VIROLOGY

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• VIROLOGY
• The smallest infectious agents and the
  smallest form of life. Viruses can be viewed only
  by Electron microscope.
• General characteristics of viruses
• 1- The viral nucleic acid become active only when
  it enters to the host cells.
• 2- Viruses are a live when they multiply in the
  host.
• Differ from other infectious agents
• They are very small.
• They need host cells for multiplication.
• Contain a single type of nucleic acid, DNA or
  RNA.
• Contain protein coat.

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• Multiply inside living cells using the synthetic
  machinery of the cell.
• Can transfer the viral nucleic acid to other
  cell.
• No ATP-generating system which is very important
  for design of antiviral drugs as the drug will be
  toxic for the virus and the host cell.
• The virus contains lipid layer so may be the
  virus become sensitive to disinfectant or
  emulsifying agents e.g.. Bile salt and
  detergents.

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• HOST RANGE
• Animal viruses
• Plant viruses
• Bacterial viruses (bacteriophage).
• The specification of the virus to infect the host
  cells depend on the requirement for the viral
  multiplication found in the host cells.
• The virus interact with specific receptors site
  on the surface of the cells-----hydrogen
  bonds----by pilli or flagella (Bacteriophage)-----
  ---Receptor sites are plasma membrane of the host
  cells (animal viruses).

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• CLASSIFICATION OF VIRUSES
• Old classification according to hot and organ
• Animal- Bacterial Plant
• Modern Classification
• Morphological classification
• Type of nucleic acid
• Size of capsid
• Number of capsomeres
• Method of transmission
• DNA----envelope Herpes, poxvirus
• Non envelope adenoviruses
• RNA-----Envelope Retrovirus (HIV)
• Non envelope Polio, Retro virus

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• isolation, cultivation and identification of
  virus
• -The requirement of living host cells for
  multiplication complicate their detection.
• -Cultivation on living cells, plant or animals.
• -Bacteriophage growing in bacteria culture.
• Growth of bacteriophage
• -Can be grown on solid bacterial culture or
  suspension.
• -Use solid medium may plaque which use for
  detection and counting viruses
• Bacteriophage host bacteria melted
  agar-----poured on a plate containing a hardened
  layer of agar----each virus infect bacterium,
  multiplies, infect all bacteria
  cells----destroyed---plaques on surface agar---
  uninfected bacteria---turbid background
• PFU---------------Plaque forming unit.

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Medically Important Virus Families

• DNA
• Adenoviridae
• Hepadnaviridae
• Herpesviridae
• Papovaviridae
• Parvoviridae
• Poxviridae

• RNA
• Arenaviridae Bunyaviridae
• Calciviridae Coronaviridae
• Flaviviridae Filoviridae
• Orthomyxoviridae
  Paramyxoviridae
• Picornaviridae Reoviridae
• Retroviridae Rhabdoviridae
• Togaviridae

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• BACTERIOPHAGE CLASSIFICATION
• 1) Virulent phage
• -Host cells-----lytic cell
• -The phage inject DNA inside host cell leaving
  the protein material outside.
• -DNA enter host cell and control the genetic
  machine of cell stimulating it to transcript the
  DNA of the virus then capsid formed around DNA
  and capsid -----rupture of bacteria---viruses---ma
  ture phage.
• 2) Temperate phage
• -Host cells-----lysogenic as no. 1 but no rupture
  of the bacteria due to DNA of virus integrated
  with bacterial chromosome.

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• REQUIREMENTS FOR VIRAL GROWTH
• All viruses are obligate intracellular parasites
  but they can survive in certain conditions as
  non-replicating particles
• Temperature
• Heat - there is great variability in the heat
  stability of
• different viruses but icosahedral viruses tend
  to be relatively stable, enveloped viruses are
  heat labile and most pathogenic viruses are
  inactivated at 55-60oC because their capsid
  protein is destroyed (an important exception is
  the hepatitis virus.
• Cold - most viruses can be preserved at
  sub-freezing temperatures, some can withstand
  lyophilisation and can be stored in the dry state
  at 4oC or even at room temperature while
  enveloped viruses tend to lose infectivity after
  prolonged storage at 90oC.

