Medical Virology

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Medical Virology

• Dept. of Medical Microbiology and Parasitology
• Shanghai Second Medical University
• Shanghai, China.
• E.mail ( xkguo_at_shsmu.edu.cn )

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General Virology

• Conception
• Viruses
• Virion
• Size and Shape
• Structure
• Replication
• Viral Variation
• Classification

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Conception

• Virology is the bioscience for study of viral
  nature,and the relationship between viruses and
  hosts. Viruses often cause serious diseases,
  relate to some cancers and congenital
  deformities, also can be used as tool for genetic
  engineering.

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• 3000BC

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History

• Smallpox was endemic in China by 1000BC. In
  response, the practice of variolation was
  developed. Recognizing that survivors of smallpox
  outbreaks were protected from subsequent
  infection, variolation involved inhalation of the
  dried crusts from smallpox lesions like snuff, or
  in later modifications, inoculation of the pus
  from a lesion into a scratch on the forearm of a
  child.

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Definition of Virus

• Viruses may be defined as acellular organisms
  whose genomes consist of nucleic acid, and which
  obligately replicate inside host cells using host
  metabolic machinery and ribosomes to form a pool
  of components which assemble into particles
  called VIRIONS, which serve to protect the genome
  and to transfer it to other cells

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Viral Properties

• Viruses are inert (nucleoprotein ) filterable
  Agents
• Viruses are obligate intracellular parasites
• Viruses cannot make energy or proteins
  independent of a host cell
• Viral genome are RNA or DNA but not both.
• Viruses have a naked capsid or envelope with
  attached proteins
• Viruses do not have the genetic capability to
  multiply by division.
• Viruses are non-living entities

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Consequences of Viral Properties

• Viruses are not living
• Viruses must be infectious to endure in nature
• Viruses must be able to use host cell processes
  to produce their components (viral messenger RNA,
  protein, and identical copies of the genome)
• Viruses must encode any required processes not
  provided by the cell
• Viral components must self-assemble

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Challenges the way we define life

• viruses do not respire,
• nor do they display irritability???
• they do not move
• they do not grow
• they do most certainly reproduce, and may adapt
  to new hosts.

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Size and Shape

• Methods
• Size of Viruses
• Shapes of Viruses

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Methods of Analysis

Electron microscopy The resolution is 5nm (1nm
10-9 m) X-ray crystallography
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Size of Viruses

• A small virus has a diameter of about 20nm.
• Parvovirus
• A large virus have a diameter of up to 400nm.
• Poxviruses

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Shape of Viruses

• Spherical
• Rod-shaped
• Brick-shaped
• Tadpole-shaped
• Bullet-shaped
• Filament

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Shapes of VirusesSpherical
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Shapes of Viruses Rod-shaped
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Shapes of Viruses Brick-shaped
.                                              
                              
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Tadpole-shaped
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Shapes of Viruses Bullet-shaped
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Shapes of Viruses Filament
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Structure of Viruses
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Virion

• the complete infectious unit of virus particle
• Structurally mature, extracellular virus
  particles.

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Virion
envelope
Capsid
Viral core
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Viral core

• Viral core
• The viral nucleic acid genome, In the center
  of the virion, Control the viral heredity and
  variation, responsible for the infectivity.

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Genome

• The genome of a virus can be either DNA or RNA
• DNA-double stranded (ds) linear or circular
• Single stranded (ss) linear or
  circular
• RNA- sssegmented or non-segmented
• sspolarity(sense) or polarity
  (non-sense)
• ds linear (only reovirus family)

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Viral Capsid

• The protein shell, or coat, that encloses the
  nucleic acid genome.
• Functions a. Protect the viral nucleic acid. b.
  Participate in the viral infection. c. Share the
  antigenicity

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Nucleocapsid

• The core of a virus particle consisting of the
  genome plus a complex of proteins.
• complex of proteins Structural proteins Non-
  Structural proteins (Enzymes Nucleic acid
  binding proteins)

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Symmetry of Nucleocapsid

• Helical
• Cubic /Icosahedral
• Complex

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Helical symmetry
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• Helical symmetry

How to assemble
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Helical

• California Encephalitis VirusCoronavirusHantavir
  usInfluenza Virus (Flu Virus)Measles Virus (
  Rubeola)Mumps VirusParainfluenza VirusRabies
  VirusRespiratory Syncytial Virus(RSV)

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Cubic or icosahedral symmetry
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Icosahedral

