Viral Cultivation/Detection

Question 1

How are viruses cultivated in the laboratory?

Answer 1

Viruses need a host system and they can be grown in:

1) Animals
2) Tissue (cell) cultures (preferred method)
3) Embryonated Eggs

Question 2

Why are animals used for viral research? What are the drawbacks?

Answer 2

1) Understanding pathogenesis (how it causes disease)
2) Understanding host immune response
3) Vaccine Development
4) Antiviral drug development Drawbacks:
* Ethical issues, genetics of different species, expensive

Question 3

Who developed a new way to culture/study viruses in the 1940’s?

Answer 3

Enders, Wellers, Robbins developed cell (tissue) culture technique

Question 4

Who are Dulbecco and Eagle?

Answer 4

Developed Dulbeco’s Modified Eagles Media (DMEM) used to culture animal cells

Question 5

What were the two immediate effects of cell culture development?

Answer 5

1) Development of the polio vaccine
* the first vaccine produced in cell culture (monkey kidney cell lines)
2) Advent of molecular virology:
* the exploitation of cell culture for the cultivation and study of viruses

Question 6

What key discoveries in molecular biology are the result of studying viruses?

Answer 6

1) Discovery of enhancers
2) Discovery of transcription factors
3) RNA editing processes
4) Cancer Biology

Question 7

What are cell cultures used for and how do we establish them?

Answer 7

Cell cultures used to propagate viruses

• Different Cell types are used to grow different viruses
• Grown in nutrient rich media (salt, AA, glucose, serum)
• Antibiotics added to prevent bacterial growth

Question 8

What are the 3 main types of cell culture?

Answer 8

1) Primary cell cultures

• prepared from animal tissues
• have a limited lifespan

2) Continuous cell lines

• single cell type can be propagated indefinitely
• commonly derived from tumor tissue
• HeLa cells from cervical tumor of H. Lacks

3) Suspension cell cultures

• do not need to attach to a substrate to divide
• large number of cells can grow in small volume

Question 9

What methods do we use to recognize that a viral infection has occurred in cell culture?

Answer 9

1. Cytopathic effects (CPE)
   * How cells change after infection
2. Hemadsorption
   * Adsorb (bind) erythrocytes

Question 10

What are Cytopathic effects?

Answer 10

Cytopathic effects (CPEs) are how the infected cell changes after infection -can be observed with inverted light microscopes

1) Rounding/detachment from plastic flask
2) Syncytia/fusion (grouping)
3) Shrinkage
4) Inclusion bodies
5) Cell lysis/death

Question 11

What are the EHV-1 Plaques we discussed in class?

Answer 11

Equine Herpes Virus-1 cultured cause plaques to form where EHV-1 destroyed the cells it infected Larger the plaque = the more virulent the strain of virus

Question 12

What are quantitative assays and what are the types we can use?

Answer 12

Used to figure out the numer of infectious particles (virions) present in sample

Types:

1. Plaque Assays
2. Hemagglutination Assays

Question 13

How is plaque assay accomplished and read?

Answer 13

Sample is diluted at different orders of magnitude and then cultured to see the numbers of plaques that arise

Plaque Forming Unit (PFU): a single virus particle that gives rise to a plaque

Question 14

Do all viruses form plaques?

Answer 14

Not all viruses form plaques, but you can do a transformation/focus assay

• Cells form piles called foci
• can count the foci present to see the number of virions present

Question 15

What are the general approaches for laboratory diagnosis of viral infections?

Answer 15

1. Direct detection
2. Virus isolation and growth in cell culture
3. Serology

Question 16

What can be used for Direct Detection of Virus?

Answer 16

1. Immuno-electron microscopy
2. Immunoflourescence

Usually not done in clinical lab

-too expensive

Question 17

What are some other methods available for virus detection besides Direct Detection?

Answer 17

Nucleic Acid Methods (Rapid Identification)

• ​PCR (DNA)
• RT-PCR (RNA)
  
  • RNA→DNA→PCR

Used to detect viruses that are not cultivatable

Can be used to manage patients w/ HIV

• Monitor T_H count + Viral Load

Question 18

What viral serology options are available to diagnose viral infections?

Answer 18

Viral Serology detects specific antibodies generated against virus

Different tests:

1. Enzyme Linked Immunosorbent Assay (ELISA)
2. Western Blot Assay

Question 19

How does ELISA work?

Answer 19

1. Coat the wells
   
   • With antigen when testing antibodies in patient’s serum
   • With Antibodies when testing viral antigen in patient’s serum
2. Incubate and wash unbound Antibodies/Antigens
3. Add Secondary Antibodies that contain enzyme indicator
4. Wash and Add substrate
5. If substrate binds secondary antibody then color change occurs and positive result

Question 20

What are some positive controls that can be used when doing an ELISA test for HIV?

Answer 20

1. Add known HIV antibody/antigen to test well(s) to ensure binding occurs
2. Add known non-HIV antibody/antigen to test well(s) to ensure binding doesn’t occur

Question 21

How does Western Blot Assay work?

Answer 21

1. Separation Gel (SDS-PAGE) to separate Viral Proteins
   
   • Big at the top and Small at the bottom
2. Proteins transferred to Blot in Blotting tank
3. Make a copy of gel proteins on membrane and immuno-stain the blot (add antibodies)
4. Autoradiography and develop bands
5. The dark bands show you which proteins present in the sample have antibodies present for them as well