The presentation consist of basic information on virus culture systems, their advantages and disadvantages.

1. CULTIVATION OF
VIRUSES
Dr. Vinod Kumar Singh
Department of Vet. Microbiology
DUVASU, Mathura

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1. Isolate and identify viruses in clinical specimens
2. maintenance of stock cultures
3. to prepare viruses for vaccines
4. for preparation of Antigen and production of Antibody
5. to do research on
• viral structure
• multiplication cycles
• genetics and effects on host cells
• studies of viral pathogenesis
Virus Cultivation - Purpose

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• Obligate intracellular parasites cannot be grown on
inanimate culture medium
• Cultivated within suitable hosts, such as a living cell
• Generally three methods are employed
1. Animal Inoculation
2. Inoculation of virus into Embryonated eggs
3. Tissue culture
Methods for Cultivation of Virus

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Animals Inoculation
• Loeffler and Frosch used cattle for studies in viral assay of
FMDV
• 1903 – Theiler uses white mice
• 1909- Landsteiner and Popper used monkeys for isolation
of Polio virus
• Reed and co worker inoculated human volunteers for their
work on Yellow Fever Disease

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Animals Inoculation
• Animal used for virus inoculation
– a) Natural host
– b) Laboratory animals
• In veterinary viruses: homologous host or heterologous host
• In case of human virus: heterologous host
• Natural host: for studies on pathogenesis, immunology, vaccine
trials, diagnosis and chemotherapy
• By animal inoculation we can observe the symptoms , lesions and
histopathology sections of infected tissues can be examined
• Animal should have no prior immunity to particular virus

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Animals Inoculation
• Laboratory animals are widely used for
routine cultivation of virus
• Should be SPF
• Monkeys, mice, rabbits, guinea pigs, ferrets
• Mice are the most widely employed

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Animal Inoculation
• Different routes of inoculation in mice are
• Intracerebral
• Subcutaneous
• Intraperitoneal
• Intranasal
• After the animal is inoculated with the virus
suspension, the animal is:
• observed for signs of disease
• visible lesions
• or is killed so that infected tissues can
be examined for virus

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Animal Inoculation Advantages
1. Gives unique insight into viral pathogenesis and host virus
relation
2. Used for the study of immune responses, epidemiology and
oncogenesis
3. More useful for those viruses that do not grow easily in cell
culture
4. Animal inoculation may be used as diagnostic procedure for
identifying and isolating a virus from a clinical specimen, such
as rabies virus inoculation of suckling mice

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Animal Inoculation Disadvantages
1. Expensive and difficulties in maintenance of animals
2. Interference of immune system
3. Individual variations
4. Difficulty in choosing of animals for particular virus
5. Some human viruses cannot be grown in animals or can be
grown but do not cause disease
6. Mice do not provide models for vaccine development
7. It will lead to generation of escape mutants
8. Issues related to animal welfare systems

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ECE Inoculation
• 1931: Woodruff and Good Pasture first used the
embryonated hen’s egg for Fowlpox virus cultivation
• Chicken, duck, and turkey eggs are the most common choices
for inoculation
• Egg used for cultivation must be sterile and the shell should
be intact and healthy
• Obtain embryonated eggs from specific pathogen-free (SPF)
flocks to avoid the possibility of the presence of maternal
antibodies (IgY) in the egg yolk sac

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ECE Inoculation Advantages
1. An intact and self-supporting unit, complete with its own sterile
environment and nourishment
2. Furnishes several embryonic tissues that readily support viral multiplication
3. Cost effective and Readily available
4. maintenance is much easier and Less labor is needed
5. Sterile and wide range of tissues and fluids
6. free from contaminating bacteria and many latent viruses.
7. Specific and non specific factors of defense are not involved
8. Sensitive to viruses which do not produce infection in adult birds.
9. Widely used method to grow virus for some vaccine production.

