ISOLATION OF VIRUS IN CELL CULTURE,EMBRYONATED

EGGS AND LABORATORY ANIMALS

Mr.Sandeep Pokhrel
CELL LINES
TYPES

PRIMARY

DIPLOID

CONTINUOUS

TISSUE

ORGAN
 Primary
▫ Animal/ human
▫ subculture once or twice
▫ Multiplication of cell ceases on contact: contact inhibition
▫ Slow met activity
▫ Less acid production
▫ Maintained for long

▫ Examples
 Rhesus monkey kidney cell culture
 Human amnion cell culture
 Chick embryo fibroblast cell culture
Disadvantages of primary cell lines
Fresh tissue sample may be difficult to obtain/require special
care in preparation

Latent viruses may be present as contaminating organ (SV40

in PRMK used for polio virus)

Organs of different individuals of same species may vary in

their ability to support replication

Costly
 Semi continuous(Cell Strain)
– 20-50 passages
– Fetal/ newborn
– Cultures may be preserved in frozen states
– Examples
• WI-38 (human embryonic lung cell strain)
• HL-8 (Rhesus embryo cell strain)
• MRC-5 (Medical Research centre,UK)
Disadvantages of semi contineous

May not support some viruses

After high passage level may lose the ability

to support viral replication
 Continuous
▫ Human/animal cancer cells
▫ Cells transformed in vitro
▫ Can grow as suspension cultures
▫ High plating efficiency
▫ Required of serum / growth factors is less
▫ Growth rate is faster
▫ Contact inhibition is less
▫ No polarity

▫ Examples
 HeLa (Human CA cervix cell line)
 HEp2 (Human epithelioma of larynx cell line)
 Vero (Vervet monkey kidney cell line)
 Disadvantages of continuous

Tends to overgrow (piling is seen)

High levels of buffering reqd due to increased acid

production as a result of high cellular metabolism
 Tissue culture
• Small tissue fragments

• Useful for latent stages

• Adherence of tissue
– Plasma drop clotting
– Warming plate
• Several cell types available for virus multiplication
 Organ culture
• 3 D structure preserved

• Good for fastidious viruses

• For transplants
•
• GROWTH CONDITIONS
• Optimum pH
– 7.1-7.5
– Hanks : closed system/ VTM
– Earles’ : open system

• Osmolality
– 280-330 mosm
– Osmometer
 Serum
▫ Fetal/ newborn calf: good for fastidious cells
▫ 5-10% growth
▫ 0-2% maintenance

▫ Horse serum : too stimulatory

▫ Human serum : toxic/ Abs

▫ Mycoplasma/ endotoxin free

▫ Store at -70°C
▫ Synthetic serum free media
 Gentamicin:
16-50
microgm/ml

microgm/ml
ANTIBIOTICS

50 U
Tetracyc
microg
EQUIPMENTS
• BSL
– II
– III: for select viruses
• Microscope
– Inverted
– Fluoroscent
• Incubators
– 35-37°C
– 33°C
– Humidified with 5% CO2
 Media
▫ Short term survival of cells during transport
▫ Long time survival
▫ Rapid cell proliferation
 VTM
 Growth media
 Maintenance media

• REAGENTS
▫ TPVG
▫ BSA
• Buffers
▫ HEPES
▫
 Surface used

• In 16*125 mm glass or
plastic round bottom screw
capped tubes

• Leighten’s tube

• Flask: prescription bottle(4 oz)/ milk dilution

bottle(6oz)
• Dram vial with coverslip
– Leightons tube

• Microwell plate: 96 well

– Flat bottomed
– 24-96
– 6-24 wells may contain cells
– Can put entire plate on
microscope stage

• Microchamber Slide culture

 Inoculation
• Centrifugation enhanced inoculation
– 1st for C. trachomatis
– Performed with shell vial
• Forced random collision b/w cell and viral particles
• Used for fastidious virus growth: hCMV, RSV etc
• Immediate early antigen of Hcmv: detected within 16-24 hrs
 Sample inoculation
– 0.1-0.5 ml added to each tissue culture system

