【作者】 沈宏辉；

【导师】 孔维； 貌盼勇；

【作者基本信息】 吉林大学 ， 生物化学和分子生物学， 2012， 博士

【摘要】 严重急性呼吸综合症（Severe acute respiratory syndrome，SARS)是2003年出现的新发传染病，其病情重，死亡率高，严重危害人类健康。虽然SARS的病原已确定为SARS相关冠状病毒（SARS-CoV），国内外研究也检测了SARS患者标本中的腺病毒、呼吸道合胞病毒、亨德拉病毒（hendra virus）、尼帕病毒（nipah virus）、流感病毒、人偏肺病毒等其他病原体，但是未曾检测过脊髓灰质炎病毒。我们在研究SARS的过程中发现SARS患者标本中可能有肠道病毒属的脊髓灰质炎病毒疫苗株SabinⅠ存在，为确证这个结果，本研究采用世界卫生组织推荐的分离脊髓灰质炎病毒的方法和人横纹肌肉瘤细胞系（human rhabdomyosarcoma，RD），对取自SARS患者的咽拭子标本进行病毒分离并成功获得病毒，通过中和试验和间接免疫荧光染色证实属于脊髓灰质炎病毒Ⅰ型，随后利用逆转录-聚合酶链反应、cDNA末端快速扩增（RACE）等分子生物学技术分段扩增病毒基因组并测序，从基因水平进一步证实其为脊髓灰质炎病毒SabinⅠ型疫苗类似株。在确定从SARS患者标本中分离获得脊髓灰质炎病毒SabinⅠ型疫苗类似株后，需要从流行病学角度进一步分析其是否在更多患者中存在，因此本研究根据获得的病毒基因组序列设计针对5′-UTR、VP3–VP1和2C-3A区3个部位的引物分别扩增SARS患者标本中提取的RNA，结果显示38.5%(5/13)的唾液标本、37.5%(3/8)的痰标本、71.4%(5/7)的尿液标本、100%(5/5)的咽拭子标本和100%(6/6)的粪便标本中可以扩增出脊髓灰质炎病毒SabinⅠ核酸，证实71.4% (15/21)的SARS患者体内存在脊髓灰质炎病毒SabinⅠ型且480和2795位点出现回复突变，其中扩增阳性患者的死亡率高于扩增阴性患者；研究结果同时显示SARS患者的肠道和肾组织中可以检测到脊髓灰质炎病毒SabinⅠ存在。为防止实验结果的假阳性，研究中设置了严格的对照并重复验证结果，其中对照样本的扩增结果显示取自25个健康人和25例肝炎患者的50份粪便标本仅1份阳性（2%），与患者组相比，其差异具有显著性意义（p﹤0.01）。虽然在患者中没有发现脊髓灰质炎相关症状体征，排除了疫苗相关麻痹型脊髓灰质炎的可能，但是由于研究组患者均为成年人，其体内脊髓灰质炎病毒SabinⅠ的存在提示在脊髓灰质炎的流行病学监测体系中，除了15岁以下的儿童，还需要加强对成人特别是严重呼吸道感染和免疫功能受损人群的病毒学检测和抗体检测，对血清脊髓灰质炎病毒中和抗体滴度显著下降的人群应该及时加强免疫。尽管标本检测得到了阳性结果，但是由于未能系统收集并检测各种标本，导致结果分析受到限制，对脊髓灰质炎病毒SabinⅠ型疫苗类似株在SARS的发生发展中是否起作用以及有何种作用尚不能确定。SARS患者体内脊髓灰质炎病毒Ⅰ型疫苗类似株的首次发现表明需要加强脊髓灰质炎病毒疫苗株尤其是SabinⅠ的实验研究，由于目前广泛应用口服减毒活疫苗，且不断出现疫苗相关麻痹型脊髓灰质炎，如何降低SabinⅠ基因组的回复突变率以减少疫苗衍生脊髓灰质炎病毒的产生是当务之急。如果能通过改造病毒的基因组使其在不降低免疫效果和安全性的前提下减少回复突变率，将有助于降低疫苗相关麻痹型脊髓灰质炎的发病率，而研究病毒基因组的功能首先需要构建感染性cDNA克隆拯救病毒。为此，在一次性扩增脊髓灰质炎病毒SabinⅠ基因组全长7441个核苷酸难度较大的情况下，本研究利用SabinⅠ基因组序列中2470位的限制性内切酶NheⅠ识别位点和5601位的限制性内切酶BglⅡ识别位点，通过分别扩增基因组1nt-2497nt、2412nt-5616nt和5588nt-7441nt核苷酸序列并插入克隆载体，然后分步酶切连接的方法成功获得脊髓灰质炎病毒SabinⅠ感染性cDNA克隆。在感染性克隆构建成功后开展了SabinⅠ病毒拯救研究，本文在转录获得病毒RNA的步骤中，没有选择常用但是昂贵、费力的体外转录，而是首次在脊髓灰质炎病毒拯救中利用近年来开始出现的真核表达载体细胞内表达T7RNA聚合酶后转录RNA的技术，结果成功拯救SabinⅠ，从而为进一步研究脊髓灰质炎病毒SabinⅠ的基因功能奠定了基础。核定位信号有助于将蛋白质从细胞胞浆内转运到细胞核内，以往有关包括脊髓灰质炎病毒在内的肠道病毒拯救的研究中，体内拯救时细胞内表达的T7RNA聚合酶都没有涉及核定位信号，为了解核定位信号对病毒拯救效果的影响，本研究在拯救病毒时分别构建了含与不含SV40大T抗原核定位信号PKKKRKV的T7RNA聚合酶真核表达质粒，然后将其分别与脊髓灰质炎病毒SabinⅠ感染性cDNA克隆共转染细胞，结果发现虽然表达含或不含核定位信号的T7RNA聚合酶都能够拯救病毒，但是表达含核定位信号的T7RNA聚合酶拯救病毒的效果要优于不含核定位信号的T7RNA聚合酶，提示脊髓灰质炎病毒SabinⅠ感染性cDNA克隆的转录优势部位可能在细胞核内，推测其与细胞核内转录因子等转录相关分子较多有关，这是在肠道病毒拯救机制研究中的首次发现。由于脊髓灰质炎病毒SabinⅠ型的自然复制主要在胞浆内，研究结果显示病毒自然复制与病毒拯救的差异，从而有助于脊髓灰质炎病毒细胞内拯救机制的研究。为寻找降低脊髓灰质炎病毒SabinⅠ回复突变率的方法，本研究采用单引物二次PCR的方法将SabinⅠ感染性cDNA克隆3D聚合酶第64位甘氨酸突变为丝氨酸并成功获得拯救病毒，初步结果显示突变后SabinⅠ感染性克隆的病毒拯救效率下降，为下一步研究突变后拯救病毒的生长特性、毒力和回复突变率奠定了基础。总之，本研究首次发现成人SARS患者体内存在脊髓灰质炎病毒疫苗株SabinⅠ，并发现在T7RNA聚合酶中插入核定位信号有助于脊髓灰质炎病毒的拯救，这些结果有助于脊髓灰质炎流行病学和脊髓灰质炎病毒拯救机制的研究。更多还原

