Immunofluorescence and Viral Disease Diagnosis

Question 1

What is a method for efficient and definitive detection of viral Ags in virus-infected cells?

Answer 1

Immunofluorescence (FA) staining in virology

Question 2

3 applications of FA

Answer 2

• Transport medium sediment from samples collected on swabs
• Cells from infected cell culture monolayers
• Shell vial monolayers

Question 3

Procedure of immunofluorescence staining

Answer 3

1. Cells are fixed to a slide
2. Monoclonal Abs applied in direct or indirect staining protocols
3. One of the Ab preparation is labeled w/ a fluorescence dye
4. Results are red w/ a fluorescence microscope

Question 4

FA stain: fluorescein isothiocyanate (FITC) is used most

• What color does it fluoresce?
• What does it stain?

Answer 4

• Fluoresces bright green

- Stains the target

Question 5

FA stain: Evans blue is used as a counterstain

• What color does it fluoresce?
• What does it stain?

Answer 5

• Fluoresces red

- Stains the background

Question 6

Steps in direct FA staining?

Answer 6

1. Fluorescein-labeled Abs are added to a microscope slide w/ smear of virus-infected cells
2. Incubate and rinse
3. If Ag/Ab binding, then green fluorescence
4. If no binding, no fluorescence

Question 7

Steps in indirect FA staining?

Answer 7

1. Abs added to microscopic slide w/ smear of virus-infected cells
2. Incubate and rinse
3. Stage 1: there will either by Ag/Ab binding or not
4. Stage 2: fluorescein-labeled anti-species globulin added
5. Incubate and rise
6. If Ag/Ab binding, then fluorescence

Question 8

In FA testing, what are smears evaluated for?

Answer 8

Intensity and distribution of fluorescence

Question 9

How do we stain cells from a cell culture monolayer?

Answer 9

• Use pipette/scraper to scrape cells off tube wall
• Spin to sediment the cells
• Make smear of sediment
• Stain, read

Question 10

How do we stain cells from cells collected on swabs submitted in transport medium?

Answer 10

• Use sediment found in transport medium after centrifugation
• Make a smear on a microscope slide
• Stain and read (at least 20-25 cells must be present to be valid test)

Question 11

Enzyme-linked virus inducible system (ELVIS) is specific for what virus?

Answer 11

Herpes simplex virus (HSV)

Question 12

How does ELVIS work?

Answer 12

• E. coli LacZ gene is cloned into cells behind an HSV promoter
• A substrate is added to infected cells. If beta galactosidase enzyme is present (only produced by HSV infected cells), it acts on the substrate to produce a color change (DARKER BLUE)

Question 13

Advantages to ELVIS

Answer 13

• Very little skill required
• Expensive monoclonal Abs not needed
• Sensitive and specific compared to cell culture

Question 14

Disadvantage to ELVIS

Answer 14

Virus-specific

Question 15

2 ways antivirals work

Answer 15

• Nucleoside analogs (allows virus to add it in and then destroy it)
• Enzyme inhibitors

Question 16

Are antivirals “virus specific” or “broad spectrum”?

Answer 16

Virus-specific

Question 17

What is CMV’s antiviral?

Answer 17

Gangciclovir

Question 18

What is HSV’s antiviral?

Answer 18

Acyclovir

Question 19

What is HIV-1’s antiviral?

Answer 19

Didanosine

Question 20

What is RSV’s antiviral?

Answer 20

Ribavirin

Question 21

What test involves using components labeled w/ active enzymes?

Answer 21

Enzyme immunoassay EIA Ag detection

Question 22

What is EIA used for?

Answer 22

To detect unknown Ags or Abs (widely used for testing fecal samples for rotavirus Ag)

Question 23

Strains of influenza A vary in regards to what?

