microbiology- part iiocw.nctu.edu.tw/course/biology/microbiology971/virus08-111819.pdf ·...
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Microbiology- Part II
http://life.nctu.edu.tw/~hlpeng/
彭慧玲 分機:56916 ([email protected])
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11/18, 11/19 Viruses I (Chapter 16, 17)11/25, 11/26 Viruses II (Chapters 18, 37)12/2, 12/3 Protist and fungi (Chapters 25, 26, 39)12/9, 12/10 Bacterial genetics and genomics (Chapters 13, 15)
12/16 Exam I (25%)
12/17, 12/23 Taxonomy, archaea and extremophiles (Chapters 19, 20)12/24, 12/30, 12/31 Bacteria (Chapters 21-24, 38)1/6, 1/7 Microbial diseases and their control (Chapters 33, 34)
1/13 Final exam (25%)
Timetable
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Chapter 16
The Viruses: Introduction and General Characteristics
11-18-200811-19-2008
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Nobel Prize 2008 Physiology or Medicine
Françoise Barré-Sinoussi and Luc Montagnierfor their discovery of "human immunodeficiency virus"
Harald zur Hausenfor his discovery of "human papilloma viruses causing cervical cancer"
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A major cause of mortalityHistorical evidence suggests that epidemics caused by measles and smallpox viruses were among the causes for the decline of the Roman EmpirePandemics and epidemics
1997~ Avian flu virus (H5N1)2003~ SARS virus2001~ Enterovirus 711996~ Foot and Mouth disease (FMD)1993~ Ebola and Hantann viruses1989~ Dengue viruses
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Viruses
Virology and virologistsAcellular and infectious agentsA filterable agent
10 nm~ 300 nmException: Mimivirus
filtrate through 0.45 μm filter 0.22 μm filter for cell culture system
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mimicking microbe virus A 2S circular DNA virus ~ 800 nm diameter1.2 Mb genome (1260 genes)No ribosome machinery
Science 306 (October 2004)
Several times bigger than the known largest virus (small poxvirus ~ 300 nm)
The giant virus- Mimivirus -discovered in 1992, nestling inside an amoeba inside a cooling tower in Bradford (Bradford coccus), UK
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Early attempts to prevent viral disease - vaccine
Lady Wortley Montagu (early 1700s)proponent of inoculation with material from smallpox lesions
Edward Jenner (1798)prevent smallpox by exposure to cowpox
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Discovery of viruses (1)Louis Pasteur- an infectious agent of rabiesCharles Chamberland (1884)- developed porcelain bacterial filters
Dimitri Ivanowski (1892)- a filterable agent of tobacco mosaic diseaseMartinus Beijerinck (1898-1900)- a filterable virus- tobacco mosaic virus (TMV)
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Discovery of viruses (2)
Loeffler and Frosch (1898-1900) a filterable virus caused hoof-and-mouth disease in cattle
Walter Reed (1900)yellow fever in humans was caused by filterable virus transmitted by mosquitoesarbovirus (arthropod-borne virus)
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Discovery of viruses (3)
Ellerman and Bang (1908)showed that leukemia in chickens was caused by a virus
Peyton Rous (1911)muscle tumors in chickens were caused by a virus ( Rous Sarcoma virus)
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Discovery of bacterial viruses (4)
Frederick Twort (1915)bacteria-infecting virus- bacteriophages
Felix d’Herelle (1917)- the existence of bacteriophages
enumeration method only reproduce in live bacteria
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The chemical nature
W. M. Stanley (1935)crystallized TMV TMV was composed mostly of protein
F. C. Bawden and N. W. Pirie (1935)TMV particles: protein and nucleic acidcomponents
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General Properties
Virion (extracellular form)consists of ≥1 molecule of DNA or RNAenclosed in protein coat- capsidnucleocapsid
nucleic acid held within protein coatprotomer: subunit of the capsid
may have additional layers- envelopea host-derived membrane structure
lipids and carbohydratespeplomers (spikes)
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Generalized Structure of Viruses
Figure 16.1
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Morphology of Selected Viruses
Figure 16.2
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Helical symmetry-TMV
Figure 16.3
