The Viruses The by vivi07

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									The Viruses

Part I: Introduction, General Characteristics and Viruses of Prokaryotes and Plants

I. Characteristics of Viruses
Different from living cells
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Simple, acellular organization Contain only DNA or RNA, but not both in the same virion (= complete virus particle) Cannot replicate independently of cells or engage in cell division

Virion = complete virus particle
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One or more molecules of DNA or RNA surrounded by a protein coat Additional layers may be present (e.g. in budding viruses) containing:
Protein from host and virus Carbohydrates from host Lipids from host

Extracellular and intracellular phases
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Extracellular phase
The virion contains few if any enzymes No replication

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Intracellular phase
High rate of nucleic acid synthesis Host protein synthesis machinery exploited/utilized to translate virion messages for various virion component Assembly of virions  exit from cell

II. Methods of Virus Cultivation
Cultivation in embryonated eggs – Animal viruses


6-8 days after chicken egg has been layered, a hole introduced into the surface using sterile technique, inoculation into appropriate region, reseal with gelatin
Continue incubation of egg Localized pock may form indicating where virus is replicating

Figure 16.1

Tissue culture – monolayers – Animal viruses
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Requires specific growth medium, growth factors and antibiotics Cell monolayer propagated first  contact inhibition
Then virus added  spreads out and settles onto cells (primary cell cultures, 4-5 passages only) Agar poured on to top to control intercellular spread
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Cell Death: Local lesions representing cellular necrosis develop  called plaques (observable with dyes that preferentially stain dead cells, e.g. trypan blue) Altered phenotype: Phenotypic changes associted with abnormalities develop  called cytopathic effects (observable macroscopically or microscopically)

Figure 16.3

Figure 16.2

Diploid cell strains
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40-50 passages Embryonic tissue origin Spontaneous mutation of diploid cell lines Immortal  often have aberrant morphology Oncogenic cell lines  suspension  immortal

Permanent cell lines
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Bacterial viruses – Bacteriophages (phages)
Requires log phase bacterial host cells for cultivation (young, dividing) Can be cultivated in either broth or agar If broth  lysis may result in clearing of broth and accumulation of cell debris in bottom of flask If agar  mix together bacterial and phage  pour onto pre-set agar base set  incubate  lawn forms  plaques form at site where one virion attached to surface and began its replication cycle  clear zone
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Bottom agar ~ 1.5% Top agar ~ 0.8%

Replication cycle: host cell infected, virus released, neighboring/adjacent cells infection, virus released, etc. Plaque morphology is extremely variable and characteristic of particular bacteriophage

Fig 16.4

Composition of bacteriophages
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Nucleic acid genome is enclosed by a protein capsid Nucleic acid is either DNA or RNA Icosahedral or helical Often have complex morphological features

Figure 16.19

Bacteriophage Life Cycle
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Adsorption
Required for infection Involves specific host receptors on cell wall (eg.g T4, complex) or on bacterial F pilus (male = F+) (filamentous phage, helical, e.g. M13)

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Adsorption is followed by migration to plasma membrane  fusion with membrane  release of DNA into cytoplasm

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Penetration
Injection of nucleic acid into cytoplasm Capsid does not enter the cell

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Replication
Following transcription of many enzymes, phage nucleic acid is replicated and structural components are made
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Host genome often degraded

Figures 17.3-17.4

Figure 17.5

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Assembly and release
Complex phage
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Head, tail and tail fibers combine in specific order Nucleic acid is inserted into capsid Host cell lysis after of assembly of 100-200 phage particles occurs (often associated with phage lysozyme) Assembly occurs at plasma membrane/outer membrane junctions No lysis occurs Continuous phage replication

Filametous phage (helical)
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Single stranded (SS) RNA phages (MS2, R17 and Qb)
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Icosahedral  adsorb to F pilus Lysis of host at end of cycle

Lysogeny
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Non-virulent stage of some phage (called temperature phage) where DNA incorporates into the host genome
Is replicated along with host DNA  called a PROPHAGE Bacterium harboring the prophage = LYSOGENIC

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Lytic state can be induced  phage replicates normally  lyses host cell

