V5

Question 1

How can we visualise viruses?

Answer 1

Viruses are tiny (100nm), so electron microscope required to aid in visualisation

Question 2

Explain how electron microscopes are used to visualise viruses

Answer 2

• Transmission EM: negative staining
• Fine resolution to visualise virus morphology
• Able to identify down to family and genus but not species level
• Rapid but requires experienced operator and expensive equipment
• “catch-all” but relatively insensitive

Question 3

Explain viral isolation

Answer 3

Viruses are obligate intracellular parasites; they do not grow on inanimate media such as agar, so lab animals or embryonate eggs used for cell culture

Question 4

What is the principle of viral serology?

Answer 4

Detect the presence of virus-specific antibodies (not the virus itself or parts of the virus).

Question 5

What are the various methods of viral serology available?

Answer 5

• Enzyme immunoassay (EIA, ELISA).
• Latex agglutination.
• Western blot (in lab only)
• Haemagglutination (in lab only)

Question 6

List the steps in ELISA

Answer 6

Well coated with antigen → Serum added to well → wash→ Only specific antibodies remain bound to antigen→ Anti-human antibody conjugated with enzyme detects the presence of bound human antibody →wash →Substrate added: colour change measured

Question 7

What does a colour change indicate in ELISA?

Answer 7

Purple colour change = positive test

Question 8

What is the diagnostic role of antibody testing?

Answer 8

• to diagnose an acute or recent infection
• to determine immune status
• To diagnose persistent, latent or chronic infection
  (IgM antibodies = early phase of infection (acute), IgG antibodies = late phase of infection (chronic))

Question 9

What are the methods for Viral Detection?

Answer 9

Detection of viral proteins, Detection of viral genome

Question 10

What is the main method of detection of a viral antigens?

Answer 10

immunofluorescence

Question 11

Explain what occurs during immunofluorescence?

Answer 11

• Uses specific antibodies directed against viral antigens
• Antigen-antibody reaction made visible by marking antibodies directly or indirectly
• directly: primary antibody as detector and reporter, stained with fluorescein
• indirectly: two antibodies, use second antibody to detect and signal

Question 12

What is the main method of viral genome detection?

Answer 12

PCR

Question 13

List the steps in PCR

Answer 13

1. Start with double-stranded DNA
2. RNA virus: first do reverse transcription (RT-PCR)
3. Denature double-stranded DNA into two single strands by heating to 95 –98 °C
4. Cool reaction to allow specific primers (part of reaction mix) to bind to each single strand
5. Warm to 72°C = optimum working temperature for polymerase enzyme so that it extends primers
6. Repeat cycle up to 30 or 40 times results in exponential amplification of target sequence