Redeem

er’s Universi

ty,,NigeriaNigeria

COURSE:COURSE: MCB 30 MCB 3088

TITLE:TITLE:ISOLATION, PURIFICATION AND ISOLATION, PURIFICATION AND

IDENTIFICATION OF VIRUSESIDENTIFICATION OF VIRUSES

Redeem

er’s Universi

ty,,NigeriaNigeria

1.1. Observe the situationObserve the situation

2.2. Ask a questionAsk a question

3.3. Turn that question into a testable hypothesisTurn that question into a testable hypothesis

4.4. Predict the outcome of your experimentPredict the outcome of your experiment

5.5. Perform your experimentPerform your experiment

6.6. Analyze the resultsAnalyze the results

7.7. Evaluate your hypothesis Evaluate your hypothesis

TH

E 7

STEP

S O

F

SC

IEN

TIF

IC M

ETH

OD

Redeem

er’s Universi

ty,,NigeriaNigeria

Viruses are?:Viruses are?:

Redeem

er’s Universi

ty,,NigeriaNigeria

Viruses .Viruses .…….1…….1• 1. Infectious agent found in virtually all life forms,

(humans, animals, plants, fungi, and bacteria)

• 2. Not considered free-living, since they cannot reproduce outside of a living cell

• 3. Replicates by transmitting their genetic information from one cell to another

Redeem

er’s Universi

ty,,NigeriaNigeria

VirusesViruses…..2…..2• 4. Consist of genetic material—either

deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)

• 5. Surrounded by a protective coating of protein, called a capsid, + or - an outer lipid envelope.

• 6. Vary in size from poxvirus (450nm - 0.000014 in) to poliovirus (30 nm - 0.000001 in) in length, so too small to be seen by light microscopy.

Redeem

er’s Universi

ty,,NigeriaNigeria

Redeem

er’s Universi

ty,,NigeriaNigeria

IdentificationIdentification3 main methods:3 main methods:

i.i. MicroscopyMicroscopy

ii.ii.IsolationIsolation

iii.iii.SerologySerology

Redeem

er’s Universi

ty,,NigeriaNigeria

Isolation & IdentificationIsolation & Identification

MicroscopyMicroscopy• Light MicroscopyLight Microscopy

• Virus size precludes use of LMVirus size precludes use of LM

• Pathogenic effect of virus on infected cellsPathogenic effect of virus on infected cells• inclusion bodies:inclusion bodies:e.g. Negri bodies in Rabies and Councilman e.g. Negri bodies in Rabies and Councilman

bodies in YF, Guanieri bodies, Smallpox. bodies in YF, Guanieri bodies, Smallpox.

• Fluorescence MicroscopyFluorescence Microscopy

• Electron MicroscopyElectron Microscopy

Redeem

er’s Universi

ty,,NigeriaNigeria

FLUORESCENCE MICROSCOPY

Redeem

er’s Universi

ty,,NigeriaNigeria

Filoviridae(Ebola, Marburg)

Arenaviridae(Lassa, Junin, Machupo, Guanarito)

Bunyaviridae(CCHF, RVF,

Hantaviruses)

Haemorrhagic Fever Viruses

Flaviviridae(dengue, yellow fever,

TBE group)

ELECTRONMICROSCOPYELECTRONMICROSCOPY

Redeem

er’s Universi

ty,,NigeriaNigeria

Viruses Structure – Viruses Structure – Small pox virusSmall pox virusA colored-transmission electron A colored-transmission electron micrograph shows a group of micrograph shows a group of Orthopoxvirus variola, the virus that Orthopoxvirus variola, the virus that causes smallpox. causes smallpox.

Once greatly feared for its ability to kill Once greatly feared for its ability to kill or disable its victims, smallpox was or disable its victims, smallpox was eradicated by 1979 through a worldwide eradicated by 1979 through a worldwide vaccination campaignvaccination campaign

Note the unique brick shaped structure.Note the unique brick shaped structure.

Redeem

er’s Universi

ty,,NigeriaNigeria

Viruses Structure - Viruses Structure - AdenovirusAdenovirus

Non enveloped icosahedral Non enveloped icosahedral virus virus

The adenovirus is one of the The adenovirus is one of the 100 virus types that causes the 100 virus types that causes the common cold.common cold.