Lysogenic conversion
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A process resulting from expression of prophage DNA genes that alters characteristics of the host bacterium Examples
Outer membrane LPS alteration Exotoxin production
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Phage encoded Diphtheria Scarlet fever Botulism

Plant Viruses
Plant tissue culture (whole plants or microscale) (in agar) Cell suspension cultures Protoplast cultures (cell wall removed enzymatically) Whole plant inoculation  full scale (leaves rubbed with abrasive material, e.g. sand) to break cell walls so that virus contacts the plasma membrane and infects exposed cells (insect vectors accomplish the same thing)
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Cell surface receptors not involved in entry  injury required

Necroses develop at site of cell death (e.g. TMV)
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Plant reoviruses cause tumors Phenotypic changes if cells don’t die
Pigmentation changes (e.g. mottling, chloroplast destruction  yellowing) Shape of leaves distorted

Figure 16.5

Most plant viruses = ssRNA (+ or -)  icosahedral or helical capsids Some viruses package segmented genomes (several strands) in separate capsids
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Examples:
Alfalfa mosaic virus Cucumoviruses (Cucumber mosaic virus) Brome mosaic virus

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Requires all capsids to enter cell to be infectious

Gemini viruses
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ssDNA  segmented genomes (two icosahedral capsids, in pairs, each containing two ssDNA strands)

Figure 18.12

III. The Purification and Enumeration of Viruses
Properties of viruses that lend themselves to isolation techniques
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Large size Often more resistant to chemicals, denaturing treatments, and enzymes (nucleases and proteases) Coated with surface proteins

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Examples of isolation techniques:
Differential centrifugation (acc. to size) Density gradient centrifugation (acc. to size and buoyant density) Rate zonal gradient centrifugation (acc. to sedimentation rates and density) Precipitation (ammonium sulfate, polyethylene glycol) Denaturation/removal of contaminants (chloroform, butanol,…. to eliminate proteins and lipids) Enzymatic digestion of cell constituents (nucleases, proteases)

Figure 16.6

Figure 16.7

Enumeration of viruses:
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Different types of assays for direct counting of particles or determination of infectious unit concentration (# particles is not equivalent to #infectious units because not all particles will attach to the correct entry site on the host cell)

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Electron microscopy (often combined with concentrating sample by centrifugation  spread on grid with latex beads, view, counted)

Figure 16.8

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Hemagglutination assay (virus clumps RBC together  agglutination  viral titer determined Plaque assay (determination of infectivity – pfu) Endpoint method
Determination of concentration (or dilution) required to damage or destroy 50% of host cells or organisms
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LD50, ID50

IV. Virus Structure
Size and Morphology
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Size of virions is variable (10 – 400nm dia) Smallest = ~ ribosome size (fX174) Largest = ~ smallest bacterium (vaccinia – poxviridae)

Figure 16.10

Structural features
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Four main morphological types of virions:
Icosahedral = polyhedron (20 equilateral triangular faces and 12 vertices) Helical – Hollow protein cylinders (rigid or flexible) Enveloped – Outer membrane – host derived; Nucleocapsid either icosahedral or helical Complex viruses – multiple components: may have tails, feet and multiple layers

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Nucleocapsids
Virions assembled around a core of nucleic acid (DNA or RNA) surrounded by a protein coat (= capsid: protein coat or shell that surrounds a virion’s nucleic acid) Basic unit of the virion structure In some cases, may constitute entire virion (e.g. papilloma virus and picornavirus), but in other cases, budding from the plasma membrane may occur (e.g. some animal viruses)

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Protein subunits = Protomers
Utilization of limited genetic material to highest efficiency possible by having repeating subunits that arrange themselves in highly ordered array Associate with each other during capsid assembly  spontaneous  self-assembly If virus is complex, then assembly of multiple pieces may be factor-dependent

Helical capsids
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Hollow cylinders surrounded by a protein coat TMV Influenza (flexible and enveloped) Size is dependent on protomers (diameter) and nucleic acid (length)

