This document discusses several methods for laboratory diagnosis of viral infections, including virus culture and isolation techniques like plaque assay and TCID50 assay, detection of viral antigens and antibodies, and detection of viral genomes through techniques like PCR. It provides an overview of each method, outlines their advantages such as speed and inexpensiveness for some techniques, and notes potential disadvantages like expense, required skill level, and variability. The goal is to detect, identify, and quantify viruses using techniques ranging from cell culture to molecular analysis.

1. BY:
KAREEM HUSSEIN

2. Lab Diagnosis of Viral Infections
Virus culture and
isolation
-CPE
-Hemagglutination
-Plaque assay
-TCID50 assay
Detection of viral
antibody
-Hemagglutination
-inhibition test
-EIA/ELISA
Detection of viral
antigen
Immunofluorescence
-EIA/ELISA
-Western Blot
Immunoprecipitation
Detection of viral
genome
-(PCR)
-southern &
northern blot

3. Lab Diagnosis of Viral Infections
Detect and identify the viruses
-Microscopy in Cell Culture
-Immunofluorescence (IF)
-Molecular Methods
Quantify the viruses
-measuring
infectivity
-examine
nucleic acid
and protein
-counting
particles

4. • Provides viable isolate of the virus
• Detects multiple viruses
• Cell culture types:
• 1. Primary cells - Monkey Kidney
• 2. Semi-continuous cells - Human embryonic kidney and skin fibroblasts
• 3. Continuous cells - HeLa, Vero, Hep2, LLC-MK2, MDCK
• Cell monolayers are then examined by lm for CPE
• (centrifugation enhanced shell vial tech)

5. • detection of virus infections by identifying virus antigens using marked Abs.
• +ves : very rapid
• -ves : very expensive d.t cost of Abs and variability may occur as a result of
non specific binding or cross reaction of the Abs

6. • amplification techniques including (PCR), (NASBA) and (LMDA) are rapid
detection and molecular identification method for most known human
viruses.
• +ves: They are extremely sensitive and rapid.
• -ves: very expensive and is useless in case of viral mutation

7. Lab Diagnosis of Viral Infections
Detect and identify the
viruses
-Microscopy in Cell Culture
-Immunofluorescence (IF)
-Molecular Methods
Quantify the viruses
-measuring
infectivity
-examine
nucleic acid
and protein
-counting
particles

8. • What is a “plaque” ?
• +ves: inexpensive
• -ves: considered time consuming, laborious and relative error may occur

9. • What is TCID50 ?
• -ves: time consuming, laborious
• +ves: inexpensive and gives more accurate results than plaque assay

10. • IFA utilizes an antibody based staining methods to detect virally infected cells.
• +ves: more sensitive and faster than traditional plaque assays or TCID50
• -ves: may be quite expensive and variability may also be introduced

11. Lab Diagnosis of Viral Infections
Detect and identify the
viruses
-Microscopy in Cell Culture
-Immunofluorescence (IF)
-Molecular Methods
Quantify the viruses
-measuring
infectivity
-examine
nucleic acid
and protein
-counting
particles

12. • detection &Amplification of viral genome.
• Reverse transcriptase PCR (RT-PCR).
• “real-time” PCR.
• +ves: very rapid, very accurate
• -ves: expensive, requiers skilled operators

13. • it employs antigens or antibodies coupled to an easily-assayed enzyme.
• Types of ELISA
• +ves: a rapid, highly sensitive method
• -ves: expensive , variability may also occur

14. • (HA) is the most common indirect method to quantify amount of virus
particles.
• +ves: fast, inexpensive with simple principle
• -ves: considered laborious

15. • For visualization of specific DNA, RNA and protein among thousands of
contaminating molecules.
• Types of blotting
• 1 ) Southern blotting ( to detect DNA )
• 2 ) Northern blotting ( to detect RNA )
• 3 ) Western blotting ( to detect protein )

16. Lab Diagnosis of Viral Infections
Detect and identify the
viruses
-Microscopy in Cell Culture
-Immunofluorescence (IF)
-Molecular Methods
Quantify the viruses
-measuring
infectivity
-examine
nucleic acid
and protein
-counting
particles

17. • It uses fluorescent dyes such as (SYBR Green I) for the DNA of viruses.
• +ves: allows acquisition of statistically reliable data with little effort
• -ves: does not provide high sensitivity
• (Virus Counter® 2100) two dyes tech.

18. • For viruses that are very small to be seen directly under light microscope.
• +ves: it doesn’t require virus-specific reagents
• -ves: high instrument cost and amount of space and facilities required