• Herpes Simplex Virus 1 (HHV1)Herpes Simplex
  Virus 2 (HHV2)Human Immunodeficiency Virus
  (HIV)Human T-lymphotrophic Virus (HTLV)Norwalk
  VirusPapilloma Virus (HPV)Polio
  virusRhinovirusRubella VirusSaint Louis
  Encephalitis VirusVaricella-Zoster Virus
  (HHV3)Western Equine Encephalitis Virus
  (WEEV)Yellow Fever Virus

• Adeno-associated Virus (AAV)AdenovirusB19Coxsac
  kievirus - ACoxsackievirus - BCytomegalovirus
  (CMV)Eastern Equine Encephalitis Virus
  (EEEV)EchovirusEpstein-Barr Virus
  (EBV)Hepatitis A Virus (HAV)Hepatitis B Virus
  (HBV)Hepatitis C Virus (HCV)Hepatitis Delta
  Virus (HDV)Hepatitis E Virus (HEV)

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Complex Virus Structures

• A well known example is the tailed bacteriophages
  such as T4.
• The head of these viruses is cubic with a
  triangulation number of 7. This is attached by a
  collar to a contractile tail with helical
  symmetry.

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T4 Bacteriophage
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Properties of naked viruses

• Stable in hostile environment
• Not damaged by drying, acid, detergent, and heat
• Released by lysis of host cells
• Can sustain in dry environment
• Can infect the GI tract and survive the acid and
  bile
• Can spread easily via hands, dust, fomites, etc
• Can stay dry and still retain infectivity
• Neutralizing mucosal and systemic antibodies are
  needed to control the establishment of infection

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Naked viruses( Non Enveloped )

• Adeno-associated Virus (AAV)AdenovirusB19Coxsac
  kievirus - ACoxsackievirus - BEchovirusHepatiti
  s A Virus (HAV)Hepatitis E Virus (HEV)Norwalk
  Virus

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Envelope

• A lipid-containing membrane that surrounds some
  viral particles.
• It is acquired during viral maturation by a
  budding process through a cellular membrane,
  Viruses-encoded glycoproteins are exposed on the
  surface of the envelope.
• Not all viruses have the envelope, and viruses
  can be divided into 2 kinds enveloped virus and
  naked virus.

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Functions of envelope

• Antigenicity
• some viruses possess neuraminidase
• Infectivity
• Resistance

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Envelope
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Properties of enveloped viruses

• Labile in dry , arid environment
• Damaged by drying, acid, detergent, and heat
• Pick up new cell membrane during multiplication
• Insert new virus-specific proteins after assembly
• Virus is released by budding

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Consequences of Properties for enveloped viruses

• Must stay moist
• Must not infect the GI tract for survival
• Must be transmitted in the protective, droplets,
  secretions, blood and body fluids
• Must reinfect another host cell to sustain
• Humoral and cell-mediated immunity are needed to
  control the infection

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Enveloped

• Herpes Simplex Virus 2 (HHV2)Human
  Immunodeficiency Virus (HIV)Human
  T-lymphotrophic Virus (HTLV)Influenza Virus (Flu
  Virus)Molluscum contagiosumPapilloma Virus
  (HPV)Polio virusRhinovirusVaricella-Zoster
  Virus (HHV3)Venezuelan Equine Encephal. Vir.
  (VEEV)Western Equine Encephalitis Virus
  (WEEV)Yellow Fever Virus

• California Encephalitis Virus
• Coronavirus
• Cytomegalovirus (CMV)
• Eastern Equine Encephalitis Virus (EEEV)
• Epstein-Barr Virus (EBV)
• HantavirusHepatitis B Virus (HBV)Hepatitis C
  Virus (HCV)Hepatitis Delta Virus (HDV)Herpes
  Simplex Virus 1 (HHV1)
• Rotavirus
• Rubella Virus
• Saint Louis Encephalitis VirusSmallpox Virus
  (Variola)Vaccinia Virus

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Spike or Peplomere
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summarize
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Virion structure

• Nucleocapsid(Naked Virus) DNA or RNA
  Structural proteins Enzymes Nucleic acid
  binding proteins
• Enveloped Virus Nucleocapsid Viral specific
  glycoproteins and Host Membrane

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Helical
Cubic
Naked Virus
Enveloped Virus
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CHEMICAL COMPOSITION OF VIRUSES

• Viral Protein
• Viral Nucleic Acid
• Viral Lipids
• Viral carbohydrate

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Viral Nucleic Acid

• DNA-double stranded (ds) linear or circular
• Single stranded (ss) linear or
  circular
• RNA- sssegmented or non-segmented
• sspolarity(sense) or polarity
  (non-sense)
• ds linear (only reovirus family)

