MPH604 Virology

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MPH604 - Virology

• Professor Kenneth L. Elkins

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Viruses - Definitions

• Obligate intracellular parasites of living cells
• Complex chemical entities possessing a complete
  genetic mechanism

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Viruses - Properties of

• Ultra microscopic size ranging from 20 nm to 450
  nm
• are filterable agents
• Are not cells structure is compact and
  economical
• Do not independently fulfill the characteristic
  of life
• Viruses are genetic parasites
• they depend upon the genetic mechanisms of the
  cell to reproduce them
• Only affect cells from an intracellular location
  intracellular parasites
• Are geometric some form crystal-like masses
• Basic structure is a protein capsid and a nucleic
  acid nucleocapsid
• some virions are enveloped
• Contain either DNA or RNA, but not both
• some possess single stranded DNA
• some possess double stranded RNA
• Lack enzymes for metabolic processes cannot
  synthesize or produce ATP (energy) independent
  from the host cell
• Viruses are assembled they do not replicate by a
  division process

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Viruses - Concept Review

• Viruses are not living
• Viruses are not cells
• Viruses are infectious they infected every known
  type of cell
• Are cell or tissue specific each virus has its
  own type of host cells
• Virus components are synthesized by the host
  cell, not by the virus
• Virus must genetically encode for processes or
  components which the cells does not already
  synthesize
• Viral synthesis and assembly usually cause cell
  death or leave the cell metabolically
  dysfunctional

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Viruses - History

• Contributions to Virus research
• Pasteur coined the name virus to mean poison
• produces a viral vaccine against rabies
• Ivanovski discovered the Tobacco Mosiac Virus
  (TMV)
• observed the filterable property of viruses
• Loeffler and Frosch first isolated an animal
  virus the foot and mouth disease virus of
  cattle
• Enders, et al. developed tissue culture methods
• Significant accomplishment in the progress of
  Virology
• electron microscopy
• animal culture
• embryonated egg culture
• tissue culture

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Virus Structure and Morphology

• Animal Viruses Virions
• Nucleocapsid
• Nucleic Acid either DNA or RNA
• Capsid structural proteins capsomeres
• protects the nucleic acid
• imparts the shape to the virus
• contributes the virus antigenicity virus specific
  antigens
• Envelope
• phospholipid/glycoprotein membrane
• derived from the host cell membrane
• modified by incorporation of viral based
  glycoproteins virus specific
• also contributes to the antigenicity of the cell
  spike

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Virus Structure and Morphology

• Bacterial Viruses
• Bacteriophages viruses whose host cell is the
  procaryotic bacterial cell
• Nucleic acid is DNA
• Protein capsid
• Lytic phage lysis and kills the host cell
• Temperate phage establishes a state of lysogeny

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Properties of Naked Virions (Nucleocapsid)

• Remains stable and infectious in the presence of
  environmental variables like
• temperature
• acids
• proteases
• detergents
• drying
• Releases from host cell by process of lysis
• Epidemiological significance
• can be transmitted easily by fomites, hands,
  droplets, dust
• can dry out and retain infectivity
• can survive the adverse conditions of the gut
• can resistant the antimicrobial effect of
  detergents and sewage treatment processes
• antibody against the capsid may be sufficient for
  protection

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Properties of Enveloped Virions

• Are destroyed or inactivated in the presence of
  environmental variables such as
• acids
• detergents
• drying
• heat
• Modifies the host cell membrane through the
  addition of viral specific antigens or
  glycoproteins
• Are released by budding from host cell or by
  lysis of host cell
• Epidemiological Significance
• must remain wet to be active and infectious
• cannot survive the harsh environment of the
  gastrointestinal tract
• does not need to kill the cell to spread or be
  released
• requires both humoral and cell-mediated immunity
  for protection and control
• illicts hypersensitivity and inflammation to
  cause immunopathogenesis

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Classification of Viruses

• Basis of Classification
• Nucleic acid type Superfamily
• Strand type and sense /-
• Capsid type virion shape
• Envelope presence or absent
• Size
• Cell trophism
• Persistence
• Enzymes in the Virion
• Families of DNA Viruses Table 6-1
• Families of RNA Viruses Table 6-3

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DNA Viruses

• Linear DNA Circular DNA
• Adenoviridae Papillomaviridae
• Parvoviridae Hepadnaviridae
• Single-stranded
• Herpesviridae
• Poxviridae
• complex

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RNA Viruses

• () RNA (-) RNA
• Picornaviridae Paramyxoviridae
• Caliciviridae Rhabdoviridae
• Coronaviridae Bunyaviridae
• Retroviridae Orthomyxoviridae
• Togaviridae Filoviridae
• Flaviviridae
• Reoviridae
• Double-stranded

