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ADVANCED VIROLOGY
METHODS IN VIROLOGY
FALL 2018
INTRODUCTION
November 3, 2018Farhad B. Hashemi, PhD 1
F. B. Hashemi, PhDDepartment of MicrobiologySchool of MedicineTehran University of Medical Sciences
Principles Molecular Virology 5th Ed.
11/3/2018
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Methods in Virology
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1. Introduction/ History
2. Living Host systems
3. Cell Culture Methods
4. Serological/Immunological assays/ Flow Cytometry
5. Ultra-structural studies
6. Molecular/genetic approaches in Virology;
Nucleic acid Technology; PCR
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1881, Louis Pasteur studied rabies in animals.
1900, Walter Reed, discovered Yellow fever virus (YFV)
as the cause of YF
1937, Max Theiler was able to propagate YFV in chick
embryos,
Produced an attenuated vaccine 17D strain; still in
use today.
The success of this approach led to many other viral
vaccines
Introduction/ History
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Cultivation of viruses
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Virology Methods
Cultivation of viruses
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Virology Methods
CsCl/Sucrose/ Percoll gradient
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Isolation of viruses:ULTRA centrifugation:
Isolation of viruses; Plaque assay
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Virology Methods
Isolation of viruses
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Virology Methods
ISOLATION OF VIRUSES
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GEL Electrophoresis:PAGE vs. Agarose
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Virology Methods
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Electrophoretic techniques
Virology Methods
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Plaque assay: Titration of virus (or anti-virus antibody)
Viral Infectivity assays
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Plaque assay: Titration of viruses (or anti-virus antibodies)
Q: What about non-adherent cells? e.g. H9 T lymphocytes
Infectivity assays
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Virology Methods
INFECTIVITY ASSAYS
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Virology Methods
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Infectivity assays
Virology
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Cell culture/ Monoclonal Antibodies:Polyclonal Ab.
Immortal cell culture/ Antibiotics!!
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3 Main Approaches
1) Direct detection
i. Electron Microscopy (EM)
ii. Cell Culture (primary vs transformed)
2) Serological Assays
i. ELISA/ Western Blot
ii. Immunofluorescence assays
i. Flow cytometry vs Microscopy
iii. HA / HI, etc
3) Nucleic Acid Based Assaysi. Hybridization
ii. Polymerase Chain reaction (PCR):
i. gel-based vs. Real time
iii. NASBA, etc
Laboratory Diagnosis of Viral Infections:
Three Main approaches:
1. DIRECT DETECTION
I. ELECTRON MICROSCOPY (EM)
II. CELL CULTURE
2. SEROLOGICAL PROCEDURES
I. ELISA/ WESTERN BLOT
II. IMMUNO-FLUORESCENCE ASSAY (DFA)
III. HA / HI, ETC
3. NUCLEIC ACID BASED
I. PCR (GEL BASED; REAL TIME)
II. REAL TIME PCR; AUTOMATION
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Detection of viruses and viruscomponents
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Structural investigations of cells and virions: Electron Microscopy
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1. Electron Microscopy (EM):
Visualize the virus
Not sensitive,
Expensive, and laborious
2. Culture the virus1. Sensitive, but not very specific
2. Viruses need living cells (animal, egg, culture) 3. High cost and time consuming 4. Many clinically relevant viruses cannot be grown in vitro
Main Approaches
1. DIRECT DETECTION
I. ELECTRON MICROSCOPY (EM)
II. CELL CULTURE
2. SEROLOGICAL PROCEDURES
I. ELISA/ Western Blot
II. Immuno-fluorescence assays
III. HA / HI, etc
3. NUCLEIC ACID BASED
I. PCR (GEL BASED; REAL TIME)
II. REAL TIME PCR; AUTOMATION
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Serological assays
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C- ELISA: Enzyme-Linked Immuno Absorbant Assay
D- Western Blot assay
Serological assays
Serological Methods : ELISA, EIA, Western Blots, H&E Histopathology
Hematoxylin and Eosin Stain (H&E)
Tissue Immuno-histochemistry 31
ELISA 96 well Microtiterplate,Alkaline Phosphotase
Hemagglutination inhibition assay (HI)
Detection of viruses and virus components
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Methods in Virology
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ELISA: Enzyme-Linked Immunosorbant Assay
ELISA. Enzyme-Linked Immuno-Adsorbant Assay
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Electron Microscopy (EM)
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Methods & Applications
1) Direct detection
i. Electron Microscopy (EM)
ii. Cell Culture (primary vs transformed)
2) Serological Assays
i. ELISA/ Western Blot
ii. Immunofluorescence assays
i. Flow cytometry vs Microscopy
iii. HA / HI, etc
3) Nucleic Acid Based Assaysi. Hybridization
ii. Polymerase Chain reaction (PCR):
i. gel-based vs. Real time
iii. NASBA, etc
Diagnosis of Viral Infections:
Methods in Virology:Hemagglutination assay (HA)
HA Binds to sialic acid receptors (on red blood cells)
Agglutinate Bird RBC’s in wells of microtiterplate. Only detects virus presence and titer. (not type)
Requires amplification in embryonated egg
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1. DIRECT Immunofluorescence assay (DFA)
Flow cytometry
Microscopy
2. Chemiluminescence (application in Western Blots)
3. Hemagglutination (HA)/ (HI:inhibition)
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• Flow Cytometry
• Various formats of data presentation
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Methods in Virology: Flow CytometryImmunofluorescence assay
Cytometry vs. FLOW Cytometry
CYTOMETRY
• Localization of antigen is possible
• Poor enumeration of cell subtypes
• Limited number of simultaneous
measurements
Flow Cytometry
• Cannot locate the antigen.
