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

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

7. 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

8. 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
101
10-1
10-2
10-3
10-4
10-5

9. 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.

10. 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

11. Quantification of CPE
Tissue Culture Infective Dose 50 (TCID50): a measure of virulence of virus
Why Quantify?
Virulence
Immunity
Strain

12. Multi-well Plates 10-2 10-3 10-4 10-1 10-5 10-6 10-7 Control 1 2 3 4 5B C D A

13. TCID50 Procedure Count wells exhibiting CPE
Ideally you would know all the dilution factors to get infection rates of zero to 100 percent
100
Decreasing Dilution
CPE

14. 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

15. 10-4 1 2 3 4 5 6
10-1
10-5
10-6
10-7
Control B C D A
10-2
10-3
TCID50
Dilution Infected % Infected / / / / / / /3 0
Log PD = x (Log10) Log PD = Log Dilution above 50 % Infection

16. 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

17. 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

18. Calculation of PFU/mL Plaques are enumerated
Plaque Counts are averaged over wells
The average is then divided by the dilution times the volume
( )/3 (10-4 x 0.1)
= 3,730,000 pfu/ml
Plaques formed per well

19. Today’s Lab Calculate PFU/ml Calculate TCID50
Observe infected histology sections
Record values and observations in lab notebook
Observe cell monolayer on inverted scope