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The Virus Cycle

• Adsorption
• binding of virion to host cell receptor(protein
  or carbohydrate)
• ability to adsorb determines the host/tissue
  range of the virus
• a specific binding between virus
  capsid/envelope(VAPs) and host cell receptor
• Penetration
• endocytosis
• pinocytosis or engulfment of complete virion
• most common mechanism for non-enveloped(naked)
  viruses
• viropexis
• hydrophobic component of capsid force the virus
  through the host cell membrane direct penetration
  for some non-enveloped viruses
• fusion
• virus merges or dissolves into the host cell
  membrane or endosome
• specific mechanism governed by pH
• neutral pH promotes fusion with cell membrane
• acid pH promotes fusion with the endosome
• the nucleocapsid in released directly into the
  cytoplasm/nucleus
• most common mechanism for enveloped viruses

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The Virus Cycle

• Uncoating releases the nucleic acid into the cell
• virus based enzyme carried in virion which
  removes the capsid
• cell based enzymes or structures within the cells
  remove the capsid
• DNA viruses are uncoated as they enter the
  nucleus
• RNA viruses are uncoated as the enter the
  cytoplasm
• Replication
• DNA viruses Fig 6-14
• RNA viruses Fig 6-12 and 6-13
• Maturation/Assembly the interaction of nucleic
  acid replicas with the capsid proteins
• DNA viruses in the nucleus/RNA viruses in the
  cytoplasm (two exceptions)
• not completely a random process, but is
  facilitated by other viral proteins/enzymes
• Release
• Budding from the cell membrane, endoplasmic
  reticulum, nuclear membrane
• Lysis

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Replication of DNA Viruses Cell Nucleus

• I. Early Proteins
• Transcription of Viral Genome
• Initially uses the cells DNA dependent RNA
  polymerase
• Results in m-RNA which is translated into
  non-structural proteins
• DNA binding proteins
• cellular growth promotors/activators
• trranscriptional activators
• viral DNA dependent DNA polymerase
• DNA synthesis primers
• II. DNA Replication
• semiconservative replication of viral DNA
• catalyzed by viral based DNA dependent DNA
  polymerase
• initiated by viral based primers
• III. Late Proteins
• transcription of viral genome to produce m-RNA
  which is translated into
• structural proteins
• capsid/capsomere proteins
• enzymes

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Replication of () RNA Viruses

• I. Positive Strand RNA functions as m-RNA
• A. Early Proteins-translated at cells ribosomes
  usually as a polyprotein
• RNA dependent RNA polymerase
• Various non-structural proteins
• Capping proteins, primers, Nucleoproteins
• Positive-sense RNA is infectious
• B. Late Proteins also translated as part of the
  ribosomal polyprotein
• Capsid Proteins

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Replication of () RNA

• I. Process occurs within the cytoplasm
• A. Virus provides the polymerase
• RNA dependent RNA polymerase carried in the
  virion
• Also synthesized as an early viral protein
• II. Requires (-) RNA template(s)
• A. pool of () RNA synthesized from (-) template
• III. Some modification of () Viral RNAs

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Replication of (-) RNA Viruses

• I. Requires a preformed source of RNA dependent
  RNA polymerase
• A. preformed in the infected cell and assembled
  and carried in the virions
• B. (-) RNA cannot function as m-RNA
• II. Requires () RNA template(s)
• A. pool of (-) RNA synthesized from templates
• Requires RNA dependent RNA polymerase
• III. Process also occurs in the cytoplasm

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Bacteriophage Cycle coliphage T-even

• Lytic Cycle Lytic Phage
• Adsorption tail fibers bind to capsid envelope
• Penetration DNA is injected into bacterium
  capsid remains outside the cell
• Replication
• early proteins regulatory enzymes inhibit
  bacterial transcription
• cell begins to make viral DNA polymerase
• bacterial DNA nuclease which degrades bacterial
  DNA
• DNA replication uses cellular nucleotides
• late proteins viral capsid proteins/lysozyme
• Assembly
• Lysis
• Lysogeny Temperate Phage
• Adsorption
• Penetration
• Recombination of Virus DNA with the bacterial DNA
• bacterium does not die, but take on new genetic
  properties

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Viral Genetics

• Basis of Genetic Change
• Mutation wild-type to genetic variant(mutant)
• copy errors
• Viruses(esp RNA) possess no checking mechanisms
• deletions/additions
• most mutations are lethal and produce inactive
  virus
• non-lethal mutation results in changes in
  virulence/antigenictiy
• Genetic Exchange
• recombination
• mixing of genes from two viruses which coinfect
  the same cell
• reassortment