– Adsorption inoculation(37 deg cel* 1 hr)

– No serum/ media
 Incubatory conditions

– 35-37°C
– Air- Hank’s medium
– CO2 – Earles’ medium
ROTATING/ROLLING RACKS (5-
STATIONARY SLANTED 12 times/hr)
RACK ROLLER DRUM METHOD

INCREASED SPEED AND

SENSITIVITY
 Interpretation

– Exam daily for 1st week & alt day for remaining period of
incubation

– CPE

– Haemadsorption test
• Influenza
• Parainfluenza
• Mumps
• Other methods of detection
– Cellular degeneration
– Plaque formation
– Metabolic inhibition
– Haemadsorption
– Interference
– Transformation
– Immunofluorescence
 Confirmation

– MAb tagging:
– FITC labelled
– Testing by IF
– Confluent/ speckled
– Nuclear/cytoplasmic
• Neutralization test
• Virus + specific Ab
• Aliquot applied to fresh cell culture

POS: VIRUS
ABSENT
• Look for CPE

NEG: VIRUS
• Cumbersome PRESENT
• Time consuming
• Viral titres required
 Advantages of culture methods

▫ Wide range of viruses grown

▫ Unanticipated viruses may be found

▫ Mixed infections easier to detect

▫ Susceptibility testing

▫ Serotyping

▫ Epidemiologic studies

▫ High sensitivity

▫ Therapeutic considerations
 Advantages of embryonated eggs

• Readily available & low cost

• Absence of latent viruses

• Absence of antibodies

• Presence of a variety of extraembryonal organs

• Suitable for the study of the behavior of many viruses in

single layers of cells.
 APPLICATION OF EMBRYONATED EGG-TECHNIQUES IN
VIROLOGY

• For the propagation in the laboratory of stock strains of virus

• For the primary isolation of virus from pathogenic material.

• For titration of viruses &antiviral sera

• For chemotherapeutic experiments

• For yielding large quantities of virus for vaccine or chemical analysis

• To study viral morphology

• To study the mode of multiplication

 HANDLING A FERTILE EGG BEFORE INOCULATION:

SUPPLY OF FERTILE EGG:

• Egg should be from healthy flocks.

• Free from natural viral & bacterial infection.

• White leghorn eggs are more suitable than dark shelled egg.

• Must be natural clean & should be fresh.

• Should not be kept longer than 8-10 days at temp. b/w 5-150c
INCUBATION OF FERTILE EGG:
Incubator:
To be equipped with forced air circulation
• Humidity 40-70 %.,
• Temp. 37.5 – 38o C but can be 39o C.

Rotation of eggs.
• In automatic incubator eggs are rotated every 4-hours of interval in
clockwise.
• Manually 2-4 times a day but not less than 2 times.

Rotation of egg is
• To prevent the adhesion of embryonic membrane.
• To keep the embryo more or less centralized.
CANDLING OF EMBRYONATED EGG:
• On 4-5 days of incubation Eggs are candled.
To examine whether
Embryo is alive or Dead

SIGN OF ALIVE EMBRYO SIGN OF DEAD EMBRYO:

- spontaneous movement. - No spontaneous movement.
- Clearly visible well developed blood - Smaller the size than expected size.
vessels. - No or barely visible enterupted blood
- The embryo is visible as a dark vessels.
shadow, often showing a dark spot
- Occasionally thick red strips.
corresponding to the Head (eye) of the
embryo.
- A diffuse paler shadow indicates
chorioallantoic membrane.
Egg –candled-day3
Egg-candled-day9-12
 STRUCTURE OF CHICK EMBRYONATD EGG
• Shell & Shell membrane.
• Chorioallantoic membrane.
• Allantoic cavity.
• Amniotic cavity.
• Yolk sac.
• Albumin.
Material required for inoculation of virus

