1、第七章 病 毒 學 孫 建 和 教 授上海交通大學農(nóng)業(yè)與生物學院TEL：34206926-MAIL：sunjhe一、病毒的發(fā)現(xiàn)和研究歷史概述1900年前后，包括口蹄疫（foot and mouth disease）在內(nèi)的多種動植物疾病病原體的濾過性特性被證明。filterable viruses（濾過性病毒）viruses（病毒）F W Twort（1915年）、F dHerelle（1917年），分別發(fā)現(xiàn)細菌（Shigella dysenteriae）病毒bacteriophagesPhago：“to eat”噬菌體（phage）1935年，W M Stanley首

2、次提純并結(jié)晶了煙草花葉病毒； Bawden等證明煙草花葉病毒的本質(zhì)為核蛋白；1940年，Kausche首先用電鏡觀察到煙草花葉病毒顆粒；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、病毒的特點和定義第一節(jié) 概述1、特點1）不具有細胞

8、結(jié)構(gòu)，具有一般化學大分子的特征。2）一種病毒的毒粒內(nèi)只含有一種核酸，DNA或者RNA。3）大部分病毒沒有酶或酶系極不完全，不含催化能量代謝 的酶，不能進行獨立的代謝作用。4）嚴格的活細胞內(nèi)寄生，沒有自身的核糖體，沒有個體生 長，也不進行二均分裂，必須依賴宿主細胞進行自身的 核酸復制，形成子代。5）個體微小，在電子顯微鏡下才能看見。6）對大多數(shù)抗生素不敏感，對干擾素敏感。例如一些簡單的病毒僅由核酸和蛋白質(zhì)外殼（coat）構(gòu)成，故可把它們視為核蛋白分子。朊病毒甚至僅由蛋白質(zhì)構(gòu)成例如利福平可抑制痘病毒復制病毒感染敏感宿主細胞后，病毒核酸進入細胞，通過其復制與表達產(chǎn)生子代病毒基因組和新的蛋白質(zhì)，然后由

9、這些新合成的病毒組分裝配（assembly）成子代毒粒，并以一定方式釋放到細胞外。病毒的這種特殊繁殖方式稱做復制（replication）。第一節(jié) 概述1、特點二、病毒的特點和定義病毒是一類既具有化學大分子屬性，又具有生物體基本特征；既具有細胞外的感染性顆粒形式，又具有細胞內(nèi)的繁殖性基因形式的獨特生物類群。超顯微的、沒有細胞結(jié)構(gòu)的、專性活細胞內(nèi)寄生的實體第一節(jié) 概述1、特點二、病毒的特點和定義表7-1 單細胞微生物與病毒性質(zhì)的比較a DNA病毒和RNA病毒中的一部分;b 利福平可抑制痘病毒復制第一節(jié) 概述2、定義二、病毒的特點和定義迄今仍無一個科學而嚴謹?shù)亩x病毒為具有獨立于其宿主的進化史的絕

10、對細胞內(nèi)寄生物，它的DNA或RNA基因組被其所編碼的蛋白質(zhì)殼體化（encapsidation）。 （Fields等，1990年）非細胞生物（真）病毒virion：至少含核酸和蛋白質(zhì)二種組分亞病毒subvirus類病毒viroid：只含具侵染性的RNA組分衛(wèi)星RNA：只含有不具侵染性的RNA組分朊病毒prion：只含蛋白質(zhì)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 病毒的分類與命名分類原則形態(tài)、結(jié)構(gòu)、基因組、化學組成、脂溶劑敏感性命名規(guī)則In

13、ternational Committee on Taxonomy of Viruses, ICTVorder, -virales（目）family, -viridae（科）, virinae（亞科）genus, -virus（屬）Species（種）/專家建議專家小組討論ICTV分會討論ICTV執(zhí)委會病毒學會大會評議確定分類報告官方刊物發(fā)布病毒的分類 2011年 第九次分類報告（2012年出版） 6個目 87個病毒科 19個亞科 348個病毒屬 2284個病毒種Dr. Andrew M.Q. KingProposals Secretary, ICTVPirbright LaboratoryI

14、nstitute for Animal HealthSunfield, Dawney Hill,Pirbright, WokingSurrey GU24 0JBUK無脊椎動物病毒7個科與獸醫(yī)學相關(guān)藻類水華噬藻體五、病毒的培養(yǎng)和純化第一節(jié) 概述同微生物學其他學科分支一樣，病毒學的進步完全得益于研究方法和技術(shù)手段的發(fā)展。通過病毒的分離與純化獲得純化的、有感染性的病毒制備物是病毒學研究和實踐的基本技術(shù)。1、病毒的培養(yǎng)病毒的培養(yǎng)：二元培養(yǎng)物法四、病毒的培養(yǎng)和純化1、病毒的培養(yǎng)噬菌體細菌培養(yǎng)物培養(yǎng)液營養(yǎng)瓊脂平板細菌培養(yǎng)液變清亮細菌平板成為殘跡平板若是噬菌體標本經(jīng)過適當稀釋再接種細菌平板，經(jīng)過一定時間培養(yǎng)

15、，在細菌菌苔上可形成圓形局部透明區(qū)域，即噬斑（plaque）。如同對細菌計數(shù)一樣，形成的噬菌斑也可用于對噬菌體的數(shù)目進行估算。四、病毒的培養(yǎng)和純化（參見P161）1、病毒的培養(yǎng)動物病毒實驗動物雞胚多種細胞培養(yǎng)絨毛尿囊膜羊膜腔卵黃囊胚胎尿囊腔Virus-induced pocks on the horioallantoic membrane of a chick embryoRoutes of Inoculation Routes of Inoculation 各種病毒在雞胚內(nèi)的繁殖與接種途徑 Growing Viruses in cell 原代細胞二倍體細胞傳代細胞系Primary cell

16、cultures（原代細胞培養(yǎng)）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)細胞反應器 “wave bag” to shake up cells四、病毒的培養(yǎng)和純化1、病毒的培養(yǎng)若標本經(jīng)過適當稀釋進行接種并輔以染色處理，病毒可在培養(yǎng)的細胞單層上形成肉眼可見的局部病損區(qū)域，即蝕斑（plaque）或稱空斑。大多數(shù)動物病毒感染敏感細胞培養(yǎng)能引起其顯微表現(xiàn)的改變，即產(chǎn)生致細胞病變效應（cytopathic effect, CPE），

18、例如細胞聚集成團、腫大、圓縮、脫落，細胞融合形成多核細胞，細胞內(nèi)出現(xiàn)包涵體（inclusion body），乃至細胞裂解等。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殼粉黑蛾(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. 蛋白質(zhì)提純方法（鹽析、等電點沉淀、有機溶劑沉淀、凝膠層析、離子交換等）四、病毒的培養(yǎng)和純化2、 病毒純化標準純化的病毒制備物應保持其感染性病毒