The adenovirus can enter a The adenovirus can enter a human host through the human host through the respiratory and GITs or the eyerespiratory and GITs or the eye

Redeem

er’s Universi

ty,,NigeriaNigeria

Techniques for Virus isolationTechniques for Virus isolation

Two major techniques - Classical and MolecularTwo major techniques - Classical and Molecular ClassicalClassical

Cell CultureCell CultureVirus isolation and quantitationVirus isolation and quantitationPreparation of AntigenPreparation of AntigenVirus PurificationVirus PurificationElectron MicroscopyElectron MicroscopyTraditional serological testsTraditional serological testsImmunoassysImmunoassysCell mediated immunityCell mediated immunity

Redeem

er’s Universi

ty,,NigeriaNigeria

Techniques for Virus isolationTechniques for Virus isolation

Two major techniques - Classical and Two major techniques - Classical and MolecularMolecular

MolecularMolecularRNA transcription, transfection & quantitationRNA transcription, transfection & quantitationDNA viruses: DNA extraction, purification & DNA viruses: DNA extraction, purification &

characterizationcharacterizationVirus mutantsVirus mutantsPolypeptidesPolypeptidesViral enzyme assaysViral enzyme assays

Redeem

er’s Universi

ty,,NigeriaNigeria

Techniques for Virus isolation: Classical Techniques for Virus isolation: Classical Cell CultureCell Culture

Primary- fresh organ of lab animal human embryo (kidney)Primary- fresh organ of lab animal human embryo (kidney)

Continuous- from primary grown continuously limited Continuous- from primary grown continuously limited subcultures generic variation, often die out 15-25 passagessubcultures generic variation, often die out 15-25 passages

Euploid- Normal human diploid cells with exact multiples of Euploid- Normal human diploid cells with exact multiples of normal chromosomal numbers, up to 50 passages, stop normal chromosomal numbers, up to 50 passages, stop dividingdividing

Aneuploid – Mouse fibroblasts, tumour cells not an exact Aneuploid – Mouse fibroblasts, tumour cells not an exact multiple of normal chromosomal numbersmultiple of normal chromosomal numbers

Insect – Insect – Aedes aegypti and A. albopictus Aedes aegypti and A. albopictus

Redeem

er’s Universi

ty,,NigeriaNigeria

Virus Isolation Virus Isolation Techniques - Techniques - Classical Classical MethodsMethods

Cell CultureCell Culture Art of growing cells in vitroArt of growing cells in vitro Over 3,200 chracterized cell lines availableOver 3,200 chracterized cell lines available Derived from 75 species (including hybridomas and plant cultures)Derived from 75 species (including hybridomas and plant cultures) Banked in cell culture banks: ATCC, ECACCBanked in cell culture banks: ATCC, ECACC Select cell type with unique characteristics for Select cell type with unique characteristics for Confirm life expectancy for study (15-20 passages or subcultures from seed stock to ensure retention of characteristics)Confirm life expectancy for study (15-20 passages or subcultures from seed stock to ensure retention of characteristics)

Types of Cell culturesTypes of Cell cultures Primary cellsPrimary cells Continuous cellsContinuous cells Insect cellsInsect cells

Propagation of cell culturesPropagation of cell cultures Media and buffersMedia and buffers Endotoxin free waterEndotoxin free water SterilizationSterilization Propagation of cells by scraping and enzyme treatmentPropagation of cells by scraping and enzyme treatment Subculture protocolSubculture protocol Suspension culturesSuspension cultures Lymphocyte culturesLymphocyte cultures Maintenance of adherent and suspension cellsMaintenance of adherent and suspension cells Quality assurance and contaminationQuality assurance and contamination Quantifying by cell counts Trypan blueQuantifying by cell counts Trypan blue Cell preservation and storageCell preservation and storage CPE due to virusesCPE due to viruses

Virus PurificationVirus Purification Electron MicroscopyElectron Microscopy Traditional serological testsTraditional serological tests ImmunoassysImmunoassys Cell mediated immunityCell mediated immunity

Redeem

er’s Universi

ty,,NigeriaNigeria

Fusion of a virus with the membrane of an endosome

Entry by fusing with plasma membrane

Redeem

er’s Universi

ty,,NigeriaNigeria

Cytopathic Effect of VirusesCytopathic Effect of VirusesVirus replication leads to cell damage Virus replication leads to cell damage

Cell Lysis

Redeem

er’s Universi

ty,,NigeriaNigeria

FIGURE 44-1 Development and progression of viral cytopathology. Human embryo skin muscle cells were infected with human cytomegalovirus and stained at selected times to demonstrate (A) uninfected cells, (B) late virus cytopathic effects (nuclear inclusions, cell enlargement), (C) cell degeneration, and (D) a focus of infected cells in a cell monolayer (i.e., a plaque), hematoxylin and eosin stain.

