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) Isolation and identification of the agent (cell culture or fertile eggs) Detection of viral nucleic acid (probes or amplification)

Infected Cells (Immunohistochemistry)

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.

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

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

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.

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

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

Infectivity Assays

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

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

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

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 10-1 3/3 100 10-2 3/3 100 10-3 3/3 100 10-4 2/3 66 10-5 1/3 33 10-6 0/3 0 10-7 0/3 0 Log PD = 66-50 x (Log10) 66-33 Log PD = 0.48 Log Dilution above 50 % Infection 10-4.48

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

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

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.

Average # Plaques = PFU/ml D x V D = Dilution factor V = Volume of diluted virus added to the well

PFU’s/ml = 42 plaques observed plaques 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

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 0 40 3 0 0 38 6 2 0 Plaques formed per well

Detection of Viral Nucleic Acid