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 !