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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

Farhad B. Hashemi, PhD 2

Methods in Virology

Farhad B. Hashemi, PhD

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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

November 3, 2018Farhad B. Hashemi, PhD

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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


GEL Electrophoresis:PAGE vs. Agarose

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Virology Methods


Electrophoretic techniques

Virology Methods

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Plaque assay: Titration of virus (or anti-virus antibody)

Viral Infectivity assays

Farhad B. Hashemi, PhD 16



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


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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Farhad B. Hashemi, PhD26

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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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FB Hashemi, PhD

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ELISA: Enzyme-Linked Immunosorbant Assay

ELISA. Enzyme-Linked Immuno-Adsorbant Assay


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FB Hashemi, PhD

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Electron Microscopy (EM)


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Methods & Applications

1) Direct detection







iii. HA / HI, etc

3) Nucleic Acid Based Assaysi. Hybridization



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)


• 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

18-Nov-3Farhad B. Hashemi, PhD

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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



Laser

SensorSample (cells)


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)


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.


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


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


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


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


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


Scatter: Forward vs. Side

FSCDetector

CollectionLens

SSCDetector

Laser Beam

Original from Purdue University Cytometry Laboratories


FLUORESCENCE DETECTORS

FSCDetector

CollectionLens

Laser Beam

FluorescenceDetector A, B, C, etc…

Source; Purdue University Cytometry Laboratories, Modified by James Marvin


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


THRESHOLD

FSCDetector

FSCDetector

Time

Time

Threshold (eg. 52)

Threshold (eg. 52)

METHODS IN VIROLOGY:

Source: Purdue University Cytometry Laboratories


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1) Direct detection







iii. HA / HI, etc

3) Nucleic Acid Based Assays (or NAATs)i. Hybridization



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):


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


Methods in Virology:


Principles of Polymerase chain Reaction (PCR)



PCR Amplification: ENDPOINT (GEL-BASED)

FB Hashemi, PhD

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Detection of viruses and virus components

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Methods in Virology

REAL TIME PCR AMPLIFICATION

FB Hashemi, PhD

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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


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

November 3, 2018Farhad B. Hashemi, PhD 71


1. Direct detection

I. Electron Microscopy (EM)

II. Cell Culture



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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1. DIRECT DETECTION

I. Electron Microscopy (EM)

II. Cell Culture



II. Immuno-fluorescence assays

III. HA / HI, etc

3. Nucleic Acid Based

I. PCR (gel based; Real time)

II. Real Time PCR; Automation

FB Hashemi, PhD 74

Methods in Virology: Summary

Documents

ADVANCED VIROLOGY - medicine.tums.ac.irmedicine.tums.ac.ir/filegallery/microbiology/...Nucleic Acid amplification tests (NAATs) Very Sensitive: The increased sensitivity of NAATs has