5. GROWTH AND CULTIVATION OF VIRUS

Introduction

Viruses are obligate intracellular parasites, meaning they cannot multiply on ordinary culture media like bacteria. They need living cells—animal cells, embryonated eggs or cell cultures—to replicate. Understanding how viruses grow and how they are cultivated is important for virus isolation, research, vaccine preparation and diagnosis.

Methods of Cultivation of Viruses

Since viruses cannot grow outside living cells, they must be cultivated using special biological systems. The three major methods are:

  1. Laboratory animals
  2. Embryonated eggs
  3. Tissue culture

1. Laboratory Animals

This is one of the earliest methods used for virus cultivation. Before the development of modern techniques, scientists relied heavily on animals to study viral diseases.

Examples and Uses

  • Monkeys: Used initially for poliovirus isolation. However, due to high cost and risk to handlers, their use is limited.
  • Mice: Most widely used; infant mice are highly susceptible to viruses like coxsackie and arboviruses.
  • Rabbits, guinea pigs, ferrets: Used selectively for specific viruses.
  • Smallpox virus: Can be inoculated on scarified skin or cornea of rabbits.

Indications of Viral Growth in Animals

  • Death of the animal
  • Appearance of disease symptoms
  • Visible lesions on skin or internal organs

Animal inoculation is also helpful for studying pathogenesis, immune responses and epidemiology of viruses.

2. Embryonated Eggs

The use of fertilized eggs for viral cultivation was introduced by Goodpasture and later refined by Burnet. This method played a key role in producing early vaccines.

Procedure

Fertile chicken eggs (5–12 days old) are disinfected and inoculated through the shell. The opening is sealed, and the egg is incubated at around 36°C. Different viruses grow in different parts of the egg.

Sites of Virus Growth in the Egg

  • Allantoic cavity: Influenza and paramyxoviruses
  • Amniotic cavity: Primary isolation of influenza viruses
  • Yolk sac: Some viruses, rickettsiae and chlamydiae
  • Chorioallantoic membrane (CAM): Pocks develop with viruses like vaccinia and herpes

Advantages

  • Easy to handle and economical
  • Readily available and bacteriologically sterile
  • No immune system to counteract viral infection
  • Provides multiple growth sites

Disadvantages

  • Some viruses do not grow in eggs on first isolation
  • Bacterial contamination can kill the embryo
  • Eggs may carry latent viruses or mycoplasma

3. Tissue Culture

Tissue culture is the most widely used and reliable method for cultivating viruses today. It involves growing human or animal cells in artificial media to support viral replication.

Historical Background

The method gained importance after Enders, Weller and Robbins (1949) successfully grew poliovirus in tissue culture, leading to a breakthrough in virology.

Types of Tissue Cultures

i. Organ Culture

Small whole organs or organ pieces are maintained in vitro. Useful for viruses that require specific organ-type cells. Example: Coronavirus isolation in tracheal ring organ culture.

ii. Explant Culture

Small pieces of minced tissue are embedded in plasma clots. Less commonly used today but historically used for adenovirus isolation.

iii. Cell Culture

The most important and commonly used technique.

Cells are separated from tissues using enzymes like trypsin, suspended in growth medium and allowed to grow on the surface of flasks or tubes as a monolayer. Based on their origin, cell cultures are classified as:

  • Primary Cell Cultures: Freshly isolated cells capable of limited divisions (e.g., monkey kidney cells, human embryonic kidney cells).
  • Diploid Cell Strains: Derived from embryonic tissues, can undergo 50–100 divisions and maintain diploid chromosome number (e.g., WI-38, HL-8).
  • Continuous Cell Lines: Derived from cancer cells, can divide indefinitely and are easy to maintain (e.g., HeLa, Hep-2, KB cell lines).

Detection of Virus Growth in Cell Culture

  • Cytopathic effect (CPE): Visible cell damage such as rounding, shrinkage or detachment.
  • Transformation: Change in cell shape or growth pattern.
  • Metabolic inhibition: Reduced metabolic activity due to viral infection.
  • Interference: Inhibition of one virus by another.
  • Hemadsorption: Red blood cells adhere to infected cell surfaces.
  • Immunofluorescence: Detection of viral antigens using fluorescent antibodies.

Key Points

  • Viruses cannot grow on ordinary media and need living systems for replication.
  • Three main cultivation methods: laboratory animals, embryonated eggs and tissue culture.
  • Tissue culture is the most common and efficient method today.
  • Growth is detected by cytopathic effects, hemadsorption and other cellular changes.

Detailed Notes:

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