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Viral Protein

• Structural protein (Capsomere)
• Enzyme
• glycoproteins (spike/viral attachment protein,
  VAP)

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Culture of Viruses
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System for the propagation of viruses

• People
• Animals cowschickens mice rats suckling
  mice
• Embryonated eggs
• Organ and tissue culture
• Organ culture
• primary tissue culture
• cell lines diploid
• Tumor or immortalized cell line

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• Cytopathic effect, CPE
• Inclusion Bodies

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CPEViral Cytopathological Effects

• Cell death
• Cell rounding/Degeneration/Aggregation
• Lass of attachments to substrate
• Inclusion bodies in the nucleus or cytoplasm,
  margination of chromatin
• Syncytia multinucleated giant cells caused by
  virus-induced cell-cell fusion
• Cell surface changes
• Viral antigen expression
• Hemadsorption (hemagglutinin expression)

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Normal cell and CPE
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Inclusions
Negri body
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TCD50,LD50,ID50

• PFU plaque forming units

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Replication of Viruses
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Replicative cycle

• As obligate intracellular parasites, Virus must
  enter and replicate in living cells in order to
  reproduce themselves. This growth cycle
  involves specific attachment of virus,
  penetration and uncoating, nucleic acid
  transcription, protein synthesis, matureation and
  assembly of the virions and their subsequent
  release from the cell by budding or lysis

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Initiation Phase

• Attachment
• Penetration
• Uncoating

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Attachment/Adsorption

• Virus attaches to the cell surface. Attachment is
  via ionic interactions which are
  temperature-independent.
• Viral attachment protein recognizes specific
  receptors on the cell surface (These may be
  protein or carbohydrate or lipid components of
  the cell surface).
• Cells without the appropriate receptors are not
  susceptible to the virus.

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PENETRATION (Virus enters the cell)

• Virions are either engulfed into vacuoles by
  endocytosis or the virus envelope fuses with
  the plasma membrane to facilitate entry
• Enveloped viruses
• Non-enveloped viruses

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Fusing

• (A) Entry by fusing with the plasma membrane.
  Some enveloped viruses fuse directly with the
  plasma membrane. Thus, the internal components of
  the virion are immediately delivered to the
  cytoplasm of the cell.

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HIV
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Endocytosis

• (B) Entry via endosomes at the cell surface

 
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influenza virus
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Enveloped viruses

• Some enveloped viruses require an acid pH for
  fusion to occur and are unable to fuse directly
  with the plasma membrane. These viruses are taken
  up by invagination of clathrin coated pits into
  endosomes. As the endosomes become acidified, the
  latent fusion activity of the virus proteins
  becomes activated by the fall in pH and the
  virion membrane fuses with the endosome membrane.
  This results in delivery of the internal
  components of the virus to the cytoplasm of the
  cell

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Non-enveloped viruses

• Non-enveloped viruses may cross the plasma
  membrane directly
• may be taken up via clathrin-coated pits into
  endosomes. They then cross (or destroy) the
  endosomal membrane.

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unenveloped viruses
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UNCOATING

• Nucleic acid has to be sufficiently uncoated that
  virus replication can begin at this stage. When
  the nucleic acid is uncoated, infectious virus
  particles cannot  be recovered  from the cell -
  this is the start of the ECLIPSE phase - which
  lasts until new infectious virions are made
• Uncoating is usually achieved by cellular
  proteases opening up the capsid

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BIOSYNTHESIS

• genome synthesis
• mRNA production
• protein synthesis

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Flow of events during the replication of Hepadna
viruses
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Flow of events during the replication of
herpesviruses
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Flow of events during the replication of
reoviruses.
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Flow of events during the replication of
togaviruses
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Flow of events during the replication of
orthomyxoviruses and paramyxoviruses.
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Flow of events during the replication of
retroviruses
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Maturation assembly release
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Maturation

• The stage of viral replication at which a virus
  particle becomes infectious nucleic acids and
  capsids are assembled together.

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ASSEMBLY

• The stage of replication during which all the
  structural components come together at one site
  in the cell and the basic structure of the virus
  particle is formed.

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RELEASE

• Disintegration naked virus cause the host
  cell lysis
• Budding enveloped viruses
• Budding viruses do not necessarily kill the cell.
  Thus, some budding viruses may be able to set up
  persistence

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Assembly
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Products of viral replication

• Virion
• DEFECTIVE VIRUS
• ABORTIVE INFECTION
• integration

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• DEFECTIVE VIRUS
• deficiency in some aspects of replication,
  but interfering the replication of normal
  viruses 
• ABORTIVE INFECTION
• When a virus infects a cell (or host), but
  cannot complete the full replication cycle ( not
  biosynthesize their components or not assemble
  virions.), i.e. a non-productive infection.

