1

Microbiology- Part II

http://life.nctu.edu.tw/~hlpeng/

彭慧玲 分機:56916 (hlpeng@mail.nctu.edu.tw)

2

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

3

Chapter 16

The Viruses: Introduction and General Characteristics

11-18-200811-19-2008

4

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"

5

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

6

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

7

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

8

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

9

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)

10

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)

11

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)

12

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

13

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

14

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)

15

Generalized Structure of Viruses

Figure 16.1

16

Morphology of Selected Viruses

Figure 16.2

17

Helical symmetry-TMV

Figure 16.3

Hollow tubes with protein walls

18

Influenza Virus – an Enveloped Virus with a Helical Nucleocapsid

Figure 16.4

19

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

20Figure 16.8

Capsid of Complex Symmetry

21Figure 16.9

Capsid of Binary symmetry

22 Figure 16.10

Enveloped viruses- many animal viruses

23

Viral EnzymesSome associated with envelope or capsidmost within the capsid- RNAP

Influenza virus

24 Table 16.1

Viral Genome Acids

25

Virus Reproduction

Figure 16.12

26Figure 16.13

Hosts for animal viruses-embryonated eggs

27Figure 16.14

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

28Figure 16.15

cytopathic effects

microscopic or macroscopic degenerative changes or abnormalities in host cells and tissues

Fibroblast cell

Adenovirus infection

Herpes virus infection

29Figure 16.17

Hosts for Plant Viruses

- plant tissue cultures- plant protoplast cultures- suitable whole plants

may cause localized necrotic lesions or generalized symptoms of infection

30

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

31

Differential centrifugation

Figure 16.18

• Size separation

32

Density gradient centrifugation

size and density

Figure 16.19 (a)

33Figure 16.19 (b)

34

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

35

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

36

Determination of LD50

Figure 16.21

37 Table 16.2

Principles of Virus Taxonomy

3838

Chapter 17

Viruses of Bacteria

3939Figure 17.1

Major phage families

- morphology- tail or tailless- nucleic acid

4040

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

4141

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

4242

free viruses

no virions –either free orwithin host

latent period –no viruses releasedfrom host

rise period –viruses released

Figure 17.2

4343 Figure 17.3

Life Cycle of T4 Phage

4444 Figure 17.4

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

4545

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

4646

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

4747

Replication Strategy of 2S DNA Viruses

Figure 17.6

4848

Synthesis of T4 DNA

contains hydroxymethyl-cytosine (HMC) instead of cytosineHMC glucosylation

protects phage DNA from host restriction endonucleases

Figure 17.9

4949

T4 DNA is terminally redundant

base sequence repeated at both endsallows for formation of concatamers

Figure 17.10

5050

Assembly of Phage Particles

Figure 17.11

5151

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

5252

Reproduction of φX174 1S (+) DNA Virus

newvirionsreleasedby lysisof host

by usualDNA replicationmethod

by rolling-circlemechanism

Figure 17.14

5353

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

5454

Replication of (+) RNA bacteriophage

Figure 17.16

5555

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

5656

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

5757

Lysogenic conversion

change in host phenotype induced by lysogeny

modification of Salmonella lipopolysaccharidestructure alter antigenic propertiesproduction of diphtheria toxin by Corynebacterium diphtheriae

5858

Lambda phage

2S DNA phagelinear genome with cohesive ends

circularizes upon entry into host

Figure 17.17

5959

Lambda Phage DNA

the DNA contains 12 base single-stranded cohesive endscircularization results from complementary base pairing

Figure 17.18

6060

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

6161

If lambda repressor CI wins…

Lysogeny is establishedlambda genome is integrated into the host genome in a reaction catalyzed by integrase

Figure 17.22

6262

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

6363

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

6464

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

6565

Chapter 18

Eucaryotic Viruses and other acellular infectious agents

66

Copyright © The McGraw-Hill

Companies, Inc. Permission requiredfor reproduction or

display 66

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

67

Copyright © The McGraw-Hill

Companies, Inc. Permission requiredfor reproduction or

display 67

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)

68

Copyright © The McGraw-Hill

Companies, Inc. Permission requiredfor reproduction or

display 68

Insect Viruses

infection often accompanied by formation of granular or polyhedral inclusion bodieshave potential as biological control agents for insect pests

Figure 18.18

69

Copyright © The McGraw-Hill

Companies, Inc. Permission requiredfor reproduction or

display 69

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

70

Copyright © The McGraw-Hill

Companies, Inc. Permission requiredfor reproduction or

display 70

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)