Figure 16.11

Figure 18.13

Icosahedral Capsids
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One or few gene products required Efficient strategy to enclose nucleic acid Hexagons and pentagons utilized most often Capsomers used to form capsids
Synthesized form knob-shaped or ring-shaped units  made up of 5 or 6 protomers
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Pentamers (pentons)  5 protomers Located at vertices of icosahedron Hexamers (hexons)  6 protomers Located at edges and triagular faces

Non-covalently associated

Figure – Icosahedral Capsid
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42 capsomers (all protomers are identical) Larger icosahedra are possible with addition of more hexons
Example: Adenovirus has 252 capsomers; protomers = different proteins (pentamers contain different subunits than hexamers)

Figure 16.13

SV40 (Simian Virus 40) – uses only pentamers
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72 cylindrical pentamers with hollow centers Flexible arms hold the pentamers together

http://rhino.bocklabs.wisc.edu/virusworld/jysart/sv40+STNV_asv2001.jpg

Figure 16.14

V. Nucleic Acids of Viruses
Four types of nucleic acid types:
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ssDNA (bacteriophage, animal viruses) dsDNA [bacteriophage (most often), animal viruses] ssRNA (most plants, bacteriophage, animal viruses) dsRNA (animal viruses)

Size of genetic material = variable
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Smallest (MS2 and Qb) ~ 1 million Daltons (34 proteins) Largest (T-even bacteriophages, Herpes virus, Vaccinia virus) ~100 – 160 million Daltons (~100 proteins) Examples:
Refer to Table I6.1

Figure 16.15

Figure 16.16

Plus (positive) strand RNA
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RNA of virus = same strand as viral mRNA Can be translated directly RNA of virus = complementary to mRNA Cannot be translated directly Several separate strands (up to 12 possible) All strands needed for infectivity Usually all segments enclosed in same nucleocapsid
Exception: Brome mosaic virus; 4 segments in 3 different virus particles

Minus (negative) strand RNA
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Segmented genomes
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Segments can be plus strand RNA, minus strand RNA and dsRNA (note that all dsRNA viruses are segmented)

VI. Viral Envelops and Enzymes
Animal virus envelopes (pleomorphic – flexible)
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Derived from nuclear or plasma membrane lipid bilayers Carbohydrate and lipid from host Some proteins from host Some proteins = virus specific
May project from surface = spikes or peplomeres Example: Influenza  hemagglutinin (HA) and neuraminidase (NA) spikes

Fig 16.17

Spikes often used for attachment to host cell surface
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Examples:
HSV type 1: binds fibroblast growth factor receptor Rabies: Binds acetyl choline receptor on neurons HIV gp120: Bind CD4 on T-cells and macrophages

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Useful for identification purposes

Some viruses = “ether sensitive”
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Lipid component dissociates  inactivation

Proteins that are embedded = glycoproteins (have sugar moieties attached to amino acid residues) Non-glycosylated proteins of virus
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Not exposed on the cell surface Found on inner surface of envelope (e.g. matrix protein of influenza)

Capsid-associated enzymes
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Usually found in enveloped viruses Often carry a nucleic acid replication enzyme Examples:
Influenza carries an RNA-dependent RNA polymerase that copies minus strand RNA to plus strand RNA for production of mRNA (RNA transcriptase) HIV carries an RNA-dependent DNA polymerase that copies plus strand RNA to single-stranded DNA (copy DNA) (Reverse Transcriptase)

VII. Complex Symmetry Capsids
Definition
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Not radially symmetrical, but contain more than one type of structure Examples
Poxviruses
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Largest  can see with phase contrast microscopy!! Complex internal structure dsDNA + proteins  contained in a nucleoid (biconcave disc) 2 elliptical or lateral bodies reside abetween nucleoid and the outer envelop Network of tubules and fibers Exterior = oval or brick-shaped

Figure 16.18

Bacteriophages
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T-even coliphages of E. coli (T2, T4, T6) Icosahedral head contains DNA Tail: collar and hollow core Helical sheath surround core Complex base plate (hexagonal) – tail pins and tail fibers (tail fibers aid in attachment to bacterial cell) Binal symmetry = combination of icosahedra and helical symmetry