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Laboratory Identification of Specific Viruses

• Cytology cytopathic effect(CPE)
• changes in cell morphology
• cell lysis
• vacuolation
• syncytia
• inclusion bodies
• Electron Microscopy is not a standard clinical
  laboratory technique, but can be used in special
  situations to find clumps of viruses
• Host Immune Response(serology)
• detection of virus specific antibody with the
  serum of patient
• IgM within the first two weeks of primary
  infection
• Acute and convalescent samples
• four-fold increase in antibody titer
• serological test
• neutralization
• hemagglutinatin-inhibition
• immunossays ELISA,FA, RIA, LA

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Cytopathetic Changes Produced by Various Viruses
FYI

• Variola virus(smallpox) cells round up
  intracytoplasmic inclusion bodies
• Herpes simplex virus cells become giant with
  multiple nucleic intranuclear inclusion
  bodies
• Adenovirus clumping of cells nuclear
  inclusions
• Poliovirus cell lysis no inclusions
• Togavirus cell lysis no inclusions
• Reovirus cell enlargement intracytoplasmic
  vacuoles and inclusion bodies
• Influenza virus cell round up no inclusions
• Rabies virus no change in cell shape
  intracytoplasmic inclusions bodies ( Negri
  bodies)
• HIV giant cells with numerous nuclei

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Laboratory Detection of Viruses

• Cytopathic effects cells from animals and
  tissue culture
• Plaques areas of cell death in tissue culture
  that are unique to a particular virus
• Inclusions bodies intracellular areas resulting
  from virus maturation and assembly that are
  unique to a particular virus
• Antigenic changes chemical changes in infected
  cells resulting from the viral modification of
  the cell membrane
• changes in cell morphology
• Shape, size, Staining properties

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Clinical Diagnosis of Viral Diseases

• viral protein detection
• Immunofluorescence detects viral antigen
  on/within cell using specific labeled
  monoclonal antibody
• enzyme immunoassay detects viral antigens
  on/within cell
• enzyme linked immunosorbent assay
• detects virus or viral antigen released from
  infected cells
• viral nucleic acid detection
• gene probes or hybridization
• northern blot, southern blot, etc
• host immune response
• detection of Virus-specific antibody

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Laboratory Culture of Viruses

• Viral Isolation and Culture
• Sources of living cells
• animals
• human fetal tissue
• embryonated eggs
• tumors
• Types of Tissue Culture
• primary cell culture fresh cells from specific
  human/animal organs
• secondary cell culture dissociated subcultured
  primary cells
• these cell lines often mutate and die (senese),
  thus are short lived
• diploid cell lines mostly human fetal
  cells(fibroblastic)
• young developing cells which are long lived, do
  not mutate, and senese
• tumor cells - malignant cells which seldom sense
  and can be subcultured for indefinite
  generations

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Tissue Cultures for Various Viruses

• Poliovirus human and primate tissue culture
• Rhinovirus human embryonic kidney and lung tissue
  culture
• Bunyavirus baby mice mosquitoes, human tissue
  culture
• Rubella virus monkey cell culture
• Influenza, mump, measles viruses chicken embryo,
  monkey or calf kidney, human tissue culture
• Rhabdovirus mice human and hamster kidney
  chicken and duck embryo
• HIV human lymphocyte cell culture chimpanzees
• Papillovirus human fetal brain tissue culture
• Herpesviruses human embryonic fibroblast culture
• Hepatitis A virus human cell culture
• Hepitatis B virus living primates( virus cannot
  be grown in cell culture)

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Viral Persistance

• Condition in which the virus in not removed or
  inactivated by the host
• Permissive Cells cell easily supports all
  aspects of the viral cycle yielding many more
  virions
• Semipermissive Cells - virus replication is slow
  or inefficient and may not occur at all
• latent persistence - virus expressed only in
  growing cell, or in cell stimulated by hormones
  or cytokines
• Non-Permissive Cells does not allow replication
  of a particular virus
• transformation or immortalization basis of
  oncogenesis
• continual unregulated growth of cells with
  changes in morphology and loss of contact
  inhibition

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Mechanisms of Viral Pathogenesis

• Inhibition of Protein synthesis
• Inhibition and degradation of cellular DNA
• Alteration of cell membrane structure
• glycoprotein insertion
• syncytia formation
• disruption of cytoskeleton
• changes in permeability
• Inclusion Body Formation
• Toxicity of Virion Component