Identification techniques virology

Question 1

Two main strategies?

Answer 1

1. Detect virus itself

2. Detect indirect / immunological evidence of infection

Question 2

Electron microscopy

Answer 2

Sensitivity : need 10 virus particles per ml for reliable detection
Prepare sample by negative staining
Thus electron micrographs ‘interpreted’ in the same way as a negative of a photograph - dark areas represent a structure on the virus ( eg cell surface protein) which the electrons could not pass through

Question 3

Culture

Answer 3

Infect known host and look for symptoms
e.g. Bacteriophages – grow on bacterial lawns
Most commonly used in the laboratory are bacteriophages of Escherichia coli
Infect known host and look for symptoms
e.g. Phages – grow on bacterial lawns
e.g. plants and animals will show classic symptoms of infection
Infect cells in ‘artificial’ culture system in the laboratory
Plant cells
Animal cells
e.g. Herpes virus in human embryonic fibroblasts

Question 4

Immunofluorescence

Answer 4

Immunofluorescence ( IF) or Immunofluorescent antibody test IFAT)
Detects virus antigen expressed on the surface of infected cells
E.g respiratory epithelial cells
Prepare smear from respiratory sample on microscope slide
(RSV)

Question 5

Enzyme immunoassay (EIA)

Answer 5

Detection of viral antigen in samples using:
plate enzyme immunoassay (EIA)
E.g. faecal sample for norovirus
Automated test in analyser based on EIA
E.g. blood sample for Hepatitis B surface antigen
Point of care/ near patient test based on EIA
E.g respiratory sample for lnfluenza A

Question 6

Molecular methods

Answer 6

1. Amplification to detect virus  
Polymerase chain reaction
Ligase chain reaction
Nucleic acid sequence based amplification 
2. Sequencing of virus  (usually after amplification)
Identification
Typing 
Epidemiology
Drug resistance 
Quantitative measurements

Question 7

Detection of immunological evidence of infection

Answer 7

Detection of antibody by e.g immunoassay; agglutination
Based on antigen and antigen binding reaction
Detects antibody produced as a result of infection

Question 8

Indirect ELISA – antibody detection

Answer 8

Thus the reaction is made up of two steps:
Antigen-antibody-antigen
Called ‘Sandwich’ or ‘indirect’ ELISA
The signal is enhanced by the extra step and it improves the quality of the result
A strong colour reaction indicates presence of antibody in patient’s serum
E.g. test to detect antibody to Rubella virus

Question 9

Competitive ELISA

Answer 9

In this case, the more of the patient’s antibody in sample, the weaker the signal from enzyme substrate
This is called a ‘competitive’ ELISA
So little or no colour indicates presence of antibody in patient’ serum
E.g. Hepatitis A antibody test

Question 10

Outline agglutination

Answer 10

Latex bead coated with antigen
Mix with patient’s serum and any antibody present will bind with antigen
Antibody binds to antigen on more than one bead; thus cross linking
Beads clump together (agglutination), so can see clumps by eye