【Abstract】 Although the causative agent of SARS has been identified as a novel coronavirus,called SARS-associated coronavirus (SARS-CoV), other pathogens, includingadenoviruses, respiratory syncytial virus, hendra virus, nipah virus, influenza virus andhuman metapneumovirus have also been detected in some patients with SARS.However, the detection of poliovirus in SARS patients has not been reported so far.In 2003, when we tried to isolate SARS-CoV in the throat swab specimen of aSARS patient, poliovirus was detected by electron microscope detection andreverse-transcriptase polymerase chain reaction (RT-PCR) assay. To confirm this result,poliovirus was isolated and purified by using human rhabdomyosarcoma (RD) cell line,which was recommended by World Health Organization as suitable manipulation forpoliovirus. The isolation was further characterized by neutralization test and indirectimmunofluorescence staining assay, as well the whole genome sequence by RT-PCRand 5’RACE amplification. All of the results indicated that our isolation was OPV-likepoliovirus type 1. In addition, to investigate whether this poliovirus type 1 wasprevalent in other SARS patients, we collected the specimens from SARS patientsincluding urine, throat swab, sputum, saliva and stool specimen. Viral RNA wasextracted and amplified by primers targeted the 5′-UTR, VP3–VP1 and 2C-3A regionsof poliovirus SabinⅠ. It indicated that 38.5%(5/13) of saliva specimens, 37.5%(3/8) ofsputum specimens, 71.4%(5/7) of urine specimens, 100%(5/5)of throat swabs and100%(6/6) of stool specimens were positive for OPV-like poliovirus 1, which showedthat 15 of 21 (71.4%) SARS patients were positive for OPV-like poliovirus 1, with thereverse mutation of 480G→A and 2795A→G. By contrast, only 1 of 50 (2%) stoolspecimens from 25 healthy individuals and 25 patients with hepatitis A or hepatitis Binfection, was positive for OPV-like poliovirus 1 (p﹤0.01). On the other side, wedemonstrated the distribution of OPV-like poliovirus 1 among the different organs inSARS patient, with that kidney and small intestine were positive and brain, lung, liver,spleen or pancreas were negative.Although the impossibility of vaccine-associated paralytic poliomyelitis in SARSpatients, the OPV-like poliovirus 1 existing in adult patients of SARS suggested that the virological testing and antibody testing should be strengthened in adults especially inpopulations with severe respiratory infection or impaired immunity, besides childrenyounger than 15-years old. It also indicated that adults with poor immunity againstpoliovirus should be inoculated with poliovirus vaccine to strengthen their immunityagainst this virus. However, the inadequate data of SARS specimens collection