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    Virus Cultivation

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    ved.g007
    This document discusses methods for cultivating viruses. Viruses are obligate intracellular parasites that cannot grow outside of host cells. They are typically cultivated within living cells or embryonated eggs. Three main cultivation methods are discussed: inoculation into animals, inoculation into embryonated eggs, and tissue culture. Embryonated eggs provide a sterile environment and suitable tissues for many viruses to grow. Specific sites of inoculation in eggs include the chorioallantoic membrane, amniotic cavity, allantoic cavity, and yolk sac. Cell cultures also support viral growth and allow detection of cytopathic effects. Identification methods include PCR, RFLP, and serological techniques.

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    Virus Cultivation

    Uploaded by

    ved.g007
    940 views33 pages
    This document discusses methods for cultivating viruses. Viruses are obligate intracellular parasites that cannot grow outside of host cells. They are typically cultivated within living cells or embryonated eggs. Three main cultivation methods are discussed: inoculation into animals, inoculation into embryonated eggs, and tissue culture. Embryonated eggs provide a sterile environment and suitable tissues for many viruses to grow. Specific sites of inoculation in eggs include the chorioallantoic membrane, amniotic cavity, allantoic cavity, and yolk sac. Cell cultures also support viral growth and allow detection of cytopathic effects. Identification methods include PCR, RFLP, and serological techniques.

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    Virus Cultivation, tissue culture, animal, embryonated egg.

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    This document discusses methods for cultivating viruses. Viruses are obligate intracellular parasites that cannot grow outside of host cells. They are typically cultivated within living cells or embryonated eggs. Three main cultivation methods are discussed: inoculation into animals, inoculation into embryonated eggs, and tissue culture. Embryonated eggs provide a sterile environment and suitable tissues for many viruses to grow. Specific sites of inoculation in eggs include the chorioallantoic membrane, amniotic cavity, allantoic cavity, and yolk sac. Cell cultures also support viral growth and allow detection of cytopathic effects. Identification methods include PCR, RFLP, and serological techniques.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    940 views33 pages

    Virus Cultivation

    Uploaded by

    ved.g007
    This document discusses methods for cultivating viruses. Viruses are obligate intracellular parasites that cannot grow outside of host cells. They are typically cultivated within living cells or embryonated eggs. Three main cultivation methods are discussed: inoculation into animals, inoculation into embryonated eggs, and tissue culture. Embryonated eggs provide a sterile environment and suitable tissues for many viruses to grow. Specific sites of inoculation in eggs include the chorioallantoic membrane, amniotic cavity, allantoic cavity, and yolk sac. Cell cultures also support viral growth and allow detection of cytopathic effects. Identification methods include PCR, RFLP, and serological techniques.

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    Viruses are obligate intracellular parasites that depend on host cells for growth and multiplication. They cannot be grown on inanimate culture media and require living cells or animals as hosts.

    The three main methods for cultivating viruses are inoculation into animals, inoculation into embryonated eggs, and tissue culture.

    Influenza viruses and mumps virus can be grown in embryonated eggs. Embryonated eggs also support a fairly wide range of animal and human viruses, making them important in diagnostic services.

    Cultivation of Viruses

    Viruses do not fall in the category of unicellular


    microorganism
    They are obligate intracellular parasites and
    lack the machinery necessary for protein and
    nucleic acid synthesis
    They depend on the host machinery for their
    growth and survival
    Unlike other microorganism, complex processes
    are involved in their multiplication
    Outside of the host cells, viruses are inactive,
    however, inside living cells, viruses show some
    of the characteristics of living things

    Cultivation of Virus
    Since the viruses are obligate intracellular
    parasites, they cannot be grown on any
    inanimate culture medium
    Viruses can be cultivated within suitable
    hosts, such as a living cell
    The primary purposes of viral cultivation are:
    1. To isolate and identify viruses in clinical
    specimens
    2. To prepare viruses for vaccines
    3. And to do detailed research on viral structure,
    multiplication cycles, genetics, and effects on
    host cells