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Parts of Embryonated Egg

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Candling of Egg
• Process of holding a strong light above or below the egg to
observe the embryo
• Candling lamp consists of a strong electric bulb covered by a
plastic or aluminum container that has an aperture

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Unfertilized Egg

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Embryonated Egg - Live

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Embryonated Egg - Live

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Embryonated Egg - Dead

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Routes of Inoculation
• Different sites of viral inoculation in Embryonated eggs are:
1. Chorioallantoic membrane(CAM)
2. Amniotic Cavity
3. Allantoic Cavity
4. Yolk sac
5. Intravenous
• Route of inoculation and age of the embryo are determined
by virus selectivity for a certain membrane or developmental
stage of the embryo

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Routes of Inoculation
Route Age of ECE Viruses
Yolk Sac 6-8 day AEV, IBV, Chlamydia, Rickettsia
CAM 10-12 day Herpes, Pox, Rous sarcoma virus
Allantoic Cavity 10-12 day Influenza virus, NDV, mumps, Avian
Adenovirus
Amniotic Cavity 10-14 day Influenza virus primary isolation, Measles,
Mumps
Intravenous 13 day BTV

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Routes of Inoculation

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Inoculation of Egg
• The egg must be injected through the
shell, usually by drilling a hole or
making a small window
• The viral suspension or suspected
virus- containing fluid is injected into
the fluid of the egg
• The exact tissue that is inoculated is guided by the type of virus
being cultivated and the goals of the experiment

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CAM Route
1. Many viruses grow readily or can be adapted to grow
• Poxviruses
• Herpes viruses
2. Virus growth and replication in the CAM is indicated by
• visible lesions (pocks)
• grey white area in transparent CAM
• morphology may vary depending on the nature of the virus
3. Each infectious virus particle can form one pock, hence this
method is suitable for plaque studies

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CAM- Route

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Allantoic Cavity Route
• Allantoic inoculation is a quick and easy method that yields large amounts
(8–15 ml) of virus-infected egg fluids
• Most of avian viruses can be isolated using this method
• Many viruses such as Newcastle disease virus can grow readily
• viruses such as influenza, may require repeated amniotic passages
before becoming adapted to the egg and grown in the allantoic cavity
• employed for influenza virus vaccine production
• Other allantoic vaccines include Yellow fever and rabies vaccines
• Duck eggs provide a better yield of rabies virus and were used for the
preparation of the inactivated non-neural rabies vaccines

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Allantoic Cavity Route

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Amniotic Cavity Route
• Volume of fluid in the infected amniotic sac is small (1-2
ml)
• Recommended for the primary isolation of human viruses:
• mumps virus
• influenza A, B and C viruses
• Little application in veterinary virology
• Newly isolated influenza viruses may require several passages
before they adapt to growth by other routes

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Yolk Sac Route
• Simplest method for growth and multiplication of virus
• Mostly mammalian viruses are isolated using this method
• Immune interference mechanism can be detected in most of
avian viruses
• This method is also used for the cultivation of some bacteria
like Chlamydiae and Rickettsiae

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Yolk Sac Route

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Detection of Viral Growth
• Viruses multiplying in embryos may or may not cause effects
visible to the naked eye
• The signs of viral growth include:
• death of the embryo
• embryo cell damage
• defects in embryonic development
• localized areas of damage in the membranes, resulting in
discrete opaque spots called pocks

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Detection of Viral Growth
• If a virus does not produce obvious changes in the developing
embryonic tissue,
• methods of detection
• Embryonic fluids and tissues can be prepared for direct
examination with an electron microscope
• Certain viruses can also be detected by
• their ability to agglutinate red blood cells
• or by their reaction with an antibody of known
specificity

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Detection of Viral Growth

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Infectious Bronchitis

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NDV- Occipital Hemorrhages

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BTV- Cherry Red Embryo

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Pock lesions on CAM

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Tissue Culture
• Tissue culture refers to the growth and maintenance of living tissue
cells in vitro.
• 1913 (1st application) : Steinhardt and colleages maintained
vaccinia virus in fragment of rabbit cornea
• Major obstracle was bacterial and fungal contamination and until the
development of antimicrobials it was not a practical technique
• 1949: Anders, Weller and Robins reported polio virus could be
grown in non-neural cells with production of CPE
• Used for: virus isolation and titration, vaccine production and
biochemical studies

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Tissue cultures
• There are two basic types:
– Explant/Organ culture
– Cell cultures
• Explant cultures
• small tissue fragment samples from the host containing different
cell types are grown in culture
• useful for virus isolation and required for the isolation of some
coronaviruses
• Demonstration of latency of alpha herpesviruses require explant of
nerve sensory ganglia

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Tissue cultures
• Cell cultures result from the breaking down of various host
tissues into individual cells
• Major three types of cell cultures are used for culturing animal
viruses:
1. Primary cell cultures
2. Diploid culture
3. Continuous cell lines