Hollow tubes with protein walls
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Influenza Virus – an Enveloped Virus with a Helical Nucleocapsid
Figure 16.4
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Icosahedral capsid structure
capsomersring- or knob-shaped units made of five or six protomerspentamers (pentons) –five subunit capsomershexamers (hexons) –six subunit capsomers
Figure 16.6
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Capsid of Complex Symmetry
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21Figure 16.9
Capsid of Binary symmetry
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Enveloped viruses- many animal viruses
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Viral EnzymesSome associated with envelope or capsidmost within the capsid- RNAP
Influenza virus
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Viral Genome Acids
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Virus Reproduction
Figure 16.12
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Hosts for animal viruses-embryonated eggs
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Host for animal viruses-tissue (cell) cultures
-monolayers cells- plaques
localized area of cellular destruction and lysis- PFU- plaque forming unit
Hosts for Bacteriophage-
Bacteria
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cytopathic effects
microscopic or macroscopic degenerative changes or abnormalities in host cells and tissues
Fibroblast cell
Adenovirus infection
Herpes virus infection
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Hosts for Plant Viruses
- plant tissue cultures- plant protoplast cultures- suitable whole plants
may cause localized necrotic lesions or generalized symptoms of infection
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Virus Purification
four commonly used methodsdifferential centrifugation and density gradient centrifugationprecipitation of viruses
commonly uses ammonium sulfate or polyethylene glycol (protein coat)
denaturation of contaminantsenzymatic digestion of cell constituents
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Differential centrifugation
Figure 16.18
• Size separation
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Density gradient centrifugation
size and density
Figure 16.19 (a)
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33Figure 16.19 (b)
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Virus assay- Particle counts
direct countsmade with an electron microscope
indirect countshemagglutination assay
determines highest dilution of virus that causes red blood cells to clump together (Fig. 35.11)
Figure 16.20
virus particles
Virus +RBC agglutination
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Measuring concentration of infectious unitsplaque assays
dilutions of virus preparation plated on lawn of host cellsnumber of plaques counted- PFU
infectious dose and lethal dose assaysdetermine smallest amount of virus infection or death of 50% of exposed host cells or organismsexpressed as ID50 or LD50
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Determination of LD50
Figure 16.21
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37 Table 16.2
Principles of Virus Taxonomy
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Chapter 17
Viruses of Bacteria
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3939Figure 17.1
Major phage families
- morphology- tail or tailless- nucleic acid
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Reproduction of 2S DNA PhagesLytic cycle
phage life cycle that culminates with host cell bursting, releasing virions
virulent phagesphages that lyse their host during the reproductive cycle
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The One-Step Growth Experimentmix bacterial host and phage
↓brief incubation
(attachment occurs)↓
dilute greatly(to release the viruses that can’t infect cells)
↓over time, collect sample and enumerate
viruses
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free viruses
no virions –either free orwithin host
latent period –no viruses releasedfrom host
rise period –viruses released
Figure 17.2
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Life Cycle of T4 Phage
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Adsorption, penetration, and DNA injection
“Receptor”specific surface
structures on hostcan be proteins, LPS
(lipopolysaccharides), techoic acids, etc.
