Virology-To Students病毒學(xué)

1、12Introducing MyselfUniversity Student: Xian Medical CollegeMaster Degree: Kunming Medical CollegeBasic Medical Teacher: FMMUPh.D. Degree: FMMUPhysician (Internal Medicine) in Xi-Jing HospitalResearch Associate: Wayne State UniversityPostdoctoral Scholar: NIHPostdoctoral Research Fellow: Wayne State

2、 UniversitySenior Scientist: VA Medical Center, Detroit, MIProfessor: SNNU345Virology and Its Importance to UsVirology:A academic field that includes the researches on virus and control the virus to protect human being and plant. Some time and for some virus, we can use the virus to make researches.

3、The roles of viruses to the development of life sciences: Virus diseases, cardiovascular diseases, and cancers are the main killers to modern human being. Excepting that, virology is very important for the development of life sciences as the follows:6The Role of Animal Viruses in Understanding the B

4、asic Outlines of Eukaryotic Gene Regulation: The first transcriptional enhancer element (acts in an orientation- and distance-independent fashion) was described in the SV40 genome, as was a distance- and orientation-dependent promoter element observed with the same virus. The transcription factors t

5、hat bind to the promoter, SP-1, or to the enhancer element, such as AP-1, AP-2, and which are essential to promote transcription along with the basal factors, were first described with SV40. Almost everything we know about the steps of messenger RNA (mRNA) processing began with observations made wit

6、h viruses. For examples, RNA splicing of new transcripts was first described with the adenoviruses. The signal for polyadenylation in the mRNA was first found using SV40. 7 The cap and methylation of bases at the 5end of mRNA was first detected using reoviruses. The discovery of the role of interfer

7、on in inducing a set of gene products that act on translational regulatory events owes its origins to virology. Posttranslational processing of proteins by proteases, carbohydrate addition to proteins in the Golgi apparatus, phosphorylation by a wide variety of important cellular protein kinases, or

8、 the addition of fatty acids to membrane-associated proteins have all been profitably studied using viruses. Indeed, a good deal of our present-day knowledge in cell biology of how protein trafficking occurs and is regulated in cells comes from the use of virus-infected cell systems. Clearly, the fi

9、eld of gene regulation has relied on virology for many of its central tenets.8The Role of Animal Viruses in the Recombinant DNA Revolution: The discovery of the enzyme reverse transcriptase in retroviruses (5,138) not only helped to prove how retroviruses replicate but also provided an essential too

10、l to produce complementary DNAs (cDNAs). The first restriction enzyme map of a chromosome, HindII plus III, was with SV40 DNA, and the first DNA to show the specificity of a restriction enzyme was SV40 DNA with EcoRI. Some of the earliest DNA cloning experiments used SV40 DNA into lambda, or human b

11、-hemoglobin genes into SV40 DNA, to construct the first mammalian expression vectors. Indeed, a debate about these very experiments led to a temporary moratorium on all such recombinant experiments. 9 From the beginning, several animal viruses had been developed into expression vectors for foreign g

12、enes, including SV40, the retroviruses, the adenoviruses, and adeno-associated virus, which has the remarkable property of site preferential integration. Modern day strategies of gene therapy will surely rely on some of these recombinant viruses. The first cDNA cloning of hemoglobin sequences utiliz

13、ed lambda vectors for the cloning and replication of these mRNA copies. In a nice twist of events, the long-elusive hepatitis virus C (non-A, non-B) was cloned from serum using recombinant DNA techniques, reverse transcriptase, and lambda phage vectors. 10The Role of Animal Virology in Oncology: It

14、is not too strong a statement to say that we owe a great proportion of our present understanding of the origins of human cancers to two major groups of animal viruses, the retroviruses and DNA tumor viruses. The oncogenes were first discovered and proven to exist in a virus and then in the host cell

15、 genome using Rous sarcoma virus. A wide variety of retroviruses have captured, altered, and delivered oncogenes to the virologists. The insertion of retroviruses into the genomes of cancerous cells also helped to locate additional oncogenes. The second group of genes that contribute to the origins

