1. Viral Detection Methodologies
Group-8
Introduction To Laboratory Medicine.
Ug-3 ,6th Semester

2. Proudly Presented By: Viral Diagnosis Sheroze Ameen Kinza Waqar
Sayeda Sarah Saleem
Sameen Ruqia
Sayeda Kashmala Zahra
Uzma Batool

3. Three General Approaches for Laboratory Diagnosis of Viral Infections
1.Direct Examination
- Microscopy
- ELISA
2.Indirect Examination
- Tissue Culture
- Chick Embryo
3. Serology
-Ab detection
- Immunofluoresecence

4. Collection of Viral Specimens
Nasal Tract Infection
G.l Tract Infection
Blood /Vesicular
Infection
Swabs , Sputum from Throat, Nasal
Stool, rectal swab. Urine.
Type Of Specimen
Skin scraps, Blood
Diarrheal and Entero -Viruses
HSV, Rubella,Pox virus, Rabies, HBV HCV, HIV, CMV
RSV, Influenza A, B
Virus Type

5. Viral Specimen Storage & Bio-safety
Personal protective equipment (PPE)
Store specimen at :
4 °C to 8 °C for short periods of time
-20 °C to - 40 °C for long term storage
Viral transport medium (VTM) collection vials
Polyester Fiber-Tipped Applicator
Viral transport medium (VTM) collection vials

6. Direct Methods of Virus Detection
Direct Methods of Viral Detection
Shehroze Ameen

7. ELISA

8. Microplate ELISA for HIV antibody:
coloured wells indicate reactivity

9. 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

10. Electron Microscopy
106 virus particles per ml required for visualization, 50, ,000
magnification normally used.
FECES
ROTA VIRUS
ADENO VIRUS
ASTRO VIRUS
CALICIVIRUS
VESICLE FLUID
HSV
VZV
SKIN SCRAPING
PAPILLOMA VIRUS
ORF
MOLLUSCUM
ADENO VIRUS
ROTA VIRUS

11. 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

12. Light Microscope

13. 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

14. PCR Advantages of PCR: Disadvantages of PCR
Extremely high sensitivity, may detect down to one viral genome per sample volume
Easy to set up
Fast turnaround time
Disadvantages of PCR
Extremely liable to contamination
High degree of operator skill required
Not easy to quantitate results
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.

15. INDIRECT METHODS
FOR
VIRAL DETECTION

16. Semi-Continuous Cell Lines
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

17. Tissue Culturing 1)Cytopathic effect: cell degeneration, Rounding,
Virus 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

18. Problems With Tissue Culturing
 Long period (up to 4 weeks)
Poor Sensitivity
Susceptible to contamination
Not Applicable on all viruses

19. Chick Embryo Inoculation
Chorioallantoic Membrane : Variola and Vaccinia Virus
Allantoic Cavity : Influenza Virus and Paramyxo virus
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.

20. Serologic Tests

21. VIRAL SEROLOGY 7-10 days Serum 7ml Red or Gold Top tube 4ºC
Turn Around Time
Specimen
Tests Principle
Levels of IgG and IgM Ab against viral antigens
7-10 days
Serum
Container
Volume
Tests Include
HIV
HSV
HBV,HCV
Rubella
Measels
CMV
7ml
Red or
Gold
Top tube
Storage
4ºC

22. 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

23. 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

24. PROCEDURE Label plates (8 or 12 wells/row) Add 50 ul PBS to all Wells
Add 50 ul AAF to 1st well
Mix & dilute (50 ul) – 1 st through last well
Add 50 ul 0.5% washed rbcs to all wells
7. Mix by shaking plates
8. Read at 20 – 30 min

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

26. Direct And Indirect Immunofluroesence
Reagents For Fixation:
Paraformaldehyde
PBS
BSA
Methanol

27. 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

28. Limitations of serological Diagnosis
Long length of time required for diagnosis
Mild local infections such as HSV genitalis may not produce a detectable humoral immune response
Extensive antigenic cross-reactivity between related viruses e.g. HSV and VZV.
Immunocompromised patients often give a reduced or absent humoral immune response.
Patients given blood or blood products may give a false positive result due to the transfer of antibody.

29. THANK YOU !