Laboratory virology

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Laboratory virology
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Diagnosis of viral infections

• Culture
• Detection of viral nucleic acids
• Detection of viral antigens
• Serology
• Test for immune response to virus

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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

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Specimens for viral diagnosis
From Medical Microbiology, 5th ed., Murray,
Rosenthal Pfaller, Mosby Inc., 2005, Table
51-1.
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Growth of virus on embryonated eggs
Davis, Duylbecco, Eisen, Ginsberg Microbiology
4th ed, J.B. Lippincott 1990, Fig. 48-1
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Primary cell culture
time
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Subculture
enzymes
time
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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)
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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

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Transformation

• Immortalization
• Loss of contact inhibition
• Anchorage independence
• Growth in soft agar
• Growth in suspension
• Tumor formation in athymic (nude) mice

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Recognition of viral growth in cell culture

• Cytopathic effect (CPE)
• Morphological changes
• Inclusion bodies
• Hemadsorbtion

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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
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CPE vaccinia
Fields Vriology (2007) 5th edition, Knipe, DM
Howley, PM, eds, Wolters Kluwer/Lippincott
Williams Wilkins, Philadelphia Fig. 2.5
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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, 5th ed., Murray, Rosenthal
Pfaller, Mosby Inc., 2005, Fig. 51-1.)
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CPE inclusion 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)
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Hemadsorbtion
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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, 5th
ed., Murray, Rosenthal Pfaller, Mosby Inc.,
2005, Fig. 51-5.)
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Assay of viruses

• Biological
• Plaque assay
• Transformation
• Endpoint Method TCID50, EID50, ID50, LD50
• 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

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Plaque formation
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Plaque
Fields Vriology (2007) 5th edition, Knipe, DM
Howley, PM, eds, Wolters Kluwer/Lippincott
Williams Wilkins, Philadelphia Fig. 2.5
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Plaque formation
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Plaque assay method
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Plaque assay
Titer 1.2 x 108 pfu/ml
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Transformationloss of contact inhibition
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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)
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Endpoint titration
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
106.3 TCID50 per ml. (TCID tissue culture
infective dose)
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Hemagglutination
RBC
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Hemagglutination test
Titer 32 HA units/ml
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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)
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Direct particle count
10 beads gt 1 ul
1.5 x 104 virus/ml
15 virus gt 1.5 x 104 virus/ml
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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)
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Assays for viral proteins and nucleic acids
From Medical Microbiology, 5th ed., Murray,
Rosenthal Pfaller, Mosby Inc., 2005, Box 51-4.
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Comparison of quantitative methods
Method Amount (per ml)
Direct electron microscope count 1010 EM particles
Quantal infectivity assay in eggs 109 egg ID50
Quantal infectivity assay by plaque formation 108 pfu
Hemagglutination assay 103 HA units
Fields Vriology (2007) 5th edition, Knipe, DM
Howley, PM, eds, Wolters Kluwer/Lippincott
Williams Wilkins, Philadelphia Table 2-4
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Serology

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

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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,
5th ed., Murray, Rosenthal Pfaller, Mosby Inc.,
2005, Fig. 51-6.
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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, 5th ed., Murray,
Rosenthal Pfaller, Mosby Inc., 2005, Fig. 51-7.
)
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Diagnostic methods for common human viruses
From Schaechters Mechanisms of Microbial
Disease 4th ed. Engleberg, DiRita Dermody
Lippincott, Williams Wilkins 2007 Table 31-3
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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