Virology

1
Virology

• Lab 6

2
Definitive Properties of Viruses

• An infectious, obligate intracellular parasite
• Viral genome comprised of either DNA or RNA
• Within an appropriate host cell, the viral genome
  is replicated and directs the synthesis
  (utilizing cellular systems) of other viral
  components
• Progeny infectious virus particles (virions) are
  formed by de novo assembly from newly synthesized
  components within the host cell
• A progeny virion assembled during the infectious
  cycle is the vehicle for transmission of the
  viral genome to the next host cell or organism,
  where its disassembly leads to the beginning of
  the next infectious cycle

3
Infected Cells (Immunohistochemistry)
4
Objectives

• Introduction to virus culture
• Isolation of virus
• Quantification of virus

5
Cell Culture

• Cell culture is still the most common method for
  the propagation of viruses.
• Tissues are dissociated into a single-cell
  suspension by mechanical disruption, followed by
  treatment with a proteolytic enzyme.
• Cells are cultured in a plastic flask in Minimum
  Essential Media (MEM). As the cells divide, they
  cover the plastic surface.
• Epithelial and fibroblastic cells attach to the
  surface of the plastic and form a monolayer.
• Two commonly used cells in fish virology- EPC -
  epitholioma papillosum carpio cells- CHSE -
  chinook salmon embryo

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Developing a cell line
Live tissue cells to be cultured
Culture vessel with appropriate growth media
Cell adhere to vessel and grow to form a monolayer
Lift cells into solution with enzyme
Seed cells into new culture vessels
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Isolation of virus from fish

• Aseptically remove the internal organs (kidney,
  liver, spleen, and intestine) and homogenize
• Take 0.5g of homogenized tissue and mix with 4.5
  ml HBSS to make 10-1 dilution
• Make 10-2 dilution of the tissue
• Filter the 10-2 dilution with a syringe and
  Swinney adapter with a 0.45 µm filter. This will
  trap bacteria in the filter but not virus
• Continue making 10-fold dilution series from
  filtered 10-2 dilution up to 10-5

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Virus Dilution
0.1 ml
0.1 ml
0.1 ml
0.1 ml
0.1 ml
1
1/10
1/100
1/1000
1/10000
1/100000
Stock
0.9 ml
0.9 ml
0.9 ml
0.9 ml
0.9 ml
10-1
10-2
10-3
10-4
10-5
101
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Cytopathic Effect

• Some viruses kill the cells in which they
  replicate, and infected cells may eventually
  detach from the cell culture plate.
• As more cells are infected, the changes become
  visible and are called cytopathic effects.

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Examples of Cytopathic Effects of Viral Infection

• Nuclear shrinking (pyknosis)
• Proliferation of nuclear membrane
• Vacuoles in cytoplasm
• Syncytia (cell fusion)

• Margination and breaking of chromosomes
• Rounding up and detachment of cultured cells
• Inclusion bodies

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Quantification of CPE

• Tissue Culture Infective Dose 50 (TCID50) a
  measure of virulence of virus
• Why Quantify?
• Virulence
• Immunity
• Strain

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Multi-well Plates
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TCID50 Procedure

• Count wells exhibiting CPE
• Ideally you would know all the dilution factors
  to get infection rates of zero to 100 percent

CPE
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Calculation of TCID50

• In any biological quantification, the most
  desirable endpoint is one representing a
  situation in which half of the inoculated
  animals or cells show the reaction (death
  in the case of animals and in CPE case
  of cells) and the other half do not.
• Reed-Muench Method of computing a 50 endpoint of
  a virus titration
• Calculates the proportionate distance between
  dilutions which infect above and below 50 of the
  wells

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TCID50
Dilution Infected Infected10-1 3/3 10010-2
3/3 10010-3 3/3 10010-4 2/3 6610-5 1/3 3
310-6 0/3 010-7 0/3 0
Log PD 66-50 x (Log10) 66-33 Log PD
0.48Log Dilution above 50
Infection 10-4.48
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Plaque Forming Units

• Areas where infected cells are being lysed by
  virus are seen as plaques, or areas of clearing
  in the cell monolayer.
• When stained with Crystal Violet these areas are
  easily identified as areas without stain

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Plaque forming Units

• A single virus infective dose can cause an area
  of cell destruction
• Movement of virus within cell is restricted by an
  agar overlay
• This causes areas of localized destruction
• Plaques are enumerated under a microscope to
  determine the plaque forming units per ml
  (PFU/ml)
• This allows comparison of different viruses in
  the same unit

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Calculation of PFU/mL

• Plaques are enumerated
• Plaque Counts are averaged over wells
• The average is then divided by the dilution times
  the volume
• (434038)/3(10-4 x 0.1)
• 3,730,000 pfu/ml

43 4 1 040 3 0 038 6 2 0Plaques formed per well
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Todays Lab

• Calculate PFU/ml
• Calculate TCID50
• Observe infected histology sections
• Record values and observations in lab notebook
• Observe cell monolayer on inverted scope