This document discusses various techniques for diagnosing and studying viruses in a laboratory setting. It describes growing viruses in cell cultures and embryonated eggs, observing cytopathic effects, and quantifying viruses using plaque assays, particle counting, and hemagglutination assays. It also covers transforming infected cells to develop continuous cell lines and detecting viral proteins and antibodies using techniques like western blotting. The goal is to isolate, propagate, quantify, and identify viruses for research and clinical diagnosis.

1. Laboratory virology www.freelivedoctor.com

2. Specimens for viral diagnosis www.freelivedoctor.com

3. Growth of virus on embryonated eggs www.freelivedoctor.com

4. Primary cell culture time www.freelivedoctor.com + enzymes

5. Subculture enzymes time www.freelivedoctor.com

6. Growth of cells in culture. A primary culture is defined as the original plating of cells from a tissue, grown to a confluent monolayer, without subculturing. A cell strain (solid line) is defined as a euploid population of cells subcultivated more than once in vitro, lacking the property of indefinite serial passage. Cell strains ultimately undergo degeneration and death, also called crisis or senescence. A cell line (dashed line) is an aneuploid population of cells that can be grown in culture indefinitely. Spontaneous transformation or alteration of a cell strain to an immortal cell line can occur at any time during cultivation of the cell strain. The time in culture and corresponding number of subcultivations or passages are shown on the abscissas. The ordinate shows the total number of cells that would accumulate if all were retained in culture. Cell culture www.freelivedoctor.com

7. Cultured cells Primary Heterogeneous – many cell types Closest to animal Technical hassle Diploid cell strain Relatively homogeneous – fewer cell types Further from animal Technically less hassle Continuous cell line Immortal Most homogeneous Genetically weird – furthest from animal Hassle free Suspension or monolayer www.freelivedoctor.com

8. Transformation Immortalization Loss of contact inhibition Anchorage independence Growth in soft agar Growth in suspension Tumor formation in athymic (nude) mice www.freelivedoctor.com

9. Cultured cell morphologies Epithelial-like (human lung carcinoma, A549 ) Fibroblast like (baby hamster kidney, BHK) www.freelivedoctor.com

10. CPE: Measles on human lung carcinoma (A549) www.freelivedoctor.com

11. CPE: vaccinia on monkey kidney (BSC40) Low multiplicity of infection (moi) single plaque High moi, 48 hr www.freelivedoctor.com

12. Hemadsorbtion www.freelivedoctor.com add red blood cells

13. Hemadsorption of erythrocytes to cells infected with influenza viruses, mumps virus, parainfluenza viruses, or togaviruses. These viruses express a hemagglutinin on their surfaces, which bind erythrocytes of selected animal species. Hemadsorption www.freelivedoctor.com

14. Syncytium formation by measles virus. Multinucleated giant cell in a histologic section of lung biopsy tissue from a measles virus-induced giant cell pneumonia in an immunocompromised child. Cytolology: CPE www.freelivedoctor.com

15. CPE: inclusion bodies Immunohistochemical staining of intra-cytoplasmic viral inclusions in the neuron of a human rabies patient. www.freelivedoctor.com Negri bodies Negri bodies neuron

16. Plaque assay: method www.freelivedoctor.com 1:100 1:10 1:10 1:10 1:10 1:10 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 virus serial dilution plate 1 ml plaques 100 10 1 (1000) (100,000) (10,000) Titer = 1 x 10 7 pfu/ml

17. Plaque assay Titer = 2.4 x 10 8 pfu/ml www.freelivedoctor.com

18. Transformation loss of contact inhibition www.freelivedoctor.com

19. Focus formation by transforming viruses Focus assay. Monolayers of the NIH3T3 mouse fibroblast cell line were infected with Maloney murine sarcoma virus. The top two panels show photomicrographs of uninfected cells (left) and a single virus-induced focus (right). The bottom two panels show stained dishes of uninfected (left) and infected (right) cells. Foci are clearly visible as darker areas on the infected dish. www.freelivedoctor.com

20. = infected = uninfected Endpoint titration Five replicate wells of cells are infected with one ml of each of four different virus dilutions, incubated, and scored for infection by looking for CPE. In this example, the final titer is 10 6.3 TCID 50 per ml. (TCID = tissue culture infective dose) www.freelivedoctor.com 10 -5 10 -6 10 -7 10 -8 5 replicate samplings 1 2 3 4 5 dilution

21. Hemagglutination RBC www.freelivedoctor.com

22. Titer = 32 HA units/ml Hemagglutination test: method 1:8 1:2 1:2 1:2 1:2 1:2 8 16 32 64 128 256 virus serial dilution mix with red blood cells side view top view www.freelivedoctor.com

23. Hemagglutination assay. Seven different samples of influenza virus, numbered 1 through 7 at the left, were serially diluted as indicated at the top, mixed with chicken red blood cells (RBC), and incubated on ice for 1 to 2 hours. Wells in the bottom row contain no virus. Agglutinated RBCs coat wells evenly, in contrast to nonagglutinated cells, which form a distinct button at the bottom of the well. The HA titer, shown at the right, is the last dilution that shows complete hemagglutination activity. Hemagglutination assay: influenza virus www.freelivedoctor.com

24. Direct particle count 10 beads => 1 ul 15 virus => 1.5 x 10 4 virus/ml 1.5 x 10 4 virus/ml www.freelivedoctor.com Beads (10 4 /ml)

25. Direct electron microscopic particle count. An electron micrograph of a spray droplet containing 15 latex beads (spheres) and 14 vaccinia virus particles (slightly smaller, brick-shaped particles). Direct particle count www.freelivedoctor.com

26. Comparison of quantitative methods www.freelivedoctor.com Method Amount (per ml) Direct electron microscope count 10 10 EM particles Quantal infectivity assay in eggs 10 9 egg ID 50 Quantal infectivity assay by plaque formation 10 8 pfu Hemagglutination assay 10 3 HA units

27. Assays for viral proteins and nucleic acids www.freelivedoctor.com

28. Neutralization, hemagglutination, and hemagglutination inhibition assays. In the assay shown, tenfold dilutions of serum were incubated with virus. Aliquots of the mixture were then added to cell cultures or erythrocytes. In the absence of antibody, the virus infected the monolayer (indicated by CPE) and caused hemagglutination (i.e., formed a gel-like suspension of erythrocytes). In the presence of the antibody, infection was blocked (neutralization), and hemagglutination was inhibited, allowing the erythrocytes to pellet. The titer of antibody in the serum was 100. pfu, Plaque-forming units.From Medical Microbiology, 5 th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-6. Antibody detection www.freelivedoctor.com

29. Western blot analysis of HIV antigens and antibody. HIV protein antigens are separated by electrophoresis and blotted onto nitrocellulose paper strips. The strip is incubated with patient antibody, washed to remove the unbound antibody, and then reacted with enzyme-conjugated antihuman antibody and chromophoric substrate. Serum from an HIV-infected person binds and identifies the major antigenic proteins of HIV. This data demonstrates the seroconversion of one HIV-infected individual with sera collected on day 0 (D0) to day 30 (D30) compared to a known positive control (PC) and negative control (NC). (From Medical Microbiology, 5 th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-7. ) Antibody detection: western blot www.freelivedoctor.com

30. Diagnostic methods for common human viruses www.freelivedoctor.com

31. Summary 4 main clinical diagnostic techniques Culture, serology, antigen detection, nucleic acid detection Virus culture Cultured cell types Cytopathic effect Not all viruses can be cultured Virus quantitation Biological Physical Basic serological techniques www.freelivedoctor.com