16、of human cancers, the tumor suppressor genes, has been shown to be intimately associated with theDNA tumor viruses. 11 Genetic alterations at the p53 locus are the single most common mutations known to occur in human cancers (60% to 65% of the time). The p53 protein was first discovered in associati

17、on with the SV40 large T-antigen . SV40, the human adenoviruses, and the human papilloma viruses all encode oncogenes that produce proteins that interact with and inactivate the functions of two tumor suppressor gene products, the retinoblastoma susceptibility gene product (Rb) and p53. The cellular

18、 oncogenes and the tumor suppressor genes in human cancers have been studied and understood most profitably using these viruses. 12 The viruses that cause cancers have provided some of the most extraordinary episodes in modern animal virology. The story of the Epstein-Barr virus and its role in seve

19、ral cancers, as well as in infectious mononucleosis, provides us with the best in detective story science. The story is not yet complete and many mysteries remain. Similarly, the identification of a new pathologic disease, adult T-cell leukemia, led to the isolation of a virus that causes the diseas

20、e and the realization that this virus human T-cell leukemia virus (HTLV-1) had been found previously. Although this discovery provided the virus, there is yet to be a satisfactory explanation of how this virus contributes to adult T-cell leukemia.13 Equally interesting is the road to the hepatitis B

21、 virus and hepatocellular carcinomas. By 1967, S. Krugman and his colleagues had good evidence distinguishing between hepatitis A and B viruses, and in the same year B. Blumberg et al. detected the Australia antigen. Through a tortuous path, it eventually became clear that the Australia antigen was

22、a diagnostic marker for hepatitis B. Although this freed the blood supply of this dangerous virus, Hilleman at Merck, Sharp and Dohme and the Chiron Corporation (which later isolated the hepatitis C virus) went on to produce the first human vaccine that prevents hepatitis B infections and very likel

23、y hepatocellular carcinomas associated with chronic virus infections. The idea of a vaccine that can prevent cancer comes some 82 to 85 years after the first discoveries of tumor viruses. At present, in many countries, newborn infants have been inoculated to prevent hepatitis B infections. Based on

24、the epidemiologic predictions, this vaccination program should result in significant reduction of liver cancer cases in 40 to 50 years from now.14Vaccines: The Salk and then Sabin poliovirus vaccines were the first beneficial products of the cell culture revolution. In the early 1950s in the United

25、States, just before the introduction of the Salk vaccine, about 21,000 cases of poliomyelitis were reportedannually. Today, the number is fewer than 10. Among the most remarkable achievements of last century is the complete eradication of smallpox, a disease with a history of over 2,000 years. In 19

26、66, the World Health Organization began a program to immunize all individuals who had come into contact with an infected person. This strategy, as opposed to trying to immunize an entire population (which simply was not possible), worked and, in October 1977, Ali Maolin of Somalia was the last perso

27、n in the world to have a naturally occurring case of smallpox. Because smallpox has no animal reservoir and requires person-to-person contact for its spread, most scientists agree that we are free of this disease. What most scientists do not agree on is whether we should store smallpox virus samples

28、 as a reference for the future. 15 The viral vaccines used in the past have included live attenuated vaccines, killed virus vaccines, and subunit vaccines. Both the killed virus vaccine (Salk) and the recombinant subunit vaccine (hepatitis B, S antigen) were new to the modern era ofvirology. In the

29、future, we will see one virus (vaccinia virus) presenting the antigens of a different virus, the injection of DNA-encoding viral antigens, and the use of specific interleukins or hormones with vaccines to stimulate immunity at specific locations in the host and to elucidate specific immunoglobulin c

30、lasses.16Chapter 2: Taxology of Virus17Classification: A coherent and workable system of classification, a taxonomy, is a critical component of the discipline of virology. However, the unique nature of viruses has defied the strict application of many of the traditional tools of taxonomy used in oth