    Cultivation of Virus
    Viruses not only need living cells to grow in but
    also they are specific about the type of cell they
    infect and grow in
    There is no universal cell that will support all
    viruses
    Viruses tend to be host specific; therefore:
    human viruses grow best in cells of human origin,
    bovine viruses in bovine cells,
    canine viruses in canine cells,
    while some viruses will not grow in vitro at all

    Therefore in the laboratory the suspected virus


    must be grown in a culture method known to
    support its growth

    Methods for Cultivation


    of Virus
    Generally three
    methods are
    employed for the virus
    cultivation
    1. Inoculation of virus
    into animals
    2. Inoculation of virus
    into embryonated
    eggs
    3. Tissue culture

    Methods for Cultivation


    of Virus
    Animals are used for studying
    viruses which do not grow in cell
    cultures or eggs, and for testing
    vaccines
    Eggs support a fairly wide range of
    animal and human viruses hence
    their importance in the diagnostic
    service
    Cell cultures; different types of cell
    lines will support different types of

    Inoculation of Virus in Animals


    Laboratory animals play an
    essential role in studies of
    viral pathogenesis
    Live animals such as
    monkeys, mice, rabbits,
    guinea pigs, ferrets are
    widely used for cultivating
    virus
    Mice are the most widely
    employed animals in virology

    Inoculation of Virus in
    Animals
    The different routes of
    inoculation in mice are:
    intracerebral
    subcutaneous
    intraperitoneal
    or 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

    ANIMAL INOCULATIONDISADVANTAGES

    Cost
    Maintenance
    Interference of immune system
    Individual variations
    Difficulty in choosing of
    animals for particular virus

    Inoculation of Virus in
    Embryonated Eggs
    Good pasture and Burnet in 1931
    first used the embryonated hens
    egg for the cultivation of virus
    The process of cultivation of
    viruses in embryonated eggs
    depends on the type of egg being
    used
    Eggs provide a suitable means for:
    the primary isolation and
    identification of viruses
    the maintenance of stock cultures
    and the production of vaccines

    Embryonated Egg
    Terms most often refer to eggs:
    Embryonated: having anembryo
    Unembryonated: not having an embryo
    De-embryonated:having lost an embryo

    Embryonated egg, referring to an


    advanced stage of development and
    not merely after fertilization

    Advantages
    An embryo is an early developmental stage of animals
    marked by rapid differentiation of cells
    Birds undergo their embryonic period within the closed
    protective case of an egg, which makes an incubating
    bird egg a nearly perfect system for viral propagation
    It is an intact and self-supporting unit, complete with
    its own sterile environment and nourishment
    It furnishes several embryonic tissues that readily
    support viral multiplication
    Defense mechanisms are not involved in embryonated
    eggs
    Cost- much less, Maintenance-easier, Less labor and
    Readily available

    Inoculation of Virus
    Chicken, duck, and turkey eggs are the
    most common choices for inoculation
    The egg used for cultivation must be
    sterile and the shell should be intact
    and healthy
    Rigorous sterile techniques must be
    used to prevent contamination by
    bacteria and fungi from the air and the
    outer surface of the shell

    Inoculation of Virus
    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

    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
    Defects in embryonic development
    and localized areas of damage in the
    membranes, resulting in discrete
    opaque spots called pocks

    Detection of Viral Growth


    If a virus does not produce obvious
    changes in the developing embryonic
    tissue, virologists have other 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

    Parts of Embryonated
    Egg
    The air sac is important to the
    developing embryo for
    respiration and for pressure
    adjustments
    The shell and shell membrane
    function both as a barrier and as
    an exchange system for gases
    and liquid molecules
    The chorioallantoic sac and its
    contents (allantoic fluid) remove
    waste products produced by the
    developing embryo
    This Membrane and its contents
    increases in size as the embryo
    grows

    Parts of Embryonated
    Egg
    The yolk sac is the source of
    nourishment for the developing Embryo
    As the embryo develops, the yolk sac
    decreases in size until it is completely
    absorbed into the digestive system of the
    mature embryo

    The amnion is a thin membrane that


    encloses the embryo and Protects it
    from physical damage
    It also serves as an exchange system and
    is best seen in the younger embryos