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• pH
• Most viruses are stable in the pH range 5-9.
  Enteroviruses that have to pass through the
  stomach can withstand low pHs. All viruses are
  destroyed by alkaline conditions.
• Radiation
• UV produce damaging results on double-stranded
  DNA that can cause inactivation of the virus.
  If conditions are right the DNA can repair
  itself.
• x-ray, gamma rays and beta particles
  inactivate viruses..
• Stabilisation by Salts
• magnesium chloride stabilises polioviruses,
  magnesium sulphate stabilises influenza viruses
  and sodium sulphate stabilises herpes virus.
  Important in the preparation of vaccines e.g..
  non-stabilised polio vaccine must be stored at
  lt0oC whereas stabilised vaccine remains potent
  for weeks at ambient temperature which is an
  advantage when immunising in rural areas

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• Ether Susceptibility and Lipid Solubility
  
  Enveloped viruses are inactivated by
  ether whereas non-enveloped ones are not (simple
  efficient test for the presence of envelopes).
• Other organic solvents and sodium deoxycholate
  also destroy the envelope.
• Detergents
• Non-ionic detergents solubilise lipid
  constituents but do not denature the proteins of
  the capsid. Anionic detergents solubilise the
  lipid constituents and disrupt the capsids into
  separated polypeptides
• 50 Glycerol
• Many viruses remain alive in 50 glycerol for
  many years. Vaccinia virus is preserved in 50
  glycerol for many years while bacteria are
  killed.

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• Formaldehyde
• Destroys viral infectivity but has minimal
  adverse affect on viral antigenicity and is used
  in the productions of inactivated viral vaccines
• Photodynamic InactivationHeterocyclic dyes like
  neutral red, proflavine and toluidine blue
  intercalate between the bases of replicating
  viral nucleic acids and on exposure to light,
  they become susceptible to inactivation
• Antibacterial Agents
• Antibacterial antibiotics and sulphonamides
  have no effect on viruses although some antiviral
  drugs have been developed. Oxidising agents are
  the most effect disinfectants e.g.. hydrogen
  peroxide, hypochlorite.

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Virus Multiplication Cycles

• Adsorption and host range
• Penetration and uncoating
• Replication and protein production
• Viral assembly
• Release of mature viruses

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Virus infection Effects on host cells

• Cytopathic effect
• Persistent infection
• Chronic latent state
• Oncogenic viruses

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• Growth of animal viruses in the lab
• 1) Embryonated eggs
• Convenient, inexpensive, the viral
  suspension injected into the fluid of the egg,
  viral growth is detected by death of the embryo,
  or lesion on the membrane of the egg.
• It is the most common method for viral
  growth and isolation , and used for preparation
  of viral vaccine.
• 2) Living animal
• e.g.. mice, rabbit, guinea pig
• Study of immunoresponse, a clinical specimen
  with virus inoculated the animal ----obseved
  signgs of disease or killing animal and examine
  the tissue.

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• 3) Cell culture
• The recent method for cultivation and growing of
• Virus
• It is a homogenous collection of cells.
• More convenient than animal or egg.
• Prepared by treating animal tissue with enzyme to
  
• separate the individual cells.
• Suspended in solution with nutrient and growth
  factors
• required by the cell
• Cells grow adhere to glass or plastic
  container to form
• a monolayer
• Virus infection leads to the deterioration of
  cells (cytopathic effect
• CPE)

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• Cell culture problems
• - Must be kept from microbial contamination.
• - Need antibiotics for preventing contamination.
• - Need experience technical worker.
• - Identification of viral isolates is not easy
  so immunological method mainly used as depend on
  antibodies.

