CULTIVATION OF VIRUSES - HOW DO VIRUSES GROW IN LABS?
CULTIVATION OF VIRUSES - GROWTH OF VIRUSES IN LABORATORY

CULTIVATION OF VIRUSES – HOW DO VIRUSES GROW IN LABORATORY?

CULTIVATION OF VIRUSES

⇒ Viruses are the obligate intracellular parasites i.e. they strictly require a living host or living cells for the growth and multiplication so they cannot be grown on an inanimate culture medium.

⇒ Three methods are employed for the cultivation of viruses:-

  • Animal inoculation
  • Embryonated egg inoculation
  • Tissue culture




ANIMAL INOCULATION

  • This is the earliest method for the cultivation of viruses & mice are the most widely used animals in virology.
  • For e.g. – Infant (suckling) mice are used in the isolation of arbovirus and coxsackieviruses, which don’t grow in any other system.
  • Mice may be inoculated by several routes: intracerebral, subcutaneous, intraperitoneal or intranasal.
  • Other animals such as Guinea pig, Rabbit & ferrets are used in some situations.
  • The growth of virus in inoculated animals may be indicated by Death, Disease or visible Lesions.
  • Later on, the test organism is sacrificed and tissues are tested for the presence of the virus.

EMBRYONATED EGG INOCULATION

⇒ Embryonated hen’s eggs (7-12 days old) are used for the cultivation of viruses in the laboratory.

⇒ It offers several sites for the cultivation of viruses as – Chorioallantoic membrane (CAM), Allantoic cavity, Amniotic Sac & Yolk Sac.

EMBRYONATED CHICK EGG

  • Chorioallantoic membrane (CAM) is inoculated for growing poxviruses. It produces visible lesions called as pocks. Pocks produced by different viruses have different morphology.
  • Allantoic cavity inoculation is employed for growing influenza virus for vaccine production.
  • Amniotic sac inoculation is mainly used for the primary isolation of influenza virus.
  • Yolk sac inoculation is used for the cultivation of some viruses and some bacteria (Chlamydia & rickettsiae).

⇒ After inoculations, eggs are incubated for 2-9 days.

TISSUE CULTURE 

⇒ Tissue culture is the most common method employed for the propagation of viruses in the laboratory.

⇒ Tissue culture & cell culture are the terms used interchangeably for this method.



⇒ Three types of tissue cultures are available:-

  • Organ culture
  • Explants culture
  • Cell culture

A.) ORGAN CULTURE

  • In this, small bits of organs are maintained in tissue culture growth medium in vitro (in labs) for days & weeks.
  • Organ cultures are useful for the isolation of some viruses which appear to be highly specialized parasites of certain organs.
  • For e.g. – trachea ring culture for the isolation of coronavirus.

B.) EXPLANT CULTURE

  • In this, fragments of minced tissue can be grown as explants. It was originally known as tissue culture.
  • For e.g. – Adenoid tissue explants cultures were used for the isolation of adenoviruses.
  • This method is rarely employed nowadays.

C.) CELL CULTURE

  • This is the type of culture technique that is routinely used for the cultivation of viruses in the laboratory.
  • In this, the tissues are dissociated into component cells by the action of proteolytic enzymes e.g. trypsin or collagenase.
  • Based on their origin, chromosomal characters and no. of cell divisions, they are classified into three types as:-
    • Primary cell culture
    • Diploid cell strains
    • Continuous cell lines

1.) Primary cell culture

  • In this, normal cells are freshly taken from the body & cultured.
  • They are capable of very limited growth in culture (5-10 divisions at most)
  • Primary cell cultures are useful for the isolation of viruses and their cultivation for vaccine production.
  • For e.g. – monkey kidney, the human kidney, human amnion, chick embryo.

2.) Diploid cell strain

  • These are the cells of the single type that retains the original diploid chromosome number during serial subcultivation for a limited number of times.
  • After about 50 serial subcultures they undergo ‘senescence’ and the cell strain is lost.
  • For e.g. – WI-38 human embryonic lung cell strain

3.) Continuous cell line

  • These are the cells of the single type, usually derived from cancer cells that are capable of indefinite growth in-vitro.
  • Some cell lines are also being used for vaccine manufacture e.g. Vero cell for rabies vaccine.



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