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INTERFERENCE

• Interferon, IFN
• Defective interfering particle, DIP

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Viral Genetics
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Genome

• The genome of a virus can be either DNA or RNA
• DNA-double stranded (ds) linear or circular
• Single stranded (ss) linear or
  circular
• RNA- sssegmented or non-segmented
• sspolarity(sense) or polarity
  (non-sense)
• ds linear (only reovirus family)

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Virus Genomes
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The Structure Complexity of Virus Genomes

• The nucleic acid comprising the genome may be
  single-stranded or double-stranded, in a
  linear, circular or segmented configuration.
  Single-stranded virus genomes may be
• positive ()sense, i.e. of the same polarity
  (nucleotide sequence) as mRNA
• negative (-)sense
• ambisense - a mixture of the two.

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The Structure Complexity of Virus Genomes

• any virus genome will usually include the
  following
• Composition - DNA or RNA, single-stranded or
  double-stranded, linear or circular.
• Size number of segments.
• Terminal structures.
• Nucleotide sequence.
• Coding capacity - open reading frames.
• Regulatory signals - transcription enhancers,
  promoters terminators.

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The Structure Complexity of Virus Genomes
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Transfection

• Infection of cells caused by nucleic acid alone

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Variation

• There are two important variation which relate
  well with medical practices
• Antigenicity variation In most viruse the
  antigenicity is stable but in some viruses such
  as influenze virus the antigenicity may vary and
  cause the disease to epidemic.
• Virulence variation(Virulent viruses) Less
  virulent viruses always used in prevention.

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Mutation

• Mutant
• Variant
• temperature sensitive(ts) mutant

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Interactionswhen two genetically distinct
viruses infect a cell 3 different phenomena can
ensue

• Recombination /Reassortment
• Complementation
• Phenotypic mixing

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• Recombination
• dsDNA viruses
• Reassortment ( segmented genomes)
• RNA viruses influenza virus

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Complementation
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Phenotypic mixing

• The genome of virus A can be coated with the
  surface protein of virus type B

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Classification of Viruses
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CLASSIFICATION OF VIRUSES-------basis of
classification

• Virion morphology
• Physicochemical properties of the virion
• Virus genome properties
• Virus protein proteries
• Genome organization and replication
• Antigenic properties
• Biologic properties

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CLASSIFICATION OF VIRUSES

• By 1995
• --71 families, 11 subfamilies
• --164 genera
• For humans and animals
• --24 families,
• --DNA 7 RNA 17 for humans

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Survey of DNA-containing Viruses

• Parvoviruses human parvovirus B19
• Papovaviruses papillomaviruses
• Adenoviruses 47 types infect humans
• Herpesviruses human herpesvirus 1-8
• Poxviruses smallpox vaccinia
• Hepadnaviruses HBV

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Survey of RNA-containing Viruses

• Retroviruses
• Bunyaviruses
• Othomyxoviruses
• Paramyxoviruses
• Rhabdovirusesrabies virus
• Bornaviruses BDV
• Filoviruses
• Other viruses
• Viroids

• Picornaviruses
• Astroviruses
• Caliciviruses
• Reoviruses
• Arboviruses
• Togaviruses
• Flaviviruses
• Arenaviruses
• Coronaviruses SARS

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DNA and RNA Viruses

• ?????(Headnasviridae)
• ?????( Retroviridae )

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Viroids(???)

• Viroids are small (200-400nt), circular RNA
  molecules with a rod-like secondary structure
  which possess no capsid or envelope which are
  associated with certain plant diseases. Their
  replication strategy like that of viruses - they
  are obligate intracellular parasites.

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Dependovirus /Virusoids????

• Viroids are small (200-400nt), circular RNA
  molecules with a rod-like secondary structure
  which possess no capsid or envelope which are
  associated with certain plant diseases. Their
  replication strategy like that of viruses - they
  are obligate intracellular parasites.

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?? (Prions)

• Prions are rather ill-defined infectious agents
  believed to consist of a single type of protein
  molecule with no nucleic acid component.
  Confusion arises from the fact that the prion
  protein the gene which encodes it are also
  found in normal 'uninfected' cells. These agents
  are associated with diseases such as
  Creutzfeldt-Jakob disease in humans, scrapie in
  sheep bovine spongiform encephalopathy (BSE) in
  cattle.