Transcript Lab Diagnosis of viral infections

Laboratory Diagnosis of
Viral Infection
Dr. Sudheer Kher
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PENETRATION
UNCOATING
HOST
FUNCTIONS
Transcription
Translation
VIRAL
LIFE
CYCLE
REPLICATION
ASSEMBLY
(MATURATION)
RELEASE
MULTIPLICATION
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

Microscopy
Electron Microscope
 Light microscope – Inclusion bodies
 Fluorescent Microscope -Fluorescent
antibody technique

Demonstration of Viral
Antigens
 Precipitation
on gel eg HBsAg
 Immunofluorescence
 Counter Immuno Electro Phoresis
(CIEP)
 Enzyme Linkes Immuno Sorbant
Assay (ELISA)
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.

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

Specimens
 According
to the disease
– Respiratory – Throat swab
– CNS – CSF
– Eyes- Conjunctival scrapings
– Liver – Blood
– PUO – Blood
– Skin - Scrapings
Viral Hemagglutination

Hemagglutination
– Originally seen with the Influenza virus by Hirst in
1941.
– A convenient method of detection & assay of
Influenza virus.
– Due to the presence of Hemagglutinin spikes on the
surface.

Reversal of hemagglutination – Elution
– Due to the presence of Neuraminidase enzyme,
Receptor Destroying Enzyme (RDE)
– Destruction of receptor – reversal of
hemagglutination – release of virus from the red cell
surface
– Found only in Myxoviuses.
Virus Culture
Embryonated Egg
Chorioallantioc membrane (CAM)
Allantoic cavity
Amniotic cavity
Yolk Sac
Cell Lines/
Tissue cultures
Primary
Diploid/ Secondary
Continuous
Animal inoculation
Suckling mice
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
Embryonated Hen’s Egg
Cell Culture


Routinely used for growing viruses
Classified into 3 types:
– Primary cell culture – normal cells freshly taken from body &
cultured, limited growth
1. Rhesus monkey kidney
2. Chick embryo fibroblast
3. Human amnion cell culture
– Diploid cell strains – cells of single type (fibroblast cells) that can
be subcultivated for limited number of times, mostly 50
1. WI-38: human embryonic lung cell
2. HL-8: Rhesus embryo cell
– Continuous cell lines – malignant cells, indefinite subcultivtion
1.
2.
3.
4.
HeLa: Human Ca of cervix cell line
HEP-2: Human epithelioma of larynx
Vero: Vervet monkey kidney
McCoy, Detroit-6, BHK-21, Kb
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
Cell Culture Bottles / Tubes
Detection of virus growth in cell cultures
1.
Cytopathic effects (CPE) – morphological
changes in cultured cells, seen under
microscope, characteristic CPE for different
groups of viruses
2.
Metabolic Inhibition – no acid production in
presence of virus
3.
Hemadsorption – influenza & parainfluenza
viruses, by adding guinea pig erythrocytes to
the culture
Detection of virus growth in cell cultures
4.
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
5.
Transformation – oncogenic viruses induce
transformation & loss of contact inhibition –
microtumors
6.
Immunofluorescence – test for viral Ag in cells
from viral infected cultures.