empty capsid remains outside of host cell
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Early mRNA resulting in production of protein factors and
enzymes involved in phage DNA synthesis (DNAP)DNA replication
synthesis of proteins that enable T4 to take over host cell
Late mRNA encode capsids and other proteins needed for
phage assembly and proteins required for cell lysisand phage release
Sequential process
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To regulate host RNA polymerase (RNAP)Strong viral promoterSynthesis of viral RNAPSynthesis of viral specific sigma factor
To modify host RNAPADP ribosylation of host RNAP
To produce enzymes needed for viral genome replication
DNA methylase and glucosylase
Early mRNA synthesis
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Replication Strategy of 2S DNA Viruses
Figure 17.6
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Synthesis of T4 DNA
contains hydroxymethyl-cytosine (HMC) instead of cytosineHMC glucosylation
protects phage DNA from host restriction endonucleases
Figure 17.9
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T4 DNA is terminally redundant
base sequence repeated at both endsallows for formation of concatamers
Figure 17.10
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Assembly of Phage Particles
Figure 17.11
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Release of Phage ParticlesT4 - E. coli system
~150 viral particles are releasedtwo proteins are involved in process
T4 lysozyme attacks the E. coli cell wallholin creates holes in the E. coli plasma membrane
Figure 17.13
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Reproduction of φX174 1S (+) DNA Virus
newvirionsreleasedby lysisof host
by usualDNA replicationmethod
by rolling-circlemechanism
Figure 17.14
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Reproduction of RNA Phages
most are plus strand RNA virusesincoming RNA acts as mRNA and directs the synthesis of phage proteins
double-stranded RNA viruses such as φ6 have also been discovered
Phage φ6 is unusual because it is enveloped
like MS2 and Qβ it attaches to the side of the F pilus but uses an envelope protein for adsorption
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Replication of (+) RNA bacteriophage
Figure 17.16
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Temperate phages and lysogenyTemperate phages have two reproductive options
reproduce lytically as virulent phages doremain within host cell without destroying it
done by many temperate phages by integration of their genome with the host genome in a relationship called lysogeny
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Lysogeny
Prophage- integrated phage genomeLysogens- infected bacterial host
they are immune to superinfectionunder appropriate conditions they will lyseand release phage particles, a process called induction
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Lysogenic conversion
change in host phenotype induced by lysogeny
modification of Salmonella lipopolysaccharidestructure alter antigenic propertiesproduction of diphtheria toxin by Corynebacterium diphtheriae
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Lambda phage
2S DNA phagelinear genome with cohesive ends
circularizes upon entry into host
Figure 17.17
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Lambda Phage DNA
the DNA contains 12 base single-stranded cohesive endscircularization results from complementary base pairing
Figure 17.18
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Infection by Lambda Phage
Two proteins appear after infectionCI the lambda repressor
product of cI geneblocks transcription of the cro gene and other genes required for the lytic cycle
Cro protein product of cro geneinhibits transcription of the lambda repressor gene
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If lambda repressor CI wins…
Lysogeny is establishedlambda genome is integrated into the host genome in a reaction catalyzed by integrase
Figure 17.22
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Cro ProteinIf Cro protein wins blocks synthesis of lambda repressor prevents integration of the lambda genome into the host chromosome lytic cycle
Figure 17.23
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λ phage Inductiontriggered by dropped levels of CI
caused by exposure to UV light and chemicals that cause DNA damage
excisionasebinds integrase and enables it to reverse integration process
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Bacteriophage genomesMosaic genomes
blocks of related sequences are shared suggests lateral gene transfer and nonhomologousrecombination have played a role in phage evolution
Figure 17.24
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Chapter 18
Eucaryotic Viruses and other acellular infectious agents
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Plant virus
entry of virus requires mechanical damage, usually caused by insects or animals that feed on hostmost plant viruses are RNA viruses
most are plus-strand RNA
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Viruses of Fungi and ProtistsFungal viruses
higher fungi infected with dsRNA viruseslower fungi infected by dsRNA or ssRNA viruses
Algal viruses4 genera recognized have linear dsDNA genomes
protozoan virusesonly 3 genera studiedGiant dsDNA virus (a Mimivirus)
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Insect Viruses
infection often accompanied by formation of granular or polyhedral inclusion bodieshave potential as biological control agents for insect pests
Figure 18.18
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Viroids
Cause plant diseases by triggering RNA silencingsome found in nucleolus, others found in chloroplast
Rodlike shape of circular, 1S-RNAs ( ~250-370 nt)unable to replicate itself (not encode gene products)
Escaped intron
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Virusoidslike viroids are covalently closed circular, ssRNA molecules capable of intrastand base paringunlike viroids, they encode one or more gene products and need a helper virus to infect host cells
Delta virus (HDV)