Figure 16.19

VIII. Principles of Virus Taxonomy
Basis of classification (International Committee for Taxonomy of Viruses)
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73 families and groups based on three properties with greatest importance
Type of nucleic acid Nucleic acid strandedness Presence or absence of an envelope

Virus family names – viridae (Poxviridae) Subfamily – virinae (Chloradopoxvirinae) Genus – (+ species) – virus (Poxviruses) Several genera within a subfamily divided based on immunologic features and host specificity
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Example: Genus – Orthopoxvirus
Species:
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Variola major (small pox) Vaccinia (cow pox)

Taxonomic group divisions based upon:
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Nature of host (animal, plant, bacteria, insect, fungus) Nucleic acid features Capsid symmetry (icosahedral, helical, binal) Envelope Diameter of virion or nucleocapsid # of capsomers in icosahedral viruses Immunologic characteristics Intracellular location of viral replication Presence or absence of DNA intermediates Disease, symptoms, transmission

Figure 18.1 - DNA Viruses of Animals

Figure 18.2 – RNA Viruses of Animals

Figure 18.3

The Viruses

Part II: Viruses of Animals

I. Life Cycle of a Virus
Adsorption of virions to host cell surface
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Mediated by random collisions followed by binding of virion to glycoprotein on host cell plasma membrane Host/tissue specificity determined by receptor phenotype  tropism
Receptors on host may project out and bind in deep depression on virion Receptors on host may bind to spikes on virion (e.g. hemagglutinin, HA)

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Virion enzymes may aid in adsorption process
Example: Neuraminidase (NA) degrades mucopolysaccharides of nasal and respiratory tract secretions

Figure 18.5

Entry into the host cell  Penetration and uncoating
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Variable – depends on whether virus is enveloped or not, whether enzymes must be co-delivered Can be fast or slow (minutes to hours)

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Three different modes of entry:
Nucleic acid may be infected/released into cell cytoplasm following capsid deformation (naked viruses) Fusion of envelope with plasma membrane
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Release of nucleocapsid into cytoplasm Transcription of viral RNA while still in the capsid by viral RNA polymerase Uncoating finishes inside Coated vesicles form and pinch off Clathrin removed, recycles Fusion with lysosomes Decreased pH aids uncoating Release of nucleic acid

Receptor-mediated endocytosis (clathrin-coated pits)
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Fig 18.4

Replication and gene transcription in DNA viruses
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Early genes transcribed  involved in DNA and RNA synthesis Host’s normal function may or may not be affected (virulent vs. non-virulent, respectively) DNA synthesis usually occurs in nucleus (not for Poxviruses)

Examples of DNA Viruses:
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Parvovirus
ssDNA ~4800 bases  3 polypeptides
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All capsid proteins, NOT enzymes Overlapping genes, different reading frames Therefore viral DNA replication occurs in nucleus during S phase of cell cycle (when host cell normally replicates its own genome) Active cell division required for virus propagationo

Host cell provides all necessary enzymes
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Herpes virus
dsDNA – enveloped, 160,000 base pairs  50-100 gene products Uncoats, transcribes by host RNA polymerase
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Early mRNA  viral DNA synthesis and regulation Viral DNA polymerase replicaes virus in the nucleus. Host DNA synthesis slows down.

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Poxvirus
dsDNA – complex virus  200 genes Receptor-mediated endocytosis in clathrin-coated pits Release of central core from lysosome into the cytoplasm DNA transcribed by virus’ DNA-dependent RNA polymerase (in CORE!)
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One of the protein products = enzyme that aids in uncoating DNA replication using viral DNA polymerase in cytoplasm Late mRNA begins  structural components for capsid

Replication and gene transcription in RNA viruses
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Four different types of RNA viruses
(+) RNA
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Example: Poliovirus  RNA genome serves as mRNA Continuous transcript Continuous polypeptide Cleaved by host and viral proteases to generate final products Viral replicase converts +RNA into +/- RNA replicative form  Correct template strand used to direct synthesis of viral +RNA genome

Figure 18.6

(-) RNA
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Example: Influenza  RNA-dependent RNA polymerase of virus = Transcriptase  mRNA generated first Viral replicase converts -RNA into +/- RNA replicative form  Correct template strand used to direct synthesis of viral -RNA genome