Redeem

er’s Universi

ty,,NigeriaNigeria

Redeem

er’s Universi

ty,,NigeriaNigeria

Transmission electron micrograph of HIV-1, budding and free  

Redeem

er’s Universi

ty,,NigeriaNigeria

Redeem

er’s Universi

ty,,NigeriaNigeria

Cytopathic Effect of VirusesCytopathic Effect of VirusesVirus replication leads to cell damage Virus replication leads to cell damage

Enlarged round refractile

cells Adenovirus

Redeem

er’s Universi

ty,,NigeriaNigeria

Enterovirus

V.stomatitis

Bov

ine

herp

es

Parainfluenza

Bovin

e a

deno

HerpesParamyxovirus

Vaccinia

Redeem

er’s Universi

ty,,NigeriaNigeria

Virus isolation and quantitationVirus isolation and quantitation Most commonly used in viral diagnosisMost commonly used in viral diagnosis

Collection of specimens – sterile, timely & preservationCollection of specimens – sterile, timely & preservation

Virus isolation and quantitationVirus isolation and quantitation Cell culture/Organ cultureCell culture/Organ culture

CPE, Hemadsorption, InterferenceCPE, Hemadsorption, Interference

Laboratory animalsLaboratory animalsMouse, rabbits, birds, sheep goats cows etcMouse, rabbits, birds, sheep goats cows etc

Fertile hen eggsFertile hen eggs

Redeem

er’s Universi

ty,,NigeriaNigeria

Diagnosis

Serology neutralization - (Neut) hemagglutination inhibition (HI) complement fixation (CFT) ELISA - IgM & IgG

Virus Detection culture, PCR, Ag detection

Histopathology

blood

liver tissue

blood or organs

Specimen neededLaboratory Test

Redeem

er’s Universi

ty,,NigeriaNigeria

Techniques for Virus isolation: Classical Techniques for Virus isolation: Classical QuantitationQuantitation

Infectivity assay(Infectivity assay( Plaque AssayPlaque Assay Pock AssayPock Assay Haemagglutinin AssayHaemagglutinin Assay

TCIDTCID5050,, EIDEID5050, LD, LD50,50, Dilution of a virus required to infect 50% of experimental Dilution of a virus required to infect 50% of experimental

animalanimal

Dilution of a virus required to kill or cause the death 50% of Dilution of a virus required to kill or cause the death 50% of experimental animalexperimental animal

To calculate we do serial 10/5/2 -fold dilution and check To calculate we do serial 10/5/2 -fold dilution and check number of animals sick or dead at end of experiment number of animals sick or dead at end of experiment

Redeem

er’s Universi

ty,,NigeriaNigeria

A plaque assay. Serial dilutions of virus have been plated on confluent monolayer cultures of cells. The cells are stained after a period of time in which a single virus infects a cell, produces new virus particles and infects surrounding cells. The white areas show areas of the culture in which the cells have been killed. Each "plaque" is the result of the presence of one original infectious virus particle.

Redeem

er’s Universi

ty,,NigeriaNigeria

Calculation of TCIDCalculation of TCID50 or LD or LD5050Reed & Muench methodReed & Muench method

(% positive above 50%) - 50%(% positive above 50%) - 50%

(% positive above 50%) – (%positive below 50%)(% positive above 50%) – (%positive below 50%)

= proportionate distance= proportionate distance

Spearman Karber method Spearman Karber method

Highest dilution giving 100% CPE + 0.5 – Highest dilution giving 100% CPE + 0.5 –

Total number of test units showing CPETotal number of test units showing CPE

Number of test units per dilution = TCIDNumber of test units per dilution = TCID5050

Redeem

er’s Universi

ty,,NigeriaNigeria

Preparation of Antigen & PurificationPreparation of Antigen & Purification Whole cell antigenWhole cell antigen