31、er disciplines of biology. Thus, scientists who concern themselves with global taxonomy of organisms have traditionally left the viruses scattered throughout the major kingdoms, reasoning that viruses have more in common with their individual hosts than they do with each other.18 Usually, based on t

32、heir hosts, you can call some viruses as animal viruses (associated with human health), plant viruses (Important to agriculture and national economy), insect viruses, avian viruses, bacterial viruses (phages), and others. Also, prokaryotic viruses (phages) and eukaryotic viruses can be often found i

33、n some books or papers. So far, more than 4,000 species of virus have be established in the world. All viruses are taxologically sorted into an internationally acceptable system named as International Virus Taxological System. This system is handled by ICTV (International Committee of Taxonomy of Vi

34、rus). The ICTV is a committee of the Virology Division of the International Union of Microbiological Societies. The objectives of the ICTV are to develop an internationally agreed-upon taxonomy and nomenclature for viruses, to maintain an index of virus names, and to communicate the proceedings of t

35、he committee to the international community of virologists. The ICTV publishes an update of the taxonomy at approximately 3-year intervals. 19 The ICTV also supports a web site (/ICTV/), which contains all its published information in a conveniently interactive format, plus

36、 links to additional sites of interest, including the universal virus database of the ICTV (ICTVdB). If you want to learn the detailed information about the system, the following websites are available for that: .au/viruses/welcome.htm http:/www.res.bbsrc.ac.uk/mirror/auz/welcome.h

37、tm /ICTVdB/welcome.htm Most important, the virus taxonomy that has been developed works well. For the trained virologist, the mention of a virus family or genus name, such as family Herpesviridae or genus Rotavirus, immediately conjures forth a set of characteristics that f

38、orm the basis for further discussion or description. Virus taxonomy serves an important practical purpose as well, in that the identification of a limited number of biologic characteristics, such as virion morphology, genome structure, or antigenic properties, quickly provides a focus for identifica

39、tion of an unknown agent for the clinician or epidemiologist and can significantly impact further investigation into treatment or prevention of a virus disease.20Virus Properties and Their Use in Taxonomy: The taxonomic method adopted for use in virology is polythetic, meaning that any given virus g

40、roup is described using a collection of individual properties. The description of a virus group is nonsystematic in that there exists no fixed list of properties that must be considered for all viruses, and no strict formula for the ordered consideration of properties. Instead, a set of properties d

41、escribing a given virus is simply compared with other viruses described in a similar fashion to formulate rational groupings. Dozens of properties can be listed for description of a virus, but they break down generally into virion morphology, including size, shape, capsid symmetry, and presence or a

42、bsence of an envelope, virion physical properties, including genome structure, sensitivity to physical or chemical insults; specific features of viral lipids, carbohydrates, and structural and nonstructural proteins; antigenic properties; and biologic properties including replication strategy, host

43、range, mode of transmission, and pathogenicity. 21The Hierarchy: The ICTV has adopted a universal classification scheme that employs the hierarchical levels of order, family, subfamily, genus, and species. Because the polythetic approach to classification introduces viruses into the middle of the hi

44、erarchy, and because the ICTV has taken a relatively conservative approach to grouping taxa, levels higher than order are not currently used. Levels lower than species, such as strains and variants, are not officially considered by the ICTV but are left to specialty groups.22Chapter 3: Structures an

45、d Life CycleI. Structures:232425Shapes and Size of Virus:nThe main shapes of virus are as the follows: 1. Spheroid or globoid virus with or without envelope. The number of virus member in this shape is biggest. Usually, they host in human being or animal. 2. Band or rod form virus. Usually, host in

46、plants or insects. 3. Brick form virus. Brick form viruses are biggest and most complex viruses. Usually, host in human being or animal. 4. Virus with a globoid head and a rod tail. Usually, host in bacteria, and we give them a different name: Phage. 5. Insect viruses inside inclusion body. One incl

47、usion body contain many virus particles inside.26nVirus size is very different between viruses from hundreds nm to ten more nm. So, usually, we use electron microscope to check or make observation on virus particles. nYou have to remember the follows in your mind: 1. The resolving power of an optica

