摘要
正链RNA病毒(Positive-stranded RNA viruses)是以RNA为遗传物质且病毒RNA可直接作为mRNA翻译产生病毒蛋白的一类病毒,其中许多成员是严重威胁与损害人类健康的病原体,或是造成经济动物患病死亡的致病因子,如丙型肝炎病毒、脊髓灰质炎病毒、SARS病毒、登革热病毒、口蹄疫病毒、牛病毒性腹泻-粘膜病病毒(简称牛腹泻病毒)等。目前,对这些危害较大的病毒基本上都缺乏快速有效的防治方法。病毒基因组的复制是病毒生存和繁殖的基础,是病毒生命周期不可或缺的重要环节,所以研究单股正链RNA病毒基因组的复制是防止相关致病因子(病原体)蔓延与传播的基础。
口蹄疫病毒(Foot-and-mouth disease virus,FMDV)是一种单股正链RNA病毒,以负链RNA作为中间体进行复制,感染敏感细胞系(如仓鼠肾细胞系BHK-21或猪肾细胞系PK-15)会引发急性感染。为了更好地研究病毒感染,需要检测单个急性感染细胞中病毒RNA量而不仅仅检测群体细胞的病毒量。在本研究中,我们建立了一种链特异性的单细胞实时荧光定量RT-PCR技术,其技术包括显微操作分离单细胞、单细胞裂解与高灵敏度的实时荧光定量RT-PCR三部分。该技术用于检测FMDV基因组RNA,总计224个急性感染FMDV的单细胞样品被分离并进行定量检测,其中185个样品为阳性(阳性率为82.6%),阳性细胞中病毒载量从几十至几十万拷贝数不等,最高达约1000000拷贝数/细胞。结果表明,在FMDV感染过程中不是每个细胞都被病毒感染上,且群体细胞中单个细胞的病毒载量存在很大的差异。该技术实现了在单细胞水平上研究病毒的复制及其与宿主细胞的关系,为阐明病毒的复制机制和进化奠定了基础,也为病毒感染细胞生物学的深入研究提供一种新的方法。另外,分离细胞和裂解细胞的技术具有广泛适用性、能用于任何其他细胞(悬浮细胞与贴壁细胞)的分离与预处理,为在单细胞水平上进行的各种研究奠定基础。
病毒基因组RNA起始复制的方式有两种：非引物依赖和引物依赖,口蹄疫病毒(FMDV)和脊髓灰质炎病毒(PV)属引物依赖,牛腹泻病毒(BVDV)是以非引物依赖起始复制。在本研究中,单细胞实时荧光定量RT-PCR技术被用于检测FMDV、PV、BVDV正链RNA与负链RNA以研究正链RNA病毒的复制规律。PV感染Vero细胞和FMDV感染BHK-21细胞、PK-15细胞的单细胞定量数据(+RNA与-RNA的对数)都具有较好的线性相关性,经过数学模型的推导分析发现一条负链可能是对应合成一条正链,只是起始+RNA量和用于合成负链的正链比例上存在一定的差异。而BVDV感染BKC细胞的单细胞定量数据表明,+RNA与-RNA的对数基本不具线性相关性,建模初步分析结果为一条负链可能是对应合成一条正链,但起始+RNA量和用于合成负链的正链比例上与FMDV和PV差异非常大。
弱碱(如NH4Cl)进入酸性的胞内体中提高与细胞内吞作用相关的细胞器的pH值,阻止依赖于低pH值的蛋白质构象变化从而影响病毒基因组释放,降低病毒感染后的增殖量。本研究用NH4Cl维持培养感染FMDV的BHK-21细胞系,存活细胞经显微操作单细胞分离、单细胞克隆培养传代,共获得17株阳性克隆细胞株。随机选取一株阳性细胞(BHK-Op)常规传代,采用实时荧光定量RT-PCR,透射电子显微镜,western blot等方法研究病毒持感细胞特性。BHK-Op48细胞切片电镜观察,病毒粒子存在于细胞质中弥散的小囊泡结构内(每一个小囊泡内仅有几个病毒粒子),直径20-25nm,不同于急性感染细胞中病毒粒子的分布,后者主要位于近核膜的囊泡中(囊泡中存在大量病毒粒子)；持感病毒感染其敏感细胞不能使之形成蚀斑但TCID50实验结果表明其毒力较野生型病毒强；序列比对结果显示,持感病毒基因组中有15个基因编码的蛋白突变全部位于非结构蛋白区,与之前的研究结果大相径庭,其中3C蛋白编码区两个保守位点的突变恰好位于其RNA结合结构域,与病毒复制相关,可能是其不能形成蚀斑的原因之一。体外持续感染的建立为研究病毒与细胞共进化以及病毒与受体相互作用提供有效模型。
The genome of positive-stranded RNA viruses is plus strand RNA, which can be used as mRNA to generate viral proteins. Many of the members are causative agents of highly contagious diseases in humans, plants and animals, e.g., HCV, poliovirus (PV), SARS virus, dengue virus, foot and mouth disease virus (FMDV), bovine viral diarrhea virus (BVDV). So far, there are no effective and rapid methods for prevention and cure of these harmful viruses. Viral genome replication is the basis of virus survival and multiplication, which is an indispensable process in virus life cycle. Studies on positive strand RNA viral replication play important roles in avoiding the spread of pathogens.