Answer 23

Hemagglutinin (H) and neuraminidiase (N)

Question 24

Herpesviridae family includes…

Answer 24

• Herpes simplex virus, types 1 and 2
• Cytomegalovirus (CMV)
• Varicella-Zoster virus
• Epstein Barr virus

Question 25

Herpesviridae family includes ____ viruses associated w/ various syndromes. All establish ____

Answer 25

DNA; latency

Question 26

Usually causes oral sores

Answer 26

HSV type 1

Question 27

Usually causes genital sores

Answer 27

HSV type 2

Question 28

Most common STD

Answer 28

HSV types 1 and 2

Question 29

Produces vesicular lesions, stress reoccurrence is common, congenital infections are the most severe manifestation

Answer 29

HSV, types 1 and 2

Question 30

HSV, types 1 and 2 | - Virus isolation

Answer 30

Grows in 24-48 hours in several cell lines

Question 31

HSV, types 1 and 2 | - Ag detection

Answer 31

Sensitivity good in moist lesions, otherwise not very sensitive (as low as 50%) compared to cell culture

Question 32

HSV, types 1 and 2 | - Serodiagnosis

Answer 32

- Not useful in diagnosis, cross reactivity b/w HSV-1 and HSV-2 - Used to determine HSV "immune status"

Question 33

HSV, types 1 and 2 | - Molecular methods

Answer 33

- Excellent | - Method of choice, especially for CSF

Question 34

HSV, types 1 and 2 | - Treatment

Answer 34

- Acyclovir, Foscarnet, others | - Very effective, few side effects

Question 35

- Asymptomatic/mild infections in most healthy (immunocompetent) individuals - Can cause a mononucleosis-like syndrome (resembles classic infectious mono clinically) in healthy people - Recurrence during stress is common - Congenital infections are possible, especially w/ new infections during pregnancy - Severe infections in immunocompromised individuals

Answer 35

Cytomegalovirus (CMV)

Question 36

Cytomegalovirus (CMV) - Virus isolation -

Answer 36

- 3 weeks or longer in traditional cell cultures | - faster results in shell vials, sensitivity varies w/ type of specimen

Question 37

Cytomegalovirus (CMV) | - Ag detection

Answer 37

- Generally NOT useful

Question 38

Cytomegalovirus (CMV) | - Serodiagnosis

Answer 38

Useful in diagnoses (seroconversion or IgM-specific detection) and for immune status testing

Question 39

Cytomegalovirus (CMV) | - Molecular diagnositcs

Answer 39

Excellent, especially for quantitative determinations

Question 40

Cytomegalovirus (CMV) | - Treatment

Answer 40

Ganciclovir

Question 41

- Agent of chickenpox, shingles

Answer 41

Varicella-Zoster virus (ZVZ)

Question 42

Varicella-Zoster virus (ZVZ) | - Virus isolation

Answer 42

6-8 days in standard cell cultures

Question 43

Varicella-Zoster virus (ZVZ) | - Ag detection

Answer 43

Highly sensitive in smears from moist lesions

Question 44

Varicella-Zoster virus (ZVZ) | - Serodiagnosis

Answer 44

Not useful in diagnosis, "immune status" only

Question 45

Varicella-Zoster virus (ZVZ) | - Molecular methods

Answer 45

- Very sensitive and accurate | - Not widely available b/c other less expensive methods are used → Ag detection

Question 46

Varicella-Zoster virus (ZVZ) | - Treatment

Answer 46

- Sometimes not treated | - High dose of acyclovir for serious cases

Question 47

Agent of infectious mononucleosis

Answer 47

Epstein Barr virus

Question 48

Epstein Barr Virus | - Ag detection

Answer 48

Not available

Question 49

Epstein Barr virus | - Virus isolation

Answer 49

Does not proliferate in standard cell cultures

Question 50

Epstein Barr Virus | - Molecular diagnosis

Answer 50

Other methods are used → Ag detection

Question 51

8 respiratory viruses detected by FA in respiratory viral Ag profile

Answer 51

- Adenovirus - Human metapneumovirus - Influenza A and B - Parainfluenza 1-3 - Respiratory Syncytial Virus (RSV)

Question 52

Appearance of positive enzyme-linked virus inducible system

Answer 52

Pos: Beta galactosidase enzyme is present and acts on the substrate causing a color change

Question 53

Strains of influenza A

Answer 53

H5N1, H7N9, H1N1, vH3N2