48、l microscope = 0.25m (250nm or 2500) 2. The resolving power of an electron microscope = 2.5 3. The diameter of a RBC = 8m, The diameter of a DNA double chain helix = 20, The diameter of an -helix = 10, The diameter of an atom = 2 - 3.27Virus Life Cycle: Viruses have to parasite in an alive cell to s

49、urvive and replicate because they are so simple that they have no any basic structure to survive independently. Replicative Cycle: Adsorption to cell Invasion into cell Remove their envelope or coat Synthesize their DNA or RNA with nucleotides from host Assemble a new virus particle with the protein

50、 and others from host Release themselves from the hosted cell to out Another adsorption to a new cell28How to culture and amplify virus?nBacterial culture for phage: E. coli strains and Plaque.nCell culture for human or animal virus: Cell culture technology.nProtoplasm culture for plant virus: Proto

51、plasm: Plant cell Remove away the shell or wall Remained part can be cultured and grow up. 29nAdsorption to cell:1. Virus Surface Adsorption Protein: The property and the electrostatic charge can affect or decide the combination of virus and cell. But, the combination mediated by electrostatic charg

52、e is reversible usually. I want to you know gp120 for HIV at least.2. Virus Receptor on Cell Surface: The receptor can be recognized by virus adsorption protein. Receptor is very important to the host and tissue specificity, and the diseases caused by virus. 3. Temperature, Concentration of ion, and

53、 pH can put effects on the electrostatic charge and the mobility of cells and virus. So, their changes will result in the virus adsorption and invasion.30nInvasion (Penetration, Insert) into cell:1. Invasion of phages: (1) Injection with anal filament, and (2) infect bacteria with sex fimbria.(1)(2)

54、312. Invasion of animal viruses: (1) Endocytosis, (2) Fusion (Envelope and Membrane), (3) Translocation.(1)(2)(3)32CellBacterium333435363738Host CellParasited CellCell ReceptorVirus ParticleRemoved EnvelopeAssembling Virus ParticleDying CellReplicating Virus DNA/RNAReleased Complete Virus Particle39

55、4041424344454647M13KE4849505152535455Hosted cellIntramolecular Recombination: ssDNA or ssRNA breakage and exchange.Copy-choice Recombination: ssRNA. RNA polymerase chooses a sequence as template and synthesize RNA.Genome re-assemble: Virus with fragmented genome. Fragments can be exchanged between v

56、irus genomesParticles of virus 1Particles of virus 2Or more than 2 virusNew virus with recombined genome56575859Your interested sequence data from a virus genomeSequence 1 (Labeled) Sequence 2 (Labeled) Mutant 1 Mutant 2Infect same cellRecombinationHigher ratio of recombinantLower ratio of recombina

57、ntShorter distance between the two sequences (or genes)Longer distance between the two sequences (or genes)Check the two sequences are from same or different genes, their function and nameRepeat the steps above to other two genesFigure out the virus gene map6061Electrophoresis to virus DNA, RNA geno

58、me fragments and protein from different serotype or sero-strain of mutated parental and filial virus particlesAnalyze the phenotype and band poly-morphology Find the fragment on that the mutation locatedCompare the protein bands poly-morphology Repeat analysis to a lot of mutants of this fragmentFig

59、ure out the mutation site and gene location62Gene Map63This is the original (Wild type) SV40 map64Modified 165Modified 26667DNA VirusesRNA VirusesMutationRecombinationMutationRecombinationReassortment68nDivergence rate of RNA viruses:697071SV40SV40 T antigen combines to ICE (Interleukin Change Enzym

60、e family)SV40 T antigen combines to p53 (a tumor suppressor gene)Apoptosisbcl-2 (a protooncogene)p35 (a apoptosis suppressor gene)Some virusesApoptosisBlock72737475IFN IIFN II767778IFN Inducing798081828384858687888990919293949596Chapter 11: The purification, detection and diagnosis of virusThe purif