BY:

KAREEM HUSSEIN

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

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

• 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)

• 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

• 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

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

• What is a “plaque” ?

• +ves: inexpensive

• -ves: considered time consuming, laborious and relative error may occur

• What is TCID50 ?

• -ves: time consuming, laborious

• +ves: inexpensive and gives more accurate results than plaque assay

• 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

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

• detection &Amplification of viral genome.

• Reverse transcriptase PCR (RT-PCR).

• “real-time” PCR.

• +ves: very rapid, very accurate

• -ves: expensive, requiers skilled operators

• 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

• (HA) is the most common indirect method to quantify amount of virus particles.

• +ves: fast, inexpensive with simple principle

• -ves: considered laborious

• 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 )

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

• 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.

• 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