Sheroze Ameen Kinza Waqar Sayeda Sarah Saleem Sameen Ruqia Sayeda Kashmala Zahra Uzma Batool Viral...

Viral Detection MethodologiesGroup-8Introduction To Laboratory Medicine.Ug-3 ,6th Semester

Proudly Presented By:

Sheroze Ameen

Kinza Waqar

Sayeda Sarah Saleem

Sameen Ruqia

Sayeda Kashmala Zahra

Uzma Batool

Viral Diagnosis

1.Direct Examination - Microscopy - ELISA

2.Indirect Examination - Tissue Culture - Chick Embryo

3. Serology -Ab detection - Immunofluoresecence

Three General Approaches for Laboratory Diagnosis of Viral Infections

Collection of Viral Specimens

Type Of Specimen

Virus Type

Nasal Tract Infection

G.l Tract Infection

Blood /VesicularInfection

Swabs , Sputum from Throat, Nasal

Stool, rectal swab. Urine.

Skin scraps, Blood

RSV, Influenza A, B

Diarrheal and Entero -Viruses

HSV, Rubella,Pox virus, Rabies, HBV HCV, HIV, CMV

Store specimen at :

4 °C to 8 °C for short periods of time

-20 °C to - 40 °C for long term storage

Viral Specimen Storage & Bio-safety

Polyester Fiber-Tipped Applicator

Personal protective equipment (PPE)

Viral transport medium (VTM) collection vials

Viral transport medium (VTM) collection vials

Direct Methods of Virus Detection

Shehroze AmeenDirect Methods

of Viral Detection

ELISA

Microplate ELISA for HIV antibody:

coloured wells indicate reactivity

Electron Microscopy

• immune electron microscopy can increase specificity of EM

• disadvantage: expensive, poor sensitivity due to high requirement of viral titres

• Virus particles are detected and identified on the basis of morphology

106 virus particles per ml required for visualization, 50,000 - 60,000

magnification normally used.

Electron Microscopy

ADENO VIRUS

ROTA VIRUS

FECES

• ROTA VIRUS• ADENO

VIRUS• ASTRO

VIRUS• CALICIVIRUS

VESICLE FLUID

• HSV• VZV

SKIN SCRAPING

• PAPILLOMA VIRUS

• ORF• MOLLUSC

UM

Light Microscope• Replicating virus often

produce histological changes in infected cells

• Viral inclusion bodies are defined as replicating virus particles either in the nucleus or cytoplasm of infected cells

Light Microscope

RT, Reverse Trancriptase & Allele Specific PCR

PCR allows the in vitro amplification of specific target DNA sequences by a factor of 106 and is thus an extremely sensitive technique. It is based on an enzymatic reaction involving the use of synthetic oligonucleotides flanking the target nucleic sequence of interest. These oligonucleotides act as primers for the thermostable Taq polymerase

PCRAdvantages of PCR:1. Extremely high

sensitivity, may detect down to one viral genome per sample volume

2. Easy to set up3. Fast turnaround time

Disadvantages of PCR1. Extremely liable to

contamination2. High degree of operator skill

required3. Not easy to quantitate

results4. A positive result may be

difficult to interpret, especially with latent viruses such as CMV, where any seropositive person will have virus present in their blood irrespective whether they have disease or not.

INDIRECT METHODS FOR

VIRAL DETECTION

Tissue Culturing

Cells from man or animal are grown as a single layer(mono-layer) on the wall of the tubes or on one side of flat bottle. Cells are incubated at 37 degree Celsius.

•Suspended in tissue culture media

Types Of Cell Lines

Primary Cell Lines

Continuous Cell

Lines

Semi-Continuous Cell

Lines

Tissue CulturingVirus growth is recognized by:

1)Cytopathic effect: cell degeneration, Rounding, shrinkage

2) Haemadsorption Test : cells acquire the ability to stick to mammalian red blood cells.

NEGRI BODIES SYNCYTIA INCLUSION BODIES

Problems With Tissue Culturing

Long period (up to 4 weeks)

Susceptible to contamination

Not Applicable on all viruses

Poor Sensitivity

Chick Embryo Inoculation1. Chorioallantoic Membrane : Variola and Vaccinia Virus 2. Allantoic Cavity : Influenza Virus and Paramyxo virus3. Amniotic Cavity: Primary isolation of Influenza virus

7-12 day old chick is used. Costs less, growth of virus is rapid and easy.

The Inoculated eggs are kept at 37 degree for 48

hours till the virus replicates.

Serologic Tests

VIRAL SEROLOGY

Tests Principle

Levels of IgG and IgM Ab against viral antigens

Tests Include

1. HIV2. HSV3. HBV,HCV4. Rubella5. Measels6. CMV

Turn Around Time

7-10 days

Specimen

Serum

Volume

7ml

Container

Red or Gold Top tube Storage

4ºC

VIRAL SEROLOGY

Diagnosing

Primary + Re-Infection

4 fold or more increase in titre of IgG or total antibody between acute and convalescent sera

Presence of IgM or IgG

Seroconversion A single high titre of

IgG (or total antibody) - very unreliable

HEMAGGLUTINATION TEST

Purpose :

-  To quantitate soluble antigens and HI titer in serum samples

-  Sensitive than CFT, simple, inexpensive, and rapid and is the

method of

choice for

Assaying antibodies to any virus that causes hemagglutination

-  Commonly used for different  strains of influenza viruses, and

parainfluenza

viruses

-  Hemagglutinins are used as antigens in influenza virus

vaccines, thus

making HI the method of choice for measuring vaccine-

induced antibodies

1.Label plates (8 or 12

wells/row)

2.Add 50 ul PBS to all

Wells

3. Add 50 ul AAF to 1st

well

4.Mix & dilute (50 ul) –

1 st through last well

5.Add 50 ul 0.5%

washed rbcs to all

wells

7. Mix by shaking

plates

8. Read at 20 – 30 min

PROCEDURE

Used For Detection of : Influenza Virus A and B Adeno Virus Herpes Simplex Virus

Direct And Indirect Immunofluroesence

Reagents For Fixation: Paraformaldehyde PBS BSA Methanol

Direct Immunoflrorescence

Indirect Immunoflrorescence

Patient has a lot of antibody Patient has little antibody

Testing using a known antibody Testing using a known antigen

To identify patient’s antigen To identify patient’s antibody

1 step test 2 step test

Limitations of serological Diagnosis1. Long length of time required for diagnosis

2. Mild local infections such as HSV genitalis may not produce a detectable humoral immune response

3. Extensive antigenic cross-reactivity between related viruses e.g. HSV and VZV.

4. Immunocompromised patients often give a reduced or absent humoral immune response.

5. Patients given blood or blood products may give a false positive result due to the transfer of antibody.

THANK YOU !