1. Laboratory Diagnosis of Viral Infection

2. VIRAL LIFE CYCLE ATTACHMENT PENETRATION HOST FUNCTIONS UNCOATING
Transcription
Translation
REPLICATION
VIRAL LIFE CYCLE
ASSEMBLY
(MATURATION)
RELEASE
MULTIPLICATION

3. Difficulties Can not be seen under light microscope
Can not be cultivated easily
Do not grow on culture media
Treatment was not available
Changed situation
Rapid techniques
Screening for Blood transfusion
Treatment available

4. Microscopy Electron Microscope Light microscope – Inclusion bodies
Fluorescent Microscope -Fluorescent antibody technique

5. Demonstration of Viral Antigens
Precipitation on gel eg HBsAg
Immunofluorescence
Counter Immuno Electro Phoresis (CIEP)
Enzyme Linkes Immuno Sorbant Assay (ELISA)

6. Isolation of Virus Laboratory animals Fertilized Hen’s Egg
Chorioallantoic membrane
Allantoic cavity
Amniotic cavity
Yolk sac
Organ/Tissue/Cell Culture
Growth identified by serological method like neutralization.

7. Serological Reactions
Rising titre of antibody in paired sample of sera
First sample – At the earliest
Second sample – After 2 weeks
Single sample IgM type of antibody detection
Techniques – Neutralization, ELISA, CFT, Haemagglutination Inhibition (HAI)Test

8. Specimens According to the disease Respiratory – Throat swab
Eyes- Conjunctival scrapings
Liver – Blood
PUO – Blood
Skin - Scrapings

9. Virus Culture Embryonated Egg Cell Lines/ Tissue cultures
Chorioallantioc membrane (CAM)
Allantoic cavity
Amniotic cavity
Yolk Sac
Cell Lines/ Tissue cultures
Primary
Diploid/ Secondary
Continuous
Animal inoculation
Suckling mice

10. Embryonated Hen’s Egg
Chorioallantoic membrane (CAM) – visible lesions called pocks. Each infectious virus particle forms one pock. e.g. Variola, Vaccinia virus
Allantoic cavity – Influenza virus (vaccine production) & paramyxoviruses
Amniotic cavity – primary isolation of Influenza virus
Yolk sac – Chlmyadia, Rickettsiae & some viruses

11. Embryonated Hen’s Egg

12. Cell Culture Routinely used for growing viruses
Classified into 3 types:
Primary cell culture – normal cells freshly taken from body & cultured, limited growth
Rhesus monkey kidney
Chick embryo fibroblast
Human amnion cell culture
Diploid cell strains – cells of single type (fibroblast cells) that can be subcultivated for limited number of times, mostly 50
WI-38: human embryonic lung cell
HL-8: Rhesus embryo cell
Continuous cell lines – malignant cells, indefinite subcultivtion
HeLa: Human Ca of cervix cell line
HEP-2: Human epithelioma of larynx
Vero: Vervet monkey kidney
McCoy, Detroit-6, BHK-21, Kb

13. Cell Culture Tissues Individual cells
trypsin & mechanical shaking
Cells are washed, counted & suspended in a growth medium.
Growth medium – Minimum Essential Medium (MEM): essential aminoacids, vitamins, salts, glucose & bicarbonate in 5% CO2 with 5% fetal calf or calf serum, antibiotics & phenol red indicator

14. Cell Culture Bottles / Tubes

15. Detection of virus growth in cell cultures
Cytopathic effects (CPE) – morphological changes in cultured cells, seen under microscope, characteristic CPE for different groups of viruses
Metabolic Inhibition – no acid production in presence of virus
Hemadsorption – influenza & parainfluenza viruses, by adding guinea pig erythrocytes to the culture

16. Detection of virus growth in cell cultures
Interference – growth of a non cytopathogenic virus can be tested by inoculating a known cytopathogenic virus: growth of first virus will inhibit the infection by second
Transformation – oncogenic viruses induce transformation & loss of contact inhibition – microtumors
Immunofluorescence – test for viral Ag in cells from viral infected cultures.