上海交通大学病毒学第二次课课件

1、第七章 病 毒 学 孙 建 和 教 授上海交通大学农业与生物学院TEL：34206926-MAIL：sunjhe一、病毒的发现和研究历史概述1900年前后，包括口蹄疫（foot and mouth disease）在内的多种动植物疾病病原体的滤过性特性被证明。filterable viruses（滤过性病毒）viruses（病毒）F W Twort（1915年）、F dHerelle（1917年），分别发现细菌（Shigella dysenteriae）病毒bacteriophagesPhago：“to eat”噬菌体（phage）1935年，W M Stanley首

2、次提纯并结晶了烟草花叶病毒； Bawden等证明烟草花叶病毒的本质为核蛋白；1940年，Kausche首先用电镜观察到烟草花叶病毒颗粒；Historical Roots1. EARLY STUDIES OF VIRAL PATHOGENESISVirology was severely constrained in the first half of the 20th century by several technical limitations, the most important of which was the lack of a cell culture system for gr

3、owing and titrating viruses. In the absence of methods for detecting viruses in tissues, observations of experimental infections were limited to clinical signs and pathological lesions that represented the endstage of disease. In spite of these adverse circumstances, extensive studies were undertake

4、n of a few infections, such as poliomyelitis and Rous sarcoma.3. THE ERA OF MOLECULAR BIOLOGYsequence viral genomes and to modify them in order to determine the genetic basis of viral variation and virulence.Applied to in vivo studies,viral genomes and their transcripts could be visualized, using in

5、 situ hybridization and the in situ polymerase chain reaction (PCR). Starting in the 1990s, it became possible to manipulate the genomes of mammalian hosts, either by ablating a specific gene function (knockout) or by inserting new or altered genes (transgenic animals)Tracking the spread of a neurot

6、ropic virus from the periphery to the central nervous system in an intact animal. Mice were infected by subcutaneous injection in the right footpad with a Sindbis virus that had been engineered to express, in addition to viral genes, luciferase under an internal promoter. Images AD respectively, wer

7、e taken at 8 hours and 1, 3 and 4 days after infection. They show that the virus used two pathways to the central nervous system, either up peripheral nerves to the spinal cord or via blood to the olfactory bulb and thence to the brain.4. PATHOGENESIS IN THE NEW MILLENNIUM二、病毒的特点和定义第一节 概述1、特点1）不具有细胞

8、结构，具有一般化学大分子的特征。2）一种病毒的毒粒内只含有一种核酸，DNA或者RNA。3）大部分病毒没有酶或酶系极不完全，不含催化能量代谢 的酶，不能进行独立的代谢作用。4）严格的活细胞内寄生，没有自身的核糖体，没有个体生 长，也不进行二均分裂，必须依赖宿主细胞进行自身的 核酸复制，形成子代。5）个体微小，在电子显微镜下才能看见。6）对大多数抗生素不敏感，对干扰素敏感。例如一些简单的病毒仅由核酸和蛋白质外壳（coat）构成，故可把它们视为核蛋白分子。朊病毒甚至仅由蛋白质构成例如利福平可抑制痘病毒复制病毒感染敏感宿主细胞后，病毒核酸进入细胞，通过其复制与表达产生子代病毒基因组和新的蛋白质，然后由

9、这些新合成的病毒组分装配（assembly）成子代毒粒，并以一定方式释放到细胞外。病毒的这种特殊繁殖方式称做复制（replication）。第一节 概述1、特点二、病毒的特点和定义病毒是一类既具有化学大分子属性，又具有生物体基本特征；既具有细胞外的感染性颗粒形式，又具有细胞内的繁殖性基因形式的独特生物类群。超显微的、没有细胞结构的、专性活细胞内寄生的实体第一节 概述1、特点二、病毒的特点和定义表7-1 单细胞微生物与病毒性质的比较a DNA病毒和RNA病毒中的一部分;b 利福平可抑制痘病毒复制第一节 概述2、定义二、病毒的特点和定义迄今仍无一个科学而严谨的定义病毒为具有独立于其宿主的进化史的绝

10、对细胞内寄生物，它的DNA或RNA基因组被其所编码的蛋白质壳体化（encapsidation）。 （Fields等，1990年）非细胞生物（真）病毒virion：至少含核酸和蛋白质二种组分亚病毒subvirus类病毒viroid：只含具侵染性的RNA组分卫星RNA：只含有不具侵染性的RNA组分朊病毒prion：只含蛋白质Viruses (Encyclopedia Britannica).infectious agents of small size and simple composition that can multiply only in living cells of animals,

11、 plants and bacteria. Viruses are obligate parasites that are metabolically inert when they are outside their hosts. They all rely, to varying extents, on the metabolic processes of their hosts to reproduce themselves. The viral diseases we see are due to the effects of this interaction between the

12、virus and its host cell (and/or the hosts response to this interaction).AdenovirusPapillomavirusHerpesvirus Hepatitis B virus Molluscum contagiosum ParvovirusInfluenza virus RotavirusParamyxoviruses Enteroviruses Rift Valley Fever Virus Filovirus Ebola virus 四 病毒的分类与命名分类原则形态、结构、基因组、化学组成、脂溶剂敏感性命名规则In

