Immunofluorescence and Viral Disease Diagnosis Flashcards
What is a method for efficient and definitive detection of viral Ags in virus-infected cells?
Immunofluorescence (FA) staining in virology
3 applications of FA
- Transport medium sediment from samples collected on swabs
- Cells from infected cell culture monolayers
- Shell vial monolayers
Procedure of immunofluorescence staining
- Cells are fixed to a slide
- Monoclonal Abs applied in direct or indirect staining protocols
- One of the Ab preparation is labeled w/ a fluorescence dye
- Results are red w/ a fluorescence microscope
FA stain: fluorescein isothiocyanate (FITC) is used most
- What color does it fluoresce?
- What does it stain?
- Fluoresces bright green
- Stains the target
FA stain: Evans blue is used as a counterstain
- What color does it fluoresce?
- What does it stain?
- Fluoresces red
- Stains the background
Steps in direct FA staining?
- Fluorescein-labeled Abs are added to a microscope slide w/ smear of virus-infected cells
- Incubate and rinse
- If Ag/Ab binding, then green fluorescence
- If no binding, no fluorescence
Steps in indirect FA staining?
- Abs added to microscopic slide w/ smear of virus-infected cells
- Incubate and rinse
- Stage 1: there will either by Ag/Ab binding or not
- Stage 2: fluorescein-labeled anti-species globulin added
- Incubate and rise
- If Ag/Ab binding, then fluorescence
In FA testing, what are smears evaluated for?
Intensity and distribution of fluorescence
How do we stain cells from a cell culture monolayer?
- Use pipette/scraper to scrape cells off tube wall
- Spin to sediment the cells
- Make smear of sediment
- Stain, read
How do we stain cells from cells collected on swabs submitted in transport medium?
- 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)
Enzyme-linked virus inducible system (ELVIS) is specific for what virus?
Herpes simplex virus (HSV)
How does ELVIS work?
- 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)
Advantages to ELVIS
- Very little skill required
- Expensive monoclonal Abs not needed
- Sensitive and specific compared to cell culture
Disadvantage to ELVIS
Virus-specific
2 ways antivirals work
- Nucleoside analogs (allows virus to add it in and then destroy it)
- Enzyme inhibitors
Are antivirals “virus specific” or “broad spectrum”?
Virus-specific
What is CMV’s antiviral?
Gangciclovir
What is HSV’s antiviral?
Acyclovir
What is HIV-1’s antiviral?
Didanosine
What is RSV’s antiviral?
Ribavirin
What test involves using components labeled w/ active enzymes?
Enzyme immunoassay EIA Ag detection
What is EIA used for?
To detect unknown Ags or Abs (widely used for testing fecal samples for rotavirus Ag)
Strains of influenza A vary in regards to what?
Hemagglutinin (H) and neuraminidiase (N)
Herpesviridae family includes…
- Herpes simplex virus, types 1 and 2
- Cytomegalovirus (CMV)
- Varicella-Zoster virus
- Epstein Barr virus
Herpesviridae family includes ____ viruses associated w/ various syndromes. All establish ____
DNA; latency
Usually causes oral sores
HSV type 1
Usually causes genital sores
HSV type 2
Most common STD
HSV types 1 and 2
Produces vesicular lesions, stress reoccurrence is common, congenital infections are the most severe manifestation
HSV, types 1 and 2
HSV, types 1 and 2
- Virus isolation
Grows in 24-48 hours in several cell lines
HSV, types 1 and 2
- Ag detection
Sensitivity good in moist lesions, otherwise not very sensitive (as low as 50%) compared to cell culture
HSV, types 1 and 2
- Serodiagnosis
- Not useful in diagnosis, cross reactivity b/w HSV-1 and HSV-2
- Used to determine HSV “immune status”
HSV, types 1 and 2
- Molecular methods
- Excellent
- Method of choice, especially for CSF
HSV, types 1 and 2
- Treatment
- Acyclovir, Foscarnet, others
- Very effective, few side effects
- 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
Cytomegalovirus (CMV)
Cytomegalovirus (CMV)
- Virus isolation
-
- 3 weeks or longer in traditional cell cultures
- faster results in shell vials, sensitivity varies w/ type of specimen
Cytomegalovirus (CMV)
- Ag detection
- Generally NOT useful
Cytomegalovirus (CMV)
- Serodiagnosis
Useful in diagnoses (seroconversion or IgM-specific detection) and for immune status testing
Cytomegalovirus (CMV)
- Molecular diagnositcs
Excellent, especially for quantitative determinations
Cytomegalovirus (CMV)
- Treatment
Ganciclovir
- Agent of chickenpox, shingles
Varicella-Zoster virus (ZVZ)
Varicella-Zoster virus (ZVZ)
- Virus isolation
6-8 days in standard cell cultures
Varicella-Zoster virus (ZVZ)
- Ag detection
Highly sensitive in smears from moist lesions
Varicella-Zoster virus (ZVZ)
- Serodiagnosis
Not useful in diagnosis, “immune status” only
Varicella-Zoster virus (ZVZ)
- Molecular methods
- Very sensitive and accurate
- Not widely available b/c other less expensive methods are used → Ag detection
Varicella-Zoster virus (ZVZ)
- Treatment
- Sometimes not treated
- High dose of acyclovir for serious cases
Agent of infectious mononucleosis
Epstein Barr virus
Epstein Barr Virus
- Ag detection
Not available
Epstein Barr virus
- Virus isolation
Does not proliferate in standard cell cultures
Epstein Barr Virus
- Molecular diagnosis
Other methods are used → Ag detection
8 respiratory viruses detected by FA in respiratory viral Ag profile
- Adenovirus
- Human metapneumovirus
- Influenza A and B
- Parainfluenza 1-3
- Respiratory Syncytial Virus (RSV)
Appearance of positive enzyme-linked virus inducible system
Pos: Beta galactosidase enzyme is present and acts on the substrate causing a color change
Strains of influenza A
H5N1, H7N9, H1N1, vH3N2
