Laboratory virology

Diagnosis of viral infections • Culture • Detection of viral nucleic acids • Detection of viral antigens • Serology

– Test for immune response to virus

Virus Culture

• Some viruses cannot be cultured • Sampling – fluids, excreta, tissue, depending on symptoms and circumstances • Laboratory animals • Embryonated eggs • Cell culture – Primary cell cultures – Diploid cell strains – Continuous cell lines – Transformation

Specimens for viral diagnosis From Medical Microbiology, 5 th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Table 51-1.

Growth of virus on embryonated eggs

Davis, Duylbecco, Eisen, Ginsberg “Microbiology” 4 th ed, J.B. Lippincott 1990, Fig. 48-1

Primary cell culture

+ enzymes time

Subculture

enzymes time

Cell culture

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. (From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.3)

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

Transformation • Immortalization • Loss of contact inhibition • Anchorage independence

– Growth in soft agar – Growth in suspension

• Tumor formation in athymic (nude) mice

Recognition of viral growth in cell culture • Cytopathic effect (CPE)

– Morphological changes – Inclusion bodies

• Hemadsorbtion

CPE: Measles on human lung carcinoma (A549) Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.5

CPE: vaccinia

Uninfected Infected Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.5

Cytolology: CPE Syncytium formation by measles virus. Multinucleated giant cell (

arrow

) visible in a histologic section of lung biopsy tissue from a measles virus-induced giant cell pneumonia in an immunocompromised child. (From Medical Microbiology, 5 th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-1.)

CPE: inclusion bodies

neuron Negri bodies Negri bodies Immunohistochemical staining of intra-cytoplasmic viral inclusions in the neuron of a human rabies patient. (Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 39.9)

Hemadsorbtion

add red blood cells

Hemadsorption 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. (From Medical Microbiology, 5 th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-5.)

Assay of viruses

– Biological • Plaque assay • Transformation • Endpoint Method: TCID 50 , EID 50 , ID 50 , LD 50 – Physical, biochemical • Hemagglutination • Direct particle count • Immunological tests for proteins • Assay for nucleic acid (Southern, PCR) • Enzymatic (reverse transcriptase for retroviruses) – Comparison of quantitative methods

Plaque formation

Plaque

Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.5

Plaque formation

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

Plaque assay

Titer = 1.2 x 10 8 pfu/ml

Transformation

loss of contact inhibition

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. (From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.7)

Endpoint titration

10 -5 dilution 10 -6 10 -7 10 -8 1 2 3 4 5 = infected = uninfected 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

per ml. (TCID = tissue culture infective dose) TCID 50

Hemagglutination

RBC

Hemagglutination test

1:8 1:2

virus

1:2 1:2 1:2 1:2

serial dilution

8 16 32 64 128 256

mix with red blood cells side view top view Titer = 32 HA units/ml

Hemagglutination assay: influenza virus 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. (From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.9)

Direct particle count

Beads (10 4 /ml) 1.5 x 10 4 virus/ml 10 beads => 1 ul 15 virus => 1.5 x 10 4 virus/ml

Direct particle count

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). (From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.8)

Assays for viral proteins and nucleic acids From Medical Microbiology, 5 th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Box 51-4.

Comparison of quantitative methods

Method Direct electron microscope count Quantal infectivity assay in eggs Quantal infectivity assay by plaque formation Hemagglutination assay Amount (per ml) 10 10 EM particles 10 9 egg ID 50 10 8 pfu 10 3 HA units Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Table 2-4

Serology • Neutralization • Hemagglutination inhibition • Western blot • ELISA, radioimmune assay (RIA)

Antibody detection 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: western blot 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. )

Diagnostic methods for common human viruses From Schaechter’s Mechanisms of Microbial Disease; 4 th ed.; Engleberg, DiRita & Dermody; Lippincott, Williams & Wilkins; 2007; Table 31-3

Summary

• Four main clinical diagnostic techniques – Culture, serology, antigen detection, nucleic acid detection • Virus culture – Not all viruses can be cultured – Cultured cell types – Cytopathic effect • Virus quantitation – Biological – Physical • Basic serological techniques