13、ternational Committee on Taxonomy of Viruses, ICTVorder, -virales（目）family, -viridae（科）, virinae（亚科）genus, -virus（属）Species（种）/专家建议专家小组讨论ICTV分会讨论ICTV执委会病毒学会大会评议确定分类报告官方刊物发布病毒的分类 2011年 第九次分类报告（2012年出版） 6个目 87个病毒科 19个亚科 348个病毒属 2284个病毒种Dr. Andrew M.Q. KingProposals Secretary, ICTVPirbright LaboratoryI

14、nstitute for Animal HealthSunfield, Dawney Hill,Pirbright, WokingSurrey GU24 0JBUK无脊椎动物病毒7个科与兽医学相关藻类水华噬藻体五、病毒的培养和纯化第一节 概述同微生物学其他学科分支一样，病毒学的进步完全得益于研究方法和技术手段的发展。通过病毒的分离与纯化获得纯化的、有感染性的病毒制备物是病毒学研究和实践的基本技术。1、病毒的培养病毒的培养：二元培养物法四、病毒的培养和纯化1、病毒的培养噬菌体细菌培养物培养液营养琼脂平板细菌培养液变清亮细菌平板成为残迹平板若是噬菌体标本经过适当稀释再接种细菌平板，经过一定时间培养

15、，在细菌菌苔上可形成圆形局部透明区域，即噬斑（plaque）。如同对细菌计数一样，形成的噬菌斑也可用于对噬菌体的数目进行估算。四、病毒的培养和纯化（参见P161）1、病毒的培养动物病毒实验动物鸡胚多种细胞培养绒毛尿囊膜羊膜腔卵黄囊胚胎尿囊腔Virus-induced pocks on the horioallantoic membrane of a chick embryoRoutes of Inoculation Routes of Inoculation 各种病毒在鸡胚内的繁殖与接种途径 Growing Viruses in cell 原代细胞二倍体细胞传代细胞系Primary cell

16、cultures（原代细胞培养）Cell culturers obtained directly from an animals organs and tissues (embryos or adult animals).(1)mechanical methods(2)chemical methods(3)enzymatic digestionCell CultureProductionby roller bottle MicrocarriersVan Wezel 1960s RIVMMonkey Kidney cellsPolioToxapheneProbes for:pH Dissolve

17、d OxygenTemperatureGas inletNitrogenOxygenCO2Gas outletHeatingViable-cell concentration (109 cells.dm-3)Culture time (hours)细胞反应器 “wave bag” to shake up cells四、病毒的培养和纯化1、病毒的培养若标本经过适当稀释进行接种并辅以染色处理，病毒可在培养的细胞单层上形成肉眼可见的局部病损区域，即蚀斑（plaque）或称空斑。大多数动物病毒感染敏感细胞培养能引起其显微表现的改变，即产生致细胞病变效应（cytopathic effect, CPE），

18、例如细胞聚集成团、肿大、圆缩、脱落，细胞融合形成多核细胞，细胞内出现包涵体（inclusion body），乃至细胞裂解等。Cytopathic EffectCytopathic effect of enterovirus 71 and HSV in cell culture: note the ballooning of cells. (Virology Laboratory, Yale-New Haven Hospital, Linda Stannard, University of Cape Town)Cytopathic effects caused by replication of

19、 poliovirus and herpes simplex virus in cultures of Vero (monkey kidney) cells. (a) Uninfected Vero cells. (b) Infected with poliovirus. (c), (d) Infected with herpes simplex virus. (a), (b) and (d) were viewed at 400 magnification; (c) was viewed at 100 magnification. The cells were stained with ha

20、ematoxylin and eosinBaculovirus occlusion bodies. (a) Transverse section through an occlusion body of a granulovirus of the Indian meal moth壳粉黑蛾(Plodia interpunctella). The nucleocapsid surrounded by its membrane can be seen at the centre, surrounded by the crystalline protein that forms the occlusi

21、on body. The bar represents 0.1 m. (b), (c) Nucleopolyhedrovirus of the leatherjacket ,(b) Light micrograph of occlusion bodies. (c) Electron micrograph of a thin section of an occlusion body. The virions are randomly embedded in the crystalline protein.Journal of Virology: October 2012, Volume 84,

22、IssueCytopathic changes involving cell membranesCell membrane fusion and syncytium formationHemadsorption and hemagglutinationCytopathic changes involving the cytoskeletonCytopathic EffectSyncytium formation in cell culture caused by RSV (top), and measles virus (bottom). (courtesy of Linda Stannard

23、, University of Cape Town, S.A.)Haemadsorption（血吸附）Syncytial formation caused by mumps virus and haemadsorption of erythrocytes onto the surface of the cell sheet. (courtesy of Linda Stannard, University of Cape Town, S.A.)Possible effects that animal viruses may have on cells they infect. Most animal viruses are lytic, and only very few are known to cause cancer. 蛋白质提纯方法（盐析、等电点沉淀、有机溶剂沉淀、凝胶层析、离子交换等）四、病毒的培养和纯化2、 病毒纯化标准纯化的病毒制备物应保持其感染性病毒