limitedthe analysis of the exact relationship between OPV-like poliovirus 1 andSARS/SARS-CoV. The role of OPV-like poliovirus 1 on SARS was unknown andneeds further research.The existence of OPV-like poliovirus 1 in SARS patients indicated that we shouldgive more attention to this virus. For the extensive use of OPV worldwide currently it isurgent to find the way to decrease the reverse mutation rate of SabinⅠgenome so as toreduce the vaccine derived poliovirus and thus vaccine-associated paralyticpoliomyelitis. If the reverse mutation rate could be lowered by modifying theSabinⅠgenome, with the same immunological effect and safety, it will facilitate thereduction of morbidity of vaccine-associated paralytic poliomyelitis. Construction ofinfectious cDNA clone to rescue virus was the important way to study the function ofvirus genome. Thus, the fragments of 1nt-2497nt, 2412nt-5616nt and 5588nt-7441ntwere amplified and cloned into T vector, followed by digestion with restriction enzymeand ligation step by step, using the NheⅠsite in 2470nt and BglⅡsite in 5601nt. Thefull-length SabinⅠinfectious cDNA clone was then constructed successfully. Instead ofexpensive and difficult extracellular transcription, we used nonviral eukaryoticexpression vector to express T7 RNA polymerase intracellularly to transcript SabinⅠRNA and then resue the virus. This successful poliovirus SabinⅠrescuing systemprovided important implications for further research on function of viral gene.Nuclear localization signals can help to transport proteins from cell plasma tonuclei. It has not been related to poliovirus rescuing in previous reports. In present studyeukaryotic vector expressing T7 RNA polymerase with/without SV40 large T antigennuclear localization signal PKKKRKV was constructed. After coinfection withSabinⅠinfectious cDNA clone on vero cells, the vector expressing T7 RNA polymerasewith nuclear localization signal showed better result of rescuing virus than that withoutnuclear localization signal. It indicated that the advantageous position of RNAtranscription with SabinⅠinfectious cDNA clone was probably existed in cell nucleus,which may due to more amount of transcription associated molecules in nucleus thanthose in cytoplasm. This is the first report on research of enterovirus rescuing mechanism.Moreover, to find the way of decreasing the reverse mutation rate, rescued viruswas acquired with Gly→Ser in 64 position in 3D polymerase region, and the primaryresults showed that the mutation contributed to decreased rescuing efficiency. It lays thefoundation for further research on characteristics of mutated rescued poliovirus.更多还原