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Primary Cell Cultures
• Derived from fresh tissues by enzymatic digestion with trypsin
or other protease to release individual cells
• differentiated
• often composed of many different cell types
• like the tissue from which they were derived, have a limited life
span, referred to as the Hayflick limit
• cultures rarely survive beyond passage 20th in vitro
• ideal for the isolation of some viruses
• Cells from subcultures are called secondary cultures

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Semi-continuous cultures
• known as diploid cell lines
• contain the normal diploid chromosome characteristic of the species
from which they were derived
• are single cell type
• dedifferentiated
• are primary cultures that have some cells that can be nurtured to
survive beyond the Hayflick limit
• die out between the 30th and 50th passage in vitro
• useful in the propagation of a wide range of viruses
• usually fibroblastic

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Continuous Cell Cultures
• Also known as heteroploid cell lines, as the cells have an
abnormal number of chromosomes
• Derived from normal or neoplastic tissue and are characterized
by their ability to be propagated in vitro indefinitely i.e.
Immortalised cells
• Reduced nutritional needs when compared to normal cells
• Not as sensitive as the other types for viral propagation
• Facilitate the large-scale growth of some viruses for vaccines
and research
• Grow as mono-layers, though a few grow in suspension
• Available from repositories, such as the American Type
Culture Collection (ATCC)

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COMMONLY USED CELL LINES
Name of Cell line Species of origin Tissue of origin Morphology1 Ploidy2
HeLa Human Carcinoma, cervix EP-L Aneu
Minnesota-EE Human Esophageal epithel. EP-L Aneu
L-132 Human Embryonic lung EP-L Aneu
J-111 Human Peripheral blood EP-L Aneu
WISH Human Amnion EP-L
BHK Hamster Kidney FB-L Eu
LLC-MK2 Monkey3 Kidney EP-L Aneu
CCRF S-180 11 Mouse Sarcoma 180 FP-L Aneu
1 FB-L: fibroblast-like; EP-L: epithelium-like
2 Aneu: aneuploid; Di: diploid; Eu: euploid
3 Macacc mulatta

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COMMONLY USED CELL LINES
Cell line Source
IBRS-2 Pig kidney
BHK-21 Baby hamster kidney
Vero Monkey kidney
PK-15 Pig kidney
RK-13 Rabbit kidney
MDBK Cow kidney
MDCK Dog kidney
EBTr Cow trachea
Dh-82 Dog macrophage like
CHO Chinese hamster ovary
Sf9 Spodoptera frugiperda, fall armyworm ovary
C6/36 Aedes albopictus, mosquito, Asian tiger

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Cell cultures- Advantages
• More convenient to work with than whole animal or eggs
• Lack immune and resistance factors of intact hosts
• Grown in chemically defined medium which is free from infection
and antibodies
• Very susceptible to infection
• mass produced to a reproducible standard and stored at -70°C
• Most viruses produce readily detectable changes in the proper host
cells
• Cultures (i.e.CPE) are easily studied with a inverted microscope

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Cell cultures- Disadvantages
• requires trained technicians
• Sophisticated laboratory
• Costly

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Identification
• In Animals
• death of the animal
• defects in animal development
• electron microscopy of infected animal tissue
• In ECE
• death of the embryo
• defects in embryonic development
• opaque spots called pocks
• electron microscopy of embryonic fluid and tissue
• Some can also be detected by their ability to agglutinate
red blood cells
• reaction with an antibody of known specificity

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Identification
• In cell culture
• viruses propagation in cell monolayer will deteriorate cells
which is called cytopathic effect (CPE)
• CPE can be detected and counted = plaques by phages (plaque
assay)
• Microscopy - CPE
- Syncytia - Cell rounding
- Membrane proliferations - Vacuolization
- Inclusion bodies - detachment
• Electron microscope (histopathology)
• Fluorescent ab (IFT/IPT)
• Haemagglutination and Heamadsorption
• NA hybridization(HPV)
• PCR/RT-PCR
• Interferences and metabolic inhibitors

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Identification
Virus plaques in a cell
layer overlaid with agar
medium and stained
with a vital dye
Uninfected cell culture Infected cell culture

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Heamadsorption
Syncytial formation caused by mumps virus
and haemadsorption of erythrocytes onto the
surface of the cell sheet

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Hemagglutination

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Immunofluorescense
HSV-infected epithelial cells from
skin lesion. (Source: Virology
Laboratory, Yale-New Haven
Hospital)
Positive immunofluorescence test for rabies
virus antigen. (Source: CDC)