Figure 18.6

dsRNA
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Example: Reovirus  (-) RNA strand transcribed by virus-associated transcriptase  then transcription continues with a new virus-encoded polymerase mRNA translated into viral proteins Viral replicase uses mRNA to produce dsRNA for virion

Figure 18.6

Retroviruses
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Example: HIV (human immunodeficiency virus)  DNA intermediates involved Reverse transcriptase (RT)-associated virus (RNAdependent DNA polymerase) + RNA associated primer +RNA  -DNA/+RNA duplex  RNAseH component of RT  Degradation of +RNA  -DNA  copied by RT into dsDNA = proviral DNA Proviral DNA converted into a circular form  Integration into host cell genome Activated host cell’s transcription factors bind to proviral Long Terminal Repeat (LTR) and promote trancription and production of +RNA +RNA is produced by transcription: two fates 1. Translation: into structural proteins, RT, glycoproteins 2. Replication: packaged into virions, assembly at cell membrane and release by budding

Figure 18.6

Figure 18.7

In general:
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Most DNA viruses replicate in the nucleus and assemble there
Exceptions:
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Poxvirus replicates in the cytoplasm because it encodes its own enzyme for DNA synthesis Poxvirus, Hepadnavirus and Iridovirus assemble in the cytoplasm

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Most RNA viruses replicate in the cytoplasm and assemble there or at membranes
Exception: Orthomyxoviruses (influenza) – replicates 8 segments of –RNA in the nucleus

Synthesis and Assembly of Viral Capsids
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Late genes are expressed  encode capsid protein components Spontaneous self assembly Icosahedral assembly
Empty procapsids assembled first followed by insertion of nucleic acid
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Often paracrystalline arrays of procapsids or completed virions are clustered at virus maturation sites Can occur in nucleus, cytoplasm or at membranes

Figure 18.8

Exit/Release from host cells
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Naked virions
Usually released following lysis of host cell

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Enveloped virions
Acquire envelope and are released simultaneously Host cell may stand up to release for long period of time  advantageous to virus!!! Process:
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Viral proteins integrate into PM (glycoproteins) Nucleocapside comes into contact with the underside of PM  membrane begins to bulge out or bud Nucleocapsid eventually surrounded by host PM  released from cell Exception: Herpesviruses use nuclear membrane

Most enveloped viruses bud off of PM
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Figure 18.9

Figure 18.10

II. Cytocidal and Cytopathic Damage Mediated by Viral Infection
Cytocidal
 

Refers to endsate = cell death Numerous mechanism that can eventually lead to cell death

Cytopathic effects (CPEs) often induced by viral infection  host cell damage

Mechanisms of host cell damage:
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Inhibition of host nucleic acid or protein synthesis Lysosomal damage  hydrolytic enzymes released into cytosol  proteolysis of host proteins Insertion of viral proteins into PM alters cell  looks different  attacked by immune system (NK cells recognize decreased MHC class I levels) Cells may fuse together  Polykaryocyte (syncytia)
Giant cells with many nuclei HIV, Respiratory syncytial virus, measles, Sendai, herpes

 

Direct cytotoxicity of viral proteins Disruption of cell structure by formation of inclusion bodies:
Negri bodies = virion clusters in rabies Ribosome clusters – arenavirus Chromatin clusters – herpesviruses

 

Disruption of host chromosomes Transformation of host cell  malignancy (cancer)
Observed with polyomavirus, herpesvirus, adenovirus, retrovirus, papillomavirus

III. Persistent, Latent and Slow Virus Infections
Definitions
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Acute infections
Rapid onset, short duration



Chronic infections
Persistent Slow reproduction Survive long periods of time (years) No obvious symptoms, but have antibodies in serum Examples: Hep B, EBV and measles



Latent infections
Reproduction terminated for period of time Dormancy followed by reactivation No antibodies or detectable virus when latent Examples: Herpes Simplex Type I (cold sores, dormant in nerve ganglia); Varicella-Zoster (Shingles following a latent chickenpox infection)



