Transcript Cell Culture

Laboratory Diagnosis of
Viral Infection
Professor Sudheer Kher
Learning Objectives

Describe the principles, techniques,
standards and recording of results and
interpretation of different methods used in
diagnosis of viral infections
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 have emerged
 Screening for Blood transfusion
 Treatment available

Techniques used
Microscopy
 Detection of Viral Antigen
 Growing and detecting viruses in

– Tissue / Organ / Cell culture
– Fertilized hen’s egg
– Laboratory animal inoculation eg mice

Detection of antobody in serum
– IgG – Rising titre in paired sample
– IgM – Indicates current / recent infection
Microscopy
Electron Microscope /
Immune Electron
Microscopy
 Light microscope –
Inclusion bodies eg Negri
Body in Rabies
 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.

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
Inoculation
Harvesting
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.
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.
Viral Assay

Viral content of a specimen: Total no. of
1. Virus particles – EM, HA
2. Infectious virions only

Assay of Infectivity: two types
1. Quantitative assays – actual no. of infectious
particle in an inoculum
2. Quantal assays – indicate the presence or
absence of infectious viruses, carried out in
animals, eggs or tissue cultures
Viral Assay
Assay of Infectivity: Quantitative
assays

–
–
Plaque assay in monolayer cell
cultures
Pock assay on CAM
*Each plaque/ pock represents one
infectious virus.
–
Plaques are clear zones that develop
on lawns of host cells.
–
The virus plaque is analogous to the
bacterial colony.
Specimens
 According
to the disease
– Respiratory – Throat swab
– CNS – CSF
– Eyes- Conjunctival scrapings
– Liver – Blood
– PUO – Blood
– Skin - Scrapings
Serological Reactions

Rising titre of antibody in paired sample of
sera is diagnostic
– First sample – At the earliest
– Second sample – After 2 weeks
Single sample - IgM type of antibody
detection. Indicates recent / current
infection.
 Techniques – Neutralization, ELISA, CFT,
Haemagglutination Inhibition (HAI)Test
