Methods used in Virology

Detection and Assay

• Assess the presence or absence of virus in
given sample or specimen
• If present- is it biologically active??
• What is the concentration (titre)

• Imp in diagnosis, vaccine etc.

Detection and assay of viruses and virus
components
• Based on virus infectivity
• Based on viral components
• Based on serology
• Based on nucleic acid
 Assays based on Infectivity
• A) Plaque Assay-
• Quantitative
• Animal virus, phages
• Monolayer of host cells> viral infection> lysis>
plaque
• PFU (a viral particle capable of initiating a
productive infection)
• Neutral red, crystal violet- better visualization
 B) Pock assay
• CAM
• Fertilized eggs> 2 week incubation> opened>
virus suspension added> sealed> incubated>
assayed

• Quantitative

• combursome
Pock formation on CAM
 C) Focus assay
• Some viruses transform host cells
• eg. Rous Sarcoma Virus
• No contact inhibition> grow as piles or foci
• Normal cells> monolayer

• Foci more dense stained

• Quantitative assay- FFU/ml

 D) Dilution End point Assay
• Quantal assay: statistical analysis
• Known number of subjects (animals, cell
culture, wells)- infected with increasing
dilutions of virus and scored (illness, death,
cytopathogenecity)
• Plot log dilution vs percentage of infected
subjects
• ID 50 (median infectious dose)
• LD 50 (median lethal dose)
• TCID50 (median tissue culture infectious dose)
 Reed-Muench method for estimating TCID50

• Dilution of virus that contains one TCID50 lies between

10−3 and 10−4
• End point can be expressed as 10−(3+x), where x is the
value to be estimated

• x = log10 dilution factor (% infection at next dilution above

50% − 50)/(% infection at next dilution above 50% − %
infection at next dilution below 50%)
= 1(69-50)/69-31
= 0.5
• End point = 10−(3+0.5) = 10−3.5
• i.e. 1 ml of a 10−3.5 dilution contains one TCID50 of virus
 Assays based on viral components
• Usually no. of viral particles ≠ infectious units

• Electron Microscopy
• Hemagglutination
 Electron Microscopy
• Direct observation
• Latex droplet method (Backus & Williams,
1950)
• Known vol of sample + known vol of
polystyrene latex droplets-> coat viral
particles> better visualization
• Free latex droplets- empty large particles
• Virus coated with latex- filled particles
TEM of Rotavirus
 Hemagglutination Assay
• Many viruses contain proteins that can bind RBC
> form lattice
• Influenza virus hemagglutinins (HA)- binds RBC N-
acetyl neuraminic acid glycoproteins
• Attaches two RBC and bridges them
• Pattern method: Normal RBC – undisturbed- fall
to the bottom of a culture well, forming a sharp
dot
• RBC + Virus- lattice (network)- coats well
• Assay is fast (30 minutes)
 Assay Based on Serology

1. Virus Neutralization
2. Hemaglutinization Inhibition
3. Complement Fixation
4. Immunostaining
5. Immunoprecipitation/Immunoblotting
6. ELISA
 Virus Neutralization
• Degree of infectivity/to detect new serotypes
• Infectivity neutralized- specific antibodies
• Dilutions of serum/monoclonal antibody
+ virus- Incubate- infectivity assayed
• Cultured cells/ embryonated eggs/animals
• End point- highest dilution inhibiting
cytopathic effect by atleast 50%
Virus neutralization assay
 Hemagglutination Inhibition
• Dilutions of serum + virus – incubated- RBC
added- incubation
• HI titre- highest dilution of serum- inhibits
hemaglutinization

• Sensitive, rapid, inexpensive

• Used to detect antibodies to viral
hemaglutinin in animal and human sera
Hemagglutination inhibition
 Complement Fixation
• Detect presence of either specific antibody
or specific antigen in a patient's serum
• Complement system: system of serum
proteins that react with antigen-antibody
complexes
Complement Fixation

Patient's serum: antibodies

against the antigen of interest,
bind to the antigen- Antigen-
antibody complexes
Complement proteins will react
with these complexes and be
depleted. Thus when the sRBC-
antibody complexes are added
in step 4, there will be no
complement left in the serum

If no antibodies against antigen

of interest are present,
complement will not be
depleted and it will react with
the sRBC-antibody complexes
added in step 4, lysing the
sRBCs and spilling their
contents into solution, thereby
turning solution pink
 Immunostaining
• Detect viral Ag
• Ab- labelled with fluorescent dyes
(fluorescein, rhodamine) or enzymes
(alkaline phosphatase; B-galactosidase)
• Methods:
– Direct immunostaining: antibody directly
coupled to indicator
– Indirect Immunostaining: second
antibody coupled to indicator; more
sensitive
 Immunofluorescense

HSV-infected epithelial cells from Positive immunofluorescence test

skin lesion. (Source: Virology for rabies virus antigen. (Source:
Laboratory, Yale-New Haven CDC)
Hospital)
 Immunoprecipitation
• technique of precipitating a protein
antigen out of solution using antibody that
specifically binds to that particular protein
• Two methods:
• Direct Capture: Specific antibodies for a
particular protein, or a group of proteins- added
directly to mixture of protein
• Beads coated with antibodies added
• Antibodies bound to their targets- stick to beads
 Immunoprecipitation
Indirect Capture:
• Antibodies immobilized on a solid-phase
substrate > such as super- paramagnetic
microbeads or on microscopic agarose beads
• Beads with bound antibodies - added to
protein mixture
• Proteins targeted by antibodies - captured
onto beads via antibodies
 Immunoblotting (Western Blot)
• Depends on reaction of antibody with
viral protein
• Proteins fractionated by electrophoresis
• Transferred to nitrocellulose - strong
affinity for proteins
• Incubate with labelled antibodies
• Detected by immunostaining
Immunoblotting
 Enzyme-linked Immunosorbent Assay
• Described by Van Weeman and Schuurs; and Engvall
and Perlman (1971)
• Combines specificity of enzymatic and immunological
reactions
• Advantages:
• Highly sensitive & specific
• Requires small amount of reagents
• Rapid
• Qualitative as well as quantitative
• Easy visual detection
• Large samples can be screened at a time
 Enzyme-linked Immunosorbent Assay

• Suitable enzyme- label

• Enzyme substrate > colourless
• product > coloured > easily detected
• Both Ab & Ag can be enzyme labelled
ELISA
 ELISA for HIV antibody

Microplate ELISA for HIV antibody: colored wells indicate reactivity

Detection of virus nucleic acids
• Hybridization methods- detect genomic
DNA, RNA, mRNA
• Using labelled probe > southern (DNA)
and northern (RNA)

• PCR based methods

Detection of virus nucleic acids
• Southern Hybridization:
• DNA digested with restriction endonucleases
• Fractionated by gel electrophoresis
• Transferred to membrane- bind strongly
• Detected by hybridization with isotopically
labelled viral nucleic acid sequence

• Northern blot hybridization: detect RNA

Hybridization
Techniques
 The
Polymerase
Chain Reaction
(PCR)