Slow virus diseases
Progressive pathological disease caused by a transmissible virus or prion Development of infection takes long period of time Symptoms often take years to present themselves Ends with disability or death

Examples:


Viruses: Measles  5-12 years later sometimes develop subacute sclerosing panencephalitis (SSPE) Papovavirus: Progressive multifocal leukoencephalopathy



Prions (Proteinaceous Infectious Particles): Creutzfeldt-Jacob Disease (CJD) – spongiform encephalopathy Kuru (Eastern New Guinea) – Dead kinsmen eaten, brain tissue handles and eaten by women and children Bovine spongiform encephalopathy (BSE, or mad cow disease) New variant CJD – in humans from eating beef that have BSE Scrapie - sheep Gerstmann-Straussler-Scheinker Syndrome (GSS) – neurodegeneration Fatal familial insomnia

IV. Viruses and Cancer
Definitions


Tumor = abnormal growth of tissue resulting in neoplasia due to loss of cell cycle regulation
Uncharacteristic cell shapes and plasma membranes Often unique antigens displayed (tumor antigens) Unorganized cell masses may form May revert to less differentiated state (anaplasia) Lumps (hard or soft) or cell suspension (leukemia)

Benign tumor
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Remains in compact mass, does not spread into surrounding tissue Can spread systemically Causing metastasis Carried by bloodstream  secondary tumors form Cancer causing genes From host or viral origin Important in cell cycle regulation

Malignant tumor
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Oncogenes
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Examples of viruses causing cancers (x6)
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Epstein-Barr virus (EBV) = Herpesvirus
EBV genomes and virions in tumor cells Nasopharyngeal carcinoma Burkitt’s lymphoma
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Malignant tumor of the jaw and abdomen of children in Central and Western Africa ~90% involve chromosome translocation of chromosome 8 to chromosome 14 (cmyc:Ig) Associated with previous infection with malaria, therefore not prevalent in USA



Hepatitis B virus
Liver cancer – hepatocellular carconoma Can integrate into host genome



Human papillomavirus
Cervical cancer



Retroviruses
Transmitted by transfusions of contaminated blood, needle sharing, sexual promiscuity, transplacental transfer, mother’s milk or by mosquitoes Human T cell lymphotrophic virus – 1 (HTLV-1)
 

Causes adult T-cell leukemia Integrates into genome  activates growth-promoting genes  dramatic amplification of WBC

HTLV-2
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 

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Causes hairy-cell leukemia Same trans-acting mechanism of cell growth activation as HTLV-1 Numerous membrane-derived protrusions on surface (hence “hairy”) Chronic, progressive lymphoproliferative disease Bone marrow, spleen and liver fill up with malignant cells  immune status declines as a result Death by blast crisis or opportunistic infection (gamma interferon provides some improvement)

Mechanism – How viruses cause cancer


Introduction of oncogenes into host genome
Rous sarcoma virus (src gene) = tyrosine kinase


Inserts into PM  phosphorylates tyrosines of many proteins  affects growth and metabolic characteristics of the cell Transactivating factor that increases gene expression of genes invovled in cell cycle regulation

HTLV-1 and HTLV-2


Efficient promoters or enhancers of virus  insert next to cellular oncogene (protoncogene)
 

Protooncogene = regulatory proteins - overexpressed Example: insertion next to cmyc

Viral products may bind to tumour suppressing proteins (e.g. p53) leading to cellular transformation

V. Prions
Definition:
 

Proteinaceous Infectious Particle Not a virus!!!
No DNA or RNA in it.



Altered prion protein causes disease
PrPc = normal, cellular prion protein, 33-35kDal hydrophobic membrane protein, function unknown PrPsc = Scrapie form of PrP = heat resistant, protease resistant, altered form of PrPc




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Contains more beta pleated sheet in secondary structure instead of alpha helix, caused by mutations in amino acid sequence When it binds to PrPc, it induces PrPc to adopt the PrPsc conformation – spreads throughout cells and adjoining tissue Spongiform encephalopathies are induced

CJD: Creutzfeld-Jacob Disease

Normal Brain Tissue

CJD Brain Tissue

Figure 18.14

18.15


								
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