 Inoculating tray
 Tuberculin syringe
 23,24,26,gauge needles
 Tincture of iodine
 sprit
 Pencil marker
 Cellotape/paraffin wax
 Dental driller
 candled box
 sterile PBS
 Rubber teat
 Material required for harvesting of virus from embryonated egg
--

Dissecting tray

Syringe

Pasture pipette

Sterile vial

Ice box

Disinfectant
 Chorioallantoic membrane(CAM)
• Used for isolation &propagation of those virus which produce pocks on
the CAM.
• Example
-Vaccinia
-Variola
-Herpex simplex
-Fowlpox
-Monkeypox
-Herpes simae(Bvirus)
.For chemotherapy trail
For viral morphological study
For production of vaccine
For antibody assay
• Embryo should be 9-14 days old , most suitable is 12days
• Place of in oculation will be located area of well-developed CAM but free of
blood vessels
• This place usually is located in the center of the whole CAM
• Amount of inoculum 0.1-0.2ml
• After inoculation rotate the egg for uniform distribution.
• Seal the hole and incubate the eggs at35 0cfor 48-72hr
Making of false air sac
Technique

Inoculation onto CAM

 Harvesting of CAM
• Before harvesting of CAM ,eggs are candled to determine whether air sac is
still displaced.
Lesions on CAM of Vaccinia virus (arrow indicate the place where shell
Was drilled)
Lesion on CAM of Rouse sarcoma virus strain B77
 ALLANTOIC CAVITY

• Route is employed for the production of virus in large quantity

required for the preparation of serological antigen,vaccine

• This route is used for the following virus

-Influenza viruses of type A,B,C
-Fowl plaque
-Sendai & Newcastle disease viruses
Technique
embryo 13-14days old
Two methods
-open method
-blind method
Inoculum-
0.1-0.2ml
Needle-
23gauge(4.5cm)
 Fig-1 Fig-2

Fig-3 Fig-4
Fig-1-drilling of shell
Fig-2-opening of the predrilled shell flap fixed with adhesive tape
Fig-3-pushing away of the shell member from the underlying CAM above embryo
Fig-4 penetration by the needle into the amniotic sac by folding technique
Blind Method
• After inoculation the puncture area and hole
sealed with a paraffin-Vaseline mixture or colloid
ion or cello tape.

• Inoculated eggs are incubated at 330-370c

depending upon the virus for 48-72hr.
 Harvesting of amniotic fluid

• eggs are 1st chilled at40c for 18hr to induce

contraction of the blood vessels

• This particularly important when working with

Myxovirus ,since release of RBC in to the
embryonic fluid may lead to loss of virus through
absorption of the cells.
Harvesting of amniotic fluid in situ
Harvesting of amniotic fluid by
Inclining the egg
Harvesting of amniotic fluid
by
Partial extrusion of the
Egg contents
 Yolk Sac inoculation

Mainly used for

- Cultivation of Rickettsiae

- Chlamydia

- Rabies virus
-
 Technique
• Embryo 5-6daysold
• Inoculum 0.1-1.0ml
• Needle 4.5cm,23gauge
• After inoculation,the hole in the shell is closed by paraffin –Vaseline mixture.
• Eggs are incubated at temp 32-37oc depending on virus inoculated

Harvesting of yolk sac

Factors influencing the susceptibility of the chick Embryo
to Virus infection

• Route of inoculation

• Age of embryo

• Temperature of incubation after virus inoculation

• Amount of virus

• Length of incubation period.

PROCESSING
Samples

Serum CSF Antemortem Brain Autopsy

Corneal impr,
skin biopsy
Detection of
antibodies
Isolation of
(unvaccinated) Rabies virus nRT- PCR DFA Seller stain
- MNT
- RFFIT (Rapid, Sensitive)

- ELISA (field test, Suckling mice Tissue culture Histopathology

rapid, low sensitivity) - Murine neuroblastoma
- BHK
Symptoms

Dissect brain

DFA RREID (field test) IHC EM nRT-PCR

 Decomposed tissue
X Formalin fixed
Formalin fixed