.15×103PFU CVB3病毒的RNA 48h后,293T細(xì)胞100%出現(xiàn)病變,而轉(zhuǎn)染72h后,HeLa細(xì)胞25%發(fā)生病變,Vero細(xì)胞未出現(xiàn)病變。使用293T細(xì)胞進(jìn)行RNA轉(zhuǎn)染拯救病毒,拯救的病毒再接種HeLa細(xì)胞產(chǎn)生的毒力最高。結(jié)果表明病毒的RNA在293T細(xì)胞內(nèi)具有更好的復(fù)制能力。RNA通過293T細(xì)胞拯救CVB3病毒的效率較HeLa細(xì)胞、Vero細(xì)胞高,同時(shí),分離病毒時(shí)間最短。在此基礎(chǔ)上,為了克服不同病毒需要特定的敏感細(xì)胞的限制,我們建立了基于293T細(xì)胞的一種通用小RNA病毒拯救方法,成功實(shí)現(xiàn)CVB3、EV71、CA16、ECHO3、HRV16、HRV86 和 EMCV 等 7 種病毒的 RNA 拯救。為了克服心臟、胰腺組織在病毒分離過程中產(chǎn)生的細(xì)胞毒性,進(jìn)一步了解組織樣本對(duì)CVB3病毒拯救方法的影響,同時(shí),評(píng)價(jià)RNA拯救方法在CVB3病毒感染的心肌炎模型中病毒的復(fù)制規(guī)律,我們將7.15×103 PFU CVB3病毒通過腹腔接種Balb/c小鼠建立CVB3感染心肌炎模型(6只/組)。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,分離血清;采集心臟和胰腺組織,一半固定,制備切片進(jìn)行HE染色,評(píng)價(jià)病毒引起的心肌和胰腺的病理變化,一半勻漿,提取病毒核酸,進(jìn)行RNA拯救,評(píng)價(jià)病毒在小鼠心肌和胰腺的復(fù)制規(guī)律。HE染色發(fā)現(xiàn)CVB3病毒感染誘導(dǎo)了典型的心肌炎變化:在感染的第5天可觀察到心肌出現(xiàn)明顯的炎癥細(xì)胞浸潤(rùn),心肌細(xì)胞出現(xiàn)玻璃樣變和纖維化;感染的第9天,病理變化達(dá)到高峰,隨后炎癥反應(yīng)逐步下降,至感染的13天接近正常水平。胰腺組織隨著感染時(shí)間的延長(zhǎng),病理?yè)p傷程度加重,感染第7天達(dá)高峰,持續(xù)至感染15天。鏡下可見間質(zhì)水腫、腺泡水腫,炎性細(xì)胞浸潤(rùn),隨病程的進(jìn)展導(dǎo)管出現(xiàn)不同程度的擴(kuò)張。通過RNA拯救方法開展了CVB3病毒在感染的小鼠心肌炎模型中的動(dòng)態(tài)分布特點(diǎn)研究,發(fā)現(xiàn)CVB3病毒在感染的早期(感染的3-5天)在心臟、胰腺和血清中的拷貝數(shù)急速升高,達(dá)到高峰,高峰維持2-3天,隨后病毒的復(fù)制拷貝數(shù)快速下降。病毒在胰腺、心臟和血清中的復(fù)制能力各不相同。感染的第5天,小鼠胰腺組織中的病毒滴度達(dá)到高峰,為826.6±28.0PFU/mg;感染的第3天,心臟和血清中的病毒滴度達(dá)到高峰,分別為24.4±1.9PFU/mg和10953.6±807 PFU/mL。我們的結(jié)果提示感染早期的心臟和胰腺組織損傷來(lái)自于CVB3病毒的直接損傷,感染后期則為炎癥反應(yīng)導(dǎo)致的損傷。綜上所述,我們通過對(duì)實(shí)驗(yàn)條件優(yōu)化,建立了 CVB3病毒RNA拯救的方法,并成功應(yīng)用于7種小RNA病毒。摸索建立了以293T細(xì)胞為轉(zhuǎn)染細(xì)胞的通用小RNA病毒拯救技術(shù)。利用該方法評(píng)價(jià)了 CVB3病毒感染小鼠心肌炎模型中病毒在心臟、胰腺和血清中的復(fù)制規(guī)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.

【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R373

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