methods++used+in+virology(c2)

Laboratory of Virology MABI: Plant Virology LecturesWAGENINGEN UR

• In virus research-diagnosis of virus diseases

• Achieve knowledge and understanding of viruses

• Focus on methods that are unique to virology

CANTHO UNIVERSITYBiRDI

Chapter 2: Methods used in virology



Chapter 2: Methods used in virology

1. Cultivation of viruses

2. Isolation of viruses

3. Centrifugation

4. Structural investigations of cells and virions

5. Electrophoretic techniques

6. Detection of viruses and virus components

7. Infectivity assays

8. Virus genetic


1. Outline methods for:– cultivation of viruses– purification of viruses– detection of viruses and their components– assay of virus infectivity– investigation of virus genome function

2. Assess the value of virus genome sequencing


Learning outcomes



Cultivation of viruses

• supply appropriate cells in which viruses can replicate: bacterialcultures, cultivated plants, protoplasts, mice, eggs, insect larvae,…..

1. Animal cell culture:

– Continuous cell lines: immortalized - subcultured indefinitely

– Medium: nutrients (animal serum) – maintenance optimum osmotic pressure and pH

– Growing on the surface of plastic vessels (contain medium withantibiotics [contamination] – specific incubator [CO2])



Isolation of viruses

* Plaques:– discrete visible zones in layers of host cells

– Infection of a cell by a single virion (plaque=clone=generally identical= isolate/strain)

– the first isolated Virus may replicate poorly / moreefficiently in numbers of cycles

– Subcultured = passaged

– After numbers of passages : virus may be geneticallydifferent to the original wild strain = lab strain


•

•

1. Differential centrifugation:Alternating cycles of low- speed centrifugation: virus in supernatant

High –speed : virus in the pellet

•

2. Density gradient centrifugationIn a solution of increasing concentration (density)

• Solutes: high solubility – sucrose

• Rate zonal centrifugation: # virus suspension on top of pre-formed gradient

• Equilibrium centrifugation: # impure virus in solution of the gradient material (density at the bottom > density of particles)

• Result: homogenous particles= sharp band (moving through gradient)


Centrifugation

• Applied in virus purification:



Structural investigations of cells and virions

1. Light microscopy– Detect virus-infected cells (cytopathic

effects/fluorescent dye link to antibody)– Confocal microscopy: pin-hole to exclude light from out-

of- focus region of specimen/used with live cells/investigate protein trafficking……….

2. X-ray crystallography– Detail information of three-dimensional structures of

virions– DNA – Protein – DNA / Protein complexes

– Require “Crystal of virions or molecules”– Crystal is diffracted = placed in a Beam of X-ray– Positions of molecules/atoms # diffraction pattern

3. Nuclear magnetic resonance: electromagnetic technology to discover essential, chemical and structural information about the molecule.

4. Atomic force microscopy



Structural investigations of cells and virions (cont.)

5. Electron microscopy• Large magnification (TEM)

• Specimen: treated (suspension of virions/ultrathin section of virus-infected cell)

• Negative staining: = heavy-metal-containing compound = dark areas around the virions (structural distortion from drying)

• Cryo-electron: wet specimen - rapidly cooled to -160C / reconstructmultiple images into three-dimensional images of virus particles


• Separate mixtures of proteins or nucleic acids

• Gel of Agarose or polyacrylamide: band

• Rate of movement >< molecular weight (measure by ladder)

• Patterns of separated molecules immobilized = blotting on a membrane [Southern (DNA) – Northern (RNA) – Western (Protein) Blot]


Electrophoretic techniques



Detection of virus and virus components

1. Detection of virions: negatively stained of specimen (high costs + limited sensitivity)

2. Detecting of infectivity using cell cultures:inoculated into a culture of cells –host > examine cell changes by

light microscopy

3. Detection of virus antigens: using virus-specific antisera / monoclonal antibodies – detect the label (enzyme / Fluorescent / Gold / Radio-active) in direct or indirect test

4. Detection of virus nucleic acids:• Hybridization: using sequence-specific DNA probes carrying

labels• PCR



Infectivity assays* Measure titre of infective virus – samples are inoculated into

suitable hosts – measure of cell response

1. Quantitative assays• Number of plaques # “lysed cells” (pfu/ml)

2. Quantal assays: detect Cytopathic effects (CPE) = TCID50 (50% Tissue

Culture Infective Dose)- (ID50/LD50)>>>cell rounding/disorientation/swelling/shrinking/death/detachment from the surface, etc. / discrete lessions =pock,…

1. One-step growth curve: burst size = final virus titre / initial virus titre• Eclipse period = viruses are replicating• m.o.i = ratio of viruses / cells (~ 5-10)• Adsorption time: ~ 2 min > stop by diluting or + antivirus

antiserum• Experiment time: Phage ~ 1 h; animal viruses ~ hours or days• Burst size >< physiological stage of cells



Virus genetics

1. Genome sequencing2. Genome manipulation

• RE analysis• Cloning• Introduce site-specific mutations into virus genome

3. Investigation of gene function and expression• block gene expression• creation of mutated genes• Reverse genetics (RNA genome)• RNAi• Introduce gene into cells and monitor transiently

expression and changes in the cells• DNA microarrays: glass slide attached to a lot of DNA

spots (=DNA probe): monitor expression of DNA, RNA genes; host cell genes•


1. Outline methods for:– cultivation of viruses– purification of viruses– detection of viruses and their components– assay of virus infectivity– investigation of virus genome function

2. Assess the value of virus genome sequencing


Learning outcomes

methods++used+in+virology(c2)

Documents

virus suspension

impure virus

virus purification

virus diseases

isolated virus

virusesdetection of

speed centrifugation

differential centrifugation