Lecture 2: Culture and Detection of Viruses

Question 1

How do you detect if someone has a virus or not?

Answer 1

1. Signs and symptoms: rash etc History
2. Immunofluorescence (fluorescent labelled antibody)- create a dye which targets a specific antibody so it will show up. ELISA
3. Nucleic Acid analysis- Polymerase chain reaction- have primers which bind to unknown DNA, and amplify this DNA, then its compared to known sequences
   DNA probe- identify specific DNA
4. Reverse Transcriptase PCR- uses RNA to creat cNDA and amplifies this
   -Used to semi quantify RNA in a solution
5. Branched DNA testing- A chemical known to bind to viral RNA is added, the brighter the test glows the more RNA present
6. Nucleic Acid sequenced Based amplification–> Viral RNA used to produce DNA which can be used to determine viral load.
7. Real time PCR- uses fluorescent probes to quantify specific DNA
   -does not give numbers but semi quantitative

Question 2

Why do we do virus cultures and how?

Answer 2

Viral culture is a laboratory test in which samples are placed with a cell type that the virus being tested for is able to infect. If the cells show changes, known as cytopathic effects, then the culture is positive.

Why? To diagnose an infection, for research and preparation of vaccines
How? Obligate intracellular parasite
-host ells

Question 3

what are the 3 lab methods of cultivation of viruses?

Answer 3

1. In cell culture
   - primary cell culture
   - secondary cell culture (cell strains)
   - continous cell culture (cell lines)
2. In embryonated eggs
3. In lab animals -

Question 4

Discuss how a primary cell culture works?

Answer 4

1. Embryonic tissue is removed from embryo using sterile technique
2. Tissue must be broken down to individual cells physically (chopped) and chemically
3. Cells are then placed into appropriate liquid medium

Embryos are removed and target organ etc removed eg liver. Hepatic tissue is then finely chopped removing undesirable tissue eg connective tissue. Tissue puree is then placed into chemical solution to further breakdown to individual cells. Cells are placed into appropriate nutrient media and incubated in warm, humid incubator with appropriate CO2/O2 supply. Culture has a limited life span (fungi/bacteria/other cells fibroblasts etc). Media needs frequent changing. Why??

Question 5

Describe how secondary cell cultures work?

Answer 5

Cells derived by re-trypsinisation and re-culture in fresh medium of successfully grown primary cells. It is generally possible to subculture or passage primary cells a few times at approximately weekly intervals but they normally have a finite life span and eventually `age’ and die. They also have a diploid chromosome number, the same as the parent cells from which they were derived.

Question 6

Immortal cell lines

Answer 6

Usually originate from tumor and are therefore cancer cells. Popular immortal cell lines include CHO cells, HeLa cells, Vero cells. These can be used as host cells or used as basis for research. However, they are very different from the non-tumor originals. Easier to grow and maintain than primary cells but limited in information gained.

Question 7

Whats something unique about immortal virus cultures?

Answer 7

• from tumor cells

- continuously turns over

Question 8

Describe the passage of cultured cells

Answer 8

Once the primary cells have divided so much that they cover the cell culture flask, they are said to have reached “confluency”: they are “confluent”.

The next step is to “passage” the cells.

1. Sterilely remove the medium.
2. Rinse the cells in sterile isotonic salt solution (e.g. PBS).
3. Trypsinisethe cells (with trypsin-EDTA).
4. Resuspend the cells in fresh sterile medium.
5. Estimate the cell concentration and % viable cells (with trypan blue).
6. Seed new cell culture flasks and incubate.

As soon as primary cells have been passaged, they become known as “secondary cells” or “cell strains”.
There are several videos of cell culture basic techniques available on the web:
e.g. Passaging cells: GIBCO® Cell Culture Basics http://www.youtube.com/watch?v=gaG15lM1t5A

Question 9

Cell lines
The process of senescence?
Immortalized?

Answer 9

Senescence: Normally, cultured cells (primary or secondary cells) can undergo a limited number of divisions (20-100) before they die.

Cultured cells trains may become transformed, such that they divide without limit- i.e. become immortalised. This happens spontaneously, but more commonly is induced by chemical, genetic or viral mutagens.
Note: Primary cells derived from cancers are often readily established continuous cell lines in culture (they are already transformed cells)

Question 10

Cell cultures
CHO cells
Vero cells

Answer 10

CHO cells:

• growing masses of human insulin receptors
• incorporate any segment of DNA from any organism into any other organism

Vero cells:
-isolated from kidney or green monkey

Question 11

Vaccine culture
-what cells can be used? moral complications or anything against them?
eggs

Answer 11

Chicken eggs:

• 11 days post fertilisation the virus strain is injected
• virus infects embryo and multiplys
• days later virus machine harvested from broken eggs (1-2 eggs per vaccine dose)
• problem- need SO MANY eggs and it takes a long time for this whole process to occur, not very useful in an epidemic
• inexpensive
• well established
• average time egg to injection -6 months - many deaths before there is enough vaccines available/

Question 12

Vaccine culture
Immortal cell lines
Advantages?

Answer 12

• scaled up quickly
• no or fewer impurities
• no albumin in case of allergy
• millions of cells stored in small containers…comparred to millions of eggs
• its an established method for polio vaccine