Foot-and-mouth disease virus is a positive-sense, single-stranded RNA virus with a negative strand as its replication intermediate, which can cause severe acute infection in sensitive cell lines (BHK-21 cells and PK-15 cells). To investigate better the actual state of virus infection, there is a need to measure the amount of FMDV RNA in a single acutely infected cell rather than in a large number of cells. Therefore, in the present study, a strand-specific single-cell quantitative real-time RT-PCR was developed to analyze the RNA of FMDV. This new method includes three techniques: a technique for isolating single cells with micromanipulators, lyses of single-cell samples and sensitive quantitative real-time RT-PCR. In the assay of acute infection, 185 of 224 (82.6%) single-cell samples were positive and contained viral genome copies ranging from several to thousands, and up to 1000000 copies. However, not all cells were infected and there were differences in the number of viral RNA copies among cells. Virus replication as well as the relationship bewteen virus and host cell can be studied in single cells basing on the technique, which helps to clarify the mechnism of virus replication and virus evolution and also provides as a new method for further research on infectious cell biology. Besides, the methods for isolation and pre-treatment of single cells are universal and can be used in other cell lines (both suspended cells and adherent cells) whether inoculated with viruses or not, which are the basis of other analyses in single cell level.
There are two mechanisms by which viral replication can be initiated:primer independent or de novo, and primer dependent. FMDV and PV belong to primer dependent while BVDV belongs to primer independent. In this study, single cell quantitative real-time RT-PCR assays were carried out for quantitation of both positive strands and negative strands of FMDV, PV and BVDV to investigate the replication of positive-stranded RNA viruses. Results showed that experimental data (Log10+RNA and Log10-RNA) of Vero cells infected with PV and BHK-21 cells/PK-15 cells infected with FMDV had good linear correlations. Mathematical modeling analysis of our data indicated that one-RNA may be used to generate one +RNA and initial+RNA copies as well as the rate of+RNAs for replication were different in two viruses (FMDV and PV). However, quantitation data of BKC cells infected with BVDV showed almost no linear correlations. Results of mathematical modeling analysis indicated that one-RNA may be used to generate one+RNA and initial+RNA copies as well as the rate of+RNAs for replication were totally different from the other two viruses.
The weak bases (ammonium chloride), which diffuse into acidic endosomes and raise the pH of endocytic organelles, prevent the required low pH-dependent proteins conformational changes leading to virus genome release, can reduce the yield of FMDV after infection. In this study, baby hamster kidney cell line (BHK-21) infected with FMDV serotype O was maintained in medium with NH4Cl weak base. Survived cells were isolated using micromanipulator to form single-cell clones,17 positive-cloned cell strains were obtained. One cell strain (BHK-Op) was selected randomly and cultured as usual for analysis with quantitative real-time RT-PCR, western blotting, transmission electron microscope. FMDV particles were observed successfully in BHK-Op48 cells, which dispersed in the cytoplasm and loacted in some small endosomes (not more than 10, usually 2 or 3) with a diameter of 20-25nm. Virions in acutely infected cells mainly located in endosomes close to the nucleus in large quantities. Infection of the persistent viruses could not form plaques in host cells but virulence was enhanced. Basing on analysis and comparison of cDNA sequences of resident viruses and wild type viruses,15 amine acid mutations were found, all of which located in nonstructural proteins rather than in structural proteins, which was inconsistent with previous studies. Two conservative amino acid mutations in 3C protein located exactly in the RNA binding domain and were relevant to virus replication, which is likely to be one of the reasons for lost plaques. The establishment of persistent infections in vitro provides an effect model for studying coevolutions of virus and host cell as well as interactions of virus and receptors. Besides, microarray experiments are underway to attempt to search host cell proteins or factors pertaining to FMDV persistence in BHK-21 cells.
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