• Can analyze many cells in a short
time frame.
• Can detect numerous parameters
at once.
Methods in Virology: Cytometry
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BD-LSR-II 561 Flow Cytometer
•FACS cell sorting method
Flow Cytometry:Flow Cytometer for phenotype studies
Forward Scatter Count (FSC) vs. side scatter count (SSC)
FSC estimates size, and SSC estimates internal structures,
Correlated measurements between these estimates
allow for differentiation of cell types in a heterogenous
cell population
FSC
SS
C
Lymphocytes
Monocytes
Granulocytes
RBCs, Debris,Dead Cells
Methods in Virology: Flow Cytometry
• Flow Cytometry
• Various formats of
data presentation
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Laser
SensorSample (cells)
Methods in Virology: Flow Cytometry
Assay principles and procedure
SIDE SCATTER
Side Scatter Channel: Laser light scattered at 90 degrees to the
axis of the laser path
The intensity of this signal is proportional to the amount of
cytosolic structure in the cell (eg. granules, cell inclusions, etc.)
i. Side Scatter=SSC=RALS=90 degree Scatter
ii. Right angle light scatter (RALS)
Methods in Virology: Flow Cytometry
Flow Cytometry: Principle; channels
Flow Cytometry: Principle; channels
As those fluorochromes leave the excited state, they
release energy (a photon) with a specific wavelength,
Emission wavelength is longer than the excitation
wavelength
Emitted photons pass through the collection lens;
split and steered down specific channels with by
optical filters.
Methods in Virology: Flow Cytometry
Flow Cytometry: Basic steps; Fluorescence Channels
1. Cells are stained with fluorochromes
2. Laser shines on the cell,
3. Fluorochromes on/in the cell (intrinsic
or extrinsic) may absorb some of the
light, and
4. Become excited and emit light
5. Device detects the emitted light
Methods in Virology: Flow Cytometry
350 457488514 610632
Forward Scatter
Light that is scattered in the forward
direction (along the same axis the laser
travels) is detected in the Forward Scatter
Channel.
The intensity of this signal has been
attributed to cell size, refractive index
(membrane permeability)
Forward Scatter=FSC=FALS=LALS
Methods in Virology: Flow Cytometry
Low pressure
High pressure
FL3
0 1024 2048 3072 4096
Count
300
280
260
240
220
200
180
160
140
120
100
80
60
40
20
0
68.70 19.16 9.56
G0/G1 CV= 2.42
G2/M
S phaseG0/G1
FL3
0 1024 2048 3072 4096
Count
340
320
300
280
260
240
220
200
180
160
140
120
100
80
60
40
20
0
74.85 9.12 15.84
GO/G1 CV= 7.79
74.85 9.12 15.84
GO/G1 CV= 7.79
Methods in Virology: Flow Cytometry
Analysis; FSC vs. SSC
Since FSC ~ size and SSC ~ internal structure, a correlated
measurement between them can allow for differentiation
of cell types in a heterogenous cell population
FSC
SS
C
Lymphocytes
Monocytes
Granulocytes
RBCs, Debris,Dead Cells
Methods in Virology: Flow Cytometry
Flow Cytometry: Spectra of Common Fluorochromes
Laser Lines (nm) 350 457488514 610632
300 400 500 600 700
PE-Texas Red
Texas Red
PI
Ethidium
PE
FITC
cis-Paranaric Acid
Source; Purdue University Cytometry Laboratories, Modified by James Marvin
FORWARD SCATTER
Source: Purdue University Cytometry Laboratories
Laser Beam
FSCDetector
Methods in Virology: Flow Cytometry
Scatter: Forward vs. Side
FSCDetector
CollectionLens
SSCDetector
Laser Beam
Original from Purdue University Cytometry Laboratories
Methods in Virology: Flow Cytometry
FLUORESCENCE DETECTORS
FSCDetector
CollectionLens
Laser Beam
FluorescenceDetector A, B, C, etc…
Source; Purdue University Cytometry Laboratories, Modified by James Marvin
Methods in Virology: Flow Cytometry
FILTERS
Many light wavelengths scatter from a cell,
Optical filters split the light into specific wavelength, so we detect them independently.