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Multiplication of animal viruses Virus---attachme
nt to the cell---penetration---uncoating---replica
tion---assembly---releaseviruses.
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USE OF FERTILE EGGS

• Largely replaced by use of TC
• Still useful in cultivation and detection of some
  viruses, flu
• Three parts of the egg are of use
• A   The amniotic cavity
• Surrounds the embryo lined by a single layer
  of epithelial cells.
• The amniotic fluid bathes the external surface of
  the embryo and comes into contact with the
  respiratory and alimentary tracts.
• B   The allantoic cavity
• Comprises an outgrowth of the hind-gut of the
  embryo and is lined with endoderm.
• Both are useful for the cultivation of viruses,
  particularly
• orthomyxoviruses (e.g., influenza) and
  paramyxoviruses (e.g., mumps).
• Membranes major source of cells in which virus
  growth occurs, but the embryo may also become
  infected.
• The allantoic cavity is routinely used because it
  is technically much simpler to inoculate.
• However, some viruses (e.g., human influenza
  isolates) may need to be egg-adapted by growth in
  the amniotic cavity before they will grow
  efficiently in the allantoic cavity the reason
  for this is not known.
• C   The chorio-allantoic membrane The membrane
  consists of an outer layer of stratified
  epithelium which constitutes the respiratory
  surface of the egg, and an inner layer of
  endoderm (the lining of the allantoic cavity).
  The membrane may be used as a cell sheet provided
  it is first dropped away from the shell membrane.
  Dermatropic viruses (poxviruses and some herpes
  viruses) will grow on this membrane, and at low
  concentrations, will give discrete foci of
  infection which consist of centres of cell
  proliferation and necrosis (pocks). The membrane
  may therefore be used to assay these viruses. In
  addition, different viruses cause pocks of
  different colour and morphology, and this is of
  diagnostic value for distinguishing between
  different poxviruses.

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The Fertile Egg

• Three parts are of use
• The amniotic cavity
• Surrounds the embryo lined by a single layer
  of epithelial cells.
• The amniotic fluid bathes the external surface of
  the embryo and comes into contact with the
  respiratory and alimentary tracts 
• The allantoic cavity
• Comprises an outgrowth of the hind-gut of the
  embryo and is lined with endoderm.
• Both useful for growing
• orthomyxo-influenza) paramyxo - mumps).

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The Fertile Egg

• Membranes major source of cells for virus growth,
  but embryo may also become infected.
• The allantoic cavity is routinely used because it
  is technically much simpler to inoculate.
• However, some viruses (e.g., human influenza
  isolates) need to be egg-adapted by growth in the
  amniotic cavity before they will grow efficiently
  in the allantoic cavity the reason for this is
  not known.

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The Fertile Egg

• The chorio-allantoic membrane
• Consists of an outer layer of
• stratified epithelium the
• respiratory surface of the egg,
• and an inner layer of endoderm
• (the lining of the allantoic cavity)
• May be used as a cell sheet, if first
• dropped away from the shell
• membrane.
• Dermatropic viruses (poxviruses
• some herpes viruses) will grow
• on this membrane
• At low concentrations, will give
• discrete foci of infection which
• consist of centres of cell
• proliferation necrosis (pocks).

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The Fertile Egg

• The CAM
• Used to assay these viruses.
• Different viruses cause pocks
• of different colour and
• morphology, of diagnostic
• value for distinguishing
• between different poxviruses.

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• Effect of animal viral infection on host
  cells
• CPE inhibition of DNA or protein
  synthesis----Damage of the cell.
• Inclusion bodies
• One type of CPE, a result of accumulation of
  viruses in the nucleus or cytoplasm of the host
  cells or both.
• Other arise at sites of earlier viral synthesis.
• Important useful for identification of the
  particular virus causing an infection.
• e.g.. Rabies----Negri bodies in cytoplasm of
  nerve cells and presented in the brain
  tissue----(Diagnostic test for rabies, also for
  measles, vaccinia, small pox, herpes.
• Polykaryocytes infection cell accumulation from
  myxoviruses.