    Routes of Viral
    Inoculation
    An embryonated egg
    offers various sites for
    the cultivation of viruses
    The different sites of
    viral inoculation in
    embryonated eggs are:
    1. Chorioallantoic
    membrane(CAM)
    2. Amniotic Cavity
    3. Allantoic Cavity
    4. Yolk sac

    Routes of Viral
    Inoculation
    The chosen route of inoculation and age of
    the embryo are determined by the given
    virus selectivity for a certain membrane or
    developmental stage of the embryo
    For example Infectious bronchitis virus is
    propagated in the yolk sac of a 5-6 day old
    embryo
    whereas Rous-sarcoma virus is inoculated on
    the chorioallantoic membrane of a 9-11 day
    old embryo and will produce pocks 5-10 days
    post-infection

    Candling of Egg
    Candling is the process
    of holding a strong light
    above or below the egg
    to observe the embryo
    A candling lamp consists
    of a strong electric bulb
    covered by a plastic or
    aluminum container that
    has a handle and an
    aperture

    Candling of Egg
    Chick egg at day 6
    Chick embryo day 11
    Chick embryo day 16

    Chorioallantoic
    Membrane (CAM)
    This method has been widely used in
    veterinary virology
    Many viruses grow readily or can be adapted
    to grow on the CAM
    Viruses produce visible foci or pocks,
    inclusion bodies, oedema or other
    abnormalities
    Each infectious virus particle forms one pock
    Viruses which can be grown include:
    Herpes viruses
    and poxviruses

    Amniotic Cavity Inoculation


    The virus is introduced directly into the
    amniotic fluid that bathes the developing
    embryo
    The volume of fluid in the infected
    amniotic sac is small (1-2 ml)
    The amniotic route is recommended for the
    primary isolation of human viruses:
    mumps virus
    and influenza A, B and C viruses

    has little application in veterinary virology

    Newly isolated influenza viruses may require


    several passages before they adapt to growth
    by other routes, such as allantoic

    Allantoic Cavity
    Inoculation

    Many viruses such as Newcastle


    disease virus can grow readily
    Other viruses such as influenza, may
    require repeated amniotic passages
    before becoming adapted to the egg
    and grown in the allantoic cavity
    Allantoic inoculation is a quick and
    easy method that yields large amounts
    (815 ml) of virus-infected egg fluids

    Yolk Sac Inoculation


    It is also a 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

    Using cell culture


    CULTIVATION/ ISOLATION
    - preferred type of growth medium for virus, more convenient than the
    previous two methods
    - use isolated cell from animal that are cultured invitro. Normal cells will
    form monolayer.

    - If viruses are present, the cells of monolayer will deteriorate as they


    multiply. Cell deterioration is called cytopathic effect (CPE). CPE can be
    detected and counted = plaques by phages (plaque assay). Microscopic
    observation via electron microscope (histopathology).
    IDENTIFICATION

    A. Normal
    B. Transformed

    Culturing of using cell culture

    Two discoveries greatly enhanced the


    usefulness of cell cultures for virologists and
    scientists
    1. The discovery and use of antibiotics made it
    possible to prevent bacterial contamination
    2. The discovery of proteolytic enzymes (e.g.
    trypsin) can free animal cells from surrounding
    tissues without injuring freed cells
    .

    Subculturing: the process by which cells from


    an existing culture are transferred to new
    containers with fresh nutrient media

    Identification of viruses
    1. PCR polymerase chain reaction
    2. Restriction fragments
    polymorphisms (RFLP)
    3. Serological method Western blot
    common method use
    4. Immunological test , ELISA,
    agglutination test if specific
    preparation of killed, inactivated or
    antibody is Aavailable

    attenuated microorganisms to induce


    artificially acquired active immunity

    Vaccine development
    1. Embryoted chicken egg one the most
    used method of viral isolation and
    growth
    2. Still used to grow viruses for some
    vaccines eg. Influenza vaccine
    3. Cell culture and animal tissue are also
    used in vaccine preparation for some

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