Optical filters: designed to absorb or reflect some wavelengths of light, while transmitting other.
3 types of filters
1. Long Pass filter
2. Short Pass filter
3. Band Pass filter
Methods in Virology: Flow Cytometry
THRESHOLD
FSCDetector
FSCDetector
Time
Time
Threshold (eg. 52)
Threshold (eg. 52)
METHODS IN VIROLOGY:
Source: Purdue University Cytometry Laboratories
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Methods & Applications
1) Direct detection
i. Electron Microscopy (EM)
ii. Cell Culture (primary vs transformed)
2) Serological Assays
i. ELISA/ Western Blot
ii. Immunofluorescence assays
i. Flow cytometry vs Microscopy
iii. HA / HI, etc
3) Nucleic Acid Based Assays (or NAATs)i. Hybridization
ii. Polymerase Chain reaction (PCR):
i. gel-based vs. Real time
iii. NASBA, etc
Diagnosis of Viral Infections:
Nucleic Acid amplification tests (NAATs)
Very Sensitive: The increased sensitivity of NAATs has revolutionized testing in clinical virology laboratory.
1. Molecular hybridization techniques:• Probes directed at a unique, conserved portion of viral
genome; highly specific; bind only to complementary DNA or RNA sequences.
Probes are particularly useful for detecting and typing viruses for which reliable culture methods are not available.
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Methods in Virology
Probe hybridization: Detection of virus components
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Methods in Virology: Molecular methods
Molecular probes: available as commercial kits for the detection of HPV, HIV, HSV,CMV,hepatitis B virus (HBV), and HCV.
If the concentration of viral genomes in patient specimen is too low to permit detection (low sensitivity)
• Commercially available probes for HSV and CMV detect only 70% to 90% of (+) specimens by culture).
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Nucleic Acid DetectionMethods in Virology
• Increased sensitivity of NAATs: revolutionized testing in clinical virology labs. Three approaches:
1. Target amplification such as PCR, strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), and transcription-mediated amplification (TMA) systems;
2. Probe amplification, including Q-beta replicase and ligase chain reaction (LCR); and
3. Signal amplification, such as branched-chain DNA (bDNA) assay and hybrid capture assay. Several commercial and in-house (“home-brew”) assays have been developed.
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Nucleic Acid DetectionMethods in Virology:
NA Amplification techniques (NAATs):
Methods & Applications
1. DIRECT detection
Electron Microscopy (EM)
Cell Culture
2. Serological Procedures
ELISA/ Western Blot
Immuno-fluorescence assays
Hemagglutination
3. Nucleic Acid Based assays
PCR (gel based; Real time)
Real Time PCR; Automation
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Methods in Virology:
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Principles of Polymerase chain Reaction (PCR)
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Methods in Virology: Molecular methods
PCR Amplification: ENDPOINT (GEL-BASED)
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Methods in Virology: Molecular methods
Detection of viruses and virus components
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Methods in Virology
REAL TIME PCR AMPLIFICATION
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iCyclerBioRad
LightCyclerRoche
7700Applied Biosystems
5700Applied Biosystems
FluorTrackerStratagene
Methods in Virology
REAL TIME PCR AMPLIFICATION
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Detect antibodies against the virus1. Highly sensitive and specific
2. Low cost, large No. of samples
3. Indirect approach
4. Dependent on host immunity
Detect viral nucleic acid (NAAT; e.g. multiplex real-time Q-PCR)
1. Highly sensitive & specific
2. Low cost, large no. of samples
3. Multiple targets/ Universally applicable4. Revolutionary technical developments
** Must know what you are looking for?
PCR-MS: Automated NA detection; e.g. MALDI-TOF
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Methods in Virology
False (+) in many viral infections (clinical vs. Laboratory diagnosis)
Exceptions:
• EBV, BK virus: False (-) in plasma during acute infection
• Influenza Virus: Virus cleared by the time of 2nd pneumonia starts
• Very useful to detect: HCV; HBV and HIV70
Method comparison: PCR vs. Culture
PCR can replace cell culture, and many serologic methods. Cannot detect unknown viruses.
Methods in Virology
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Methods & Applications
1. Direct detection
I. Electron Microscopy (EM)
II. Cell Culture
2. Serological Procedures
I. ELISA/ Western Blot
II. Immuno-fluorescence assay (DFA)
III. HA / HI, etc
3. Nucleic Acid Based
I. PCR (gel based; Real time)
II. Real Time PCR; Automation
Methods in Virology: Review
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Viral GenomicsMethods in Virology: Molecular methods
Virus genetics
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Methods in Virology: Molecular methods
1. DIRECT DETECTION
I. Electron Microscopy (EM)
II. Cell Culture
2. Serological Procedures
I. ELISA/ Western Blot
II. Immuno-fluorescence assays
III. HA / HI, etc
3. Nucleic Acid Based
I. PCR (gel based; Real time)
II. Real Time PCR; Automation
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Methods in Virology: Summary