Lecture 3 - Viral Agents 2 Flashcards
Why obtain lab identification of a virus? 1) 2) 3) 4)
1) Treatment options
2) Public health measures
3) Surveillance
4) Research
Methods of viral identification 1) 2) 3) 4) 5)
1) Direct visualisation (EM)
2) Viral cultivation (gold standard)
3) Viral protein detection
4) Serology
5) Viral nucleic acid detection
How stable are viruses?
Typically very susceptible to environmental, chemical inactivation.
Particularly enveloped viruses
Ways to preserve viral infectivity for cultivation 1) 2) 3) 4) 5)
1) 4 degrees for a day or so
2) -70 for long-term storage
3) -196 (liquid N2) for permanent storage
4) Freeze drying
5) Buffered transfer medium (pH buffered)
Freeze drying preservation
Particularly useful for naked viruses.
Dehydration of a frozen suspension in a vacuum.
Used for some live-viral vaccines.
How are PC2 or PC3 viral samples handled?
Class II biohazard cabinets
Why should freeze-thaw be avoided?
Forms crystals which shear viruses.
Particularly enveloped viruses
What is buffering used for?
Maintain pH, to maintain a metastable viral state
Types of samples that are EM’d
Messy, crudely prepared samples.
EG: ID’ing rotavirus from faecal samples
Ways to cultivate virus
1)
2)
3)
1) Suckling mice
2) Embryonised chicken egg
3) Mammalian cell line
Why were suckling mice used?
Had no significant immune system
Places in an embryonised chicken egg that can be inocculated with virus 1) 2) 3) 4)
1) Chorioallantoic membrane
2) Amnotic
3) Yolk sac
4) Allantoic
Where is influenza best cultivated in a chicken egg?
Allantoic innoculation
Problem with egg innoculation
Not all human viruses can replicate in avian cells
How do viral inocculations of chicken eggs present?
As ‘pocks’, which are collections of chicken leukocytes on membranes.
Three mammalian cell culture types used to culture virus
1) Primary cells
2) Diploid cell culture
3) Transformed cells
Primary cell lines
Tissue culture, often from animals.
Can only divide up to ten times
Diploid cell lines
Up to 100 generations in culture.
Which cell line are most vaccines cultured in?
Diploid cell lines
Transformed cell lines
Aneuploid (abnormal chromosomes)
Often have an activated oncogene
Can divide indefinitely in culture
Not used for human vaccines
Why aren’t transformed cell lines used for human vaccines?
Could transfer an oncogene to human
Example of primary human cell line
Foreskin fibroblast line
Examples of continuous cell lines
HeLa, Vero
How are viral cell cultures prepared?
Cells are inoculated, incubated at 37 degrees for a few days.
Inspected for cytopathic effects
Cytopathic effects 1) 2) 3) 4) 5)
1) Nuclear inclusions
2) Cytoplasmic inclusions
3) Lysis
4) Multinucleate syncitia
5) Transformed cells
Cytopathic effects from polio
Rounding, nuclear pyknosis
Inclusion bodies
Represent areas of viral protein production.
DNA viruses cause nuclear inclusions.
RNA viruses cause cytoplasmic inclusions.
Viruses that can cause intranuclear inclusion bodies
Herpesvirus, adenovirus
Cytopathic effects of measlesvirus
Synsitia, cell inclusions
Viruses that cause multinucleate syncitia
HIV, measlesvirus
How are multinucleate syncitia formed?
Measles fusion protein expressed on cell membrane, which causes cells to stick together
How is a plaque assay performed?
Monolayer of mammalian cells, inoculated with virus.
Sandwich monolayer between two agar gels to immobilise virus.
Observe areas of cell lysis
How can cell transformation occur?
Some viruses encode oncogenes (EG: retroviruses)
Direct detection of viral antigen
Fluorescently tagged anti-antigen antibody binds immobilised viral antigen
Indirect detection of viral antigen
Antibody against viral antigen immobilised. Binds viral antigen. Another antibody (fluorescently tagged) against viral antigen is washed over sample.
Haemadsorption
Some viral antigens cause red blood cells to agglutinate
Name for agglutination of red blood cells
Haemadsorption
Haemagglutination assay
Often used for influenza.
Measures ability of viral particles to agglutinate red blood cells.
Serial dilution of viral sample with constant concentration of red blood cells.
The maximum dilution that results in a shield (rather than a dot) is the haemagglutination titre
Haemagglutination inhibition assay
Constant virus, red blood cell concentration.
Serial dilutions of antibody. See when it inhibits haemagglutination (dot)
What is haemagglutination inhibition used for?
Serotyping
How do viral titres vary between assays?
In descending order of viral particles: Electron microscopy Quantal assay in eggs Plaque assay in mammalian cells Haemagglutination asssay
Electron microscopy influenza viral titres per mL
10^10 virions
Quantal assay in eggs influenza viral titres per mL
10^9 egg ID50
Plaque assay in mammalian cells influenza viral titres per mL
10^8 pfu
Haemagglutination assay influenza viral titres per mL
10^3 HA units
Antigen capture assay
Known antibody affixed to plate. Binds to antigen of interest. Sample washed over antibodies. Second antibody - with a fluorescent tag - which binds to antigen is washed over plate.
Anti-viral antibody assay
Viral antigen affixed to plate. Patient serum washed over plate. If patient has antibodies against antigen, these will bind. Fluorescently-tagged anti human Fc antibody washed over.
Antigen capture assay vs anti-viral antibody assay
Antigen capture has high sensitivity
Anti-viral antibody assay has high specificity
Example of antigen capture assay
HIV p24 assay
Example of anti-viral antibody assay
HIV western blot
Western blotting 1) 2) 3) 4)
1) Run antigen samples on a gel
2) Blot gel onto nitrocellulose gel
3) Immunostaining of blot with a tagged antibody
4) Autoradiography of blot
HIV western blot asssay
1)
2)
3)
1) Detergents used to solubilise cells with potential HIV infection, vs wild-type HIV proteins
2) Western blot performed with anti-HIV antibodies
3) High specificity.
Detection of viral antigen vs detection of host antibody assays
Detection of viral proteins has a high sensitivity, therefore false positives
Detection of host antibodies has a high specificity, therefore false negatives
Problems with infectivity or serology assays
Generally slow. Too slow for clinical use
Viral nucleic acid detection assays 1) 2) 3) 4) 5) 6)
1) Southern blot for viral DNA
2) Northern blot for viral RNA
3) PCR for DNA
4) Reverse transcriptase PCR for RNA
5) Viral nucleic acid sequencing
6) DNA microarray
PCR assay pros and cons
1) Highly sensitive, specific
2) Fast
3) Cheap
4) Prone to contamination
5) Low amount of sample needed
Difference in PCR technique for DNA vs RNA virus
For DNA virus, normal PCR
For RNA virus, need a viral reverse transcriptase enzyme
Stages of PCR
1) Denaturation (DNA splits)
2) Annealing (primers bind)
3) Extension (DNA polymerase)
How was it initially known that SARS was novel
No other human coronavirus could grow in Vero cells
