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Methods in Virology

Methods in Virology. Techniques. Detection of a meaningful immune response to the virus (antibody or cell-mediated) by immunologic assay(s) Identification of the agent by staining of specimens or sections of tissue (light and electron microscopy)

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Methods in Virology

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  1. Methods in Virology

  2. Techniques • Detection of a meaningful immune response to the virus (antibody or cell-mediated) by immunologic assay(s) • Identification of the agent by staining of specimens or sections of tissue (light and electron microscopy) • Isolation and identification of the agent (cell culture or fertile eggs) • Detection of viral nucleic acid (probes or amplification)

  3. Infected Cells (Immunohistochemistry)

  4. 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.

  5. Developing a cell line 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

  6. 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

  7. 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.

  8. 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 Examples of Cytopathic Effects of Viral Infection

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

  10. Infectivity Assays

  11. 1 2 3 4 5 6 10-3 10-1 10-4 10-2 10-5 A B 10-6 C 10-7 D Control Multi-well Plates

  12. 100 0 Decreasing Dilution TCID50 Procedure • Count wells exhibiting CPE • Ideally you would know all the dilution factors to get infection rates of zero to 100 percent CPE

  13. 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

  14. 1 2 3 4 5 6 10-1 10-5 A 10-2 10-6 B C 10-3 10-7 D 10-4 Control TCID50 Dilution Infected % Infected10-1 3/3 10010-2 3/3 10010-3 3/3 10010-4 2/3 6610-5 1/3 3310-6 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

  15. 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

  16. 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

  17. Calculating viral titer based on the plaque assay method • The viral titer is a quantitative measurement of the biological activity of a virus and is expressed as plaque forming units (pfu) per ml. • To calculate the viral titer, count the number of well isolated plaques. • Then use the following formula to determine the titer (pfu/ml) of your viral stock.

  18. Average # Plaques = PFU/ml D x V D = Dilution factor V = Volume of diluted virusadded to the well

  19. Wells observed 7 days after inoculation with of 0.1ml viral solution PFU’s/ml = 42 plaques observed plaques (10-7 dilution factor)(0.1ml virus added) PFU’s/ml = 4.2 x109

  20. Calculation of PFU/mL • Plaques are enumerated • Plaque Counts are averaged over wells • The average is then divided by the dilution times the volume (43+40+38)/3(10-4 x 0.1) = 3,730,000 pfu/ml 43 4 1 040 3 0 038 6 2 0Plaques formed per well

  21. Detection of Viral Nucleic Acid

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