Freezing and ThawingFreezing and Thawing SonicationSonication Alkaline glycine treatmentAlkaline glycine treatment Fractionation of antigensFractionation of antigens

Tween 80Tween 80Beta-propiolactone extraction -Beta-propiolactone extraction -Non-ionic detergent – separates envelope proteins from Non-ionic detergent – separates envelope proteins from

capsidcapsid

Virus PurificationVirus Purification Adsorption/elution from erythrocytes based on Adsorption/elution from erythrocytes based on

hemagglutinanility of virushemagglutinanility of virus

Adsorption/elution from CaHPOAdsorption/elution from CaHPO4.4.2H2H220 brushite (DNA from RNA 0 brushite (DNA from RNA Toga from Flavi)Toga from Flavi)

Redeem

er’s Universi

ty,,NigeriaNigeria

Preparation of Antigen & PurificationPreparation of Antigen & Purification Chromatographic proceduresChromatographic procedures

Gel filtrationGel filtration Ion exchange or affinity Ion exchange or affinity

UltracentrifugationUltracentrifugation

Precipitationntechnique using PEG 600 in a cold high –salt environment Precipitationntechnique using PEG 600 in a cold high –salt environment gentler than other techsgentler than other techs

Sucrose gradient produces bands of pure virusSucrose gradient produces bands of pure virus CsCL gradientCsCL gradient

FiltrationFiltration

Electron MicroscopyElectron Microscopy Negative staining virus particle do not scatter ions under EM, so carbon Negative staining virus particle do not scatter ions under EM, so carbon

coating in plastic films coating in plastic films

Redeem

er’s Universi

ty,,NigeriaNigeria

Traditional serological testsTraditional serological tests

Neutralization TestNeutralization Test

Complement foxation TestComplement foxation Test

Haemagglutination-inhibition testHaemagglutination-inhibition test

Single radial hemolysisSingle radial hemolysis

Particle agglutination test with latex or labelled erythrocytesParticle agglutination test with latex or labelled erythrocytes

Immunodiffusion testImmunodiffusion test

Countercurrent Immunoelectrophoresis CIEOPCountercurrent Immunoelectrophoresis CIEOP

Redeem

er’s Universi

ty,,NigeriaNigeria

ImmunoassaysImmunoassays

ELISAELISAFIAFIARIARIA

ImmunoassysImmunoassysCell mediated immunityCell mediated immunity

Redeem

er’s Universi

ty,,NigeriaNigeria

Medical Medical VirologyVirology• Initial in vitro screening of drug

candidates for potential antiviral activities

• Detection of virus and viral antigen• Detection of viral nucleic acid• Serological methods• Disinfection

Redeem

er’s Universi

ty,,NigeriaNigeria

Virus Structure Virus Structure ……1……1

• Individual virus particle is called a virion

• Contains genetic material, or genomes, in one of several forms

• Viral genes may consist of either DNA or RNA. (cellular organisms genes only DNA)

• Almost all viral DNA is double-stranded, with either a circular or a linear arrangement

Redeem

er’s Universi

ty,,NigeriaNigeria

Redeem

er’s Universi

ty,,NigeriaNigeria

Redeem

er’s Universi

ty,,NigeriaNigeria

Redeem

er’s Universi

ty,,NigeriaNigeria

Redeem

er’s Universi

ty,,NigeriaNigeria

ASSIGNMENTASSIGNMENT

SPEND TIME ON ICTV:CRITERIA FOR VIRUS CLASSIFICATION

MORPHOLOGYSEROLOGYNUCLEIC ACID DISEASE SYNDROME

Redeem

er’s Universi

ty,,NigeriaNigeria

AcknowledgementAcknowledgement

Information contained in this slide were obtained from various sources, including CELLS ALIVE, WIKIPEDIA, IMMUNOLOGY TUTORIALS, PERSONAL LECTURE NOTES AND VIROLOGY METHODS MANUAL (Mahy & Kangro)

Redeem

er’s Universi

ty,,NigeriaNigeria

AcknowledgementAcknowledgement

Information contained in this presentation was obtained from various sources, including:CELLS ALIVE, WIKIPEDIA, IMMUNOLOGY TUTORIALS, PERSONAL LECTURE NOTES AND VIROLOGY METHODS MANUAL (Mahy & Kangro)