Embryonated Egg Cultures for Viruses

Embryonated chicken egg is used for the culture of some viruses. The viruses grow in the cells of the embryo and membranes.

Specimens are inoculated into pathogen-free fertilized eggs of 10-11 days and are incubated for 2-9 days before harvesting the viruses. Growth and multiplication of the viruses are indicated by the death of the embryo, or by the formation of typical pocks or lesions on the egg membranes.

The embryonated chicken egg was first used for the cultivation of viruses by Good Pasteur and Burnet (1931).

Handling of eggs in a influenza vaccine plant

Before the development of cell lines for the culture of viruses, egg inoculation was one of the preferred methods of virus cultivation. Until now, egg culture is a preferred method for manufacturing both inactivated influenza vaccine (“flu shot”) and live attenuated (weakened) vaccine (“nasal spray flu vaccine”). Many manufacturers are also producing cell-based influenza vaccine.

Egg Inoculation

Specimens are injected into different sites (different cells) within fertilized eggs. These sites are different for different viruses (specificity of the virus).
Inoculation sites are;

  • Yolk Sac
  • Amniotic Sac
  • Chorioallantoic Membrane
  • Allantoic Sac

Egg inoculation sites are selected based on the virus yield which in turn depends on the susceptibility and permissiveness of these cells.

The eggs used for cultivation must be sterile and the shell should be intact and healthy. The handling of the eggs should be done in sterilized conditions.

Instead of manual injection by laboratory workers, vaccine manufacturing plants use automated machines to inoculate eggs and to harvest viruses after incubation.

Candling of embryonated eggs to see chicken embryo inside
Inoculation methodName of viruses
Allantoic inoculationInfluenza virus, Mumps virus, Newcastle disease virus, Avian adenovirus
Amniotic inoculationInfluenza virus, Mumps virus
Chorioallantoic membrane inoculationHerpes simplex virus, Poxvirus, Rous sarcoma irus
Yolk sac inoculationHerpes simplex virus
Allantoic inoculation

Allantoic Sac

The allantoic cavity is a larger cavity found in fertilized eggs and contains about 10 mL fluid per egg. It is lined with cells and after virus inoculation, the virus replicates in those cells. It is the most convenient method for the propagation of Newcastle disease virus.

An allantoic cavity is also commonly used for manufacturing viral vaccines such as influenza vaccine, yellow fever vaccine, and rabies vaccine.

Procedure of Allantoic Cavity Inoculation

For propagation of influenza virus

  • Take pathogen-free fertilized chicken eggs of 11-12 days.
  • Locate a non-veined area of the allantoic cavity which is located just below the air sac placing the egg in front of a light source. Mark the area with a pencil
  • Make a small nick in the marked area using a jeweler’s scribe.
  • Drill a hole at the top of the egg with a Dremel motorized tool. This is done to decrease the pressure of the air sac to prevent the leakage of inoculum.
  • Inoculate the eggs using a tuberculin syringe (a 1 ml syringe fitted with a 1/2 inch, 27 gauge needle).
  • Pass the needle through the hole in the shell, through the chorioallantoic membrane, and inject the inoculum in the allantoic cavity, which is filled with allantoic fluid.
  • Seal these two holes of the shell with melted paraffin.
  • Incubate the eggs at 37 degrees C for 48 hours.

During the incubation period, the virus replicates in the cells and virus particles are released by budding into the allantoic fluid.

Harvesting the virus

  • Remove the top of the eggshell (the part covering the air sac).
  • Pierce the shell membrane and chorioallantoic membrane with a pipette.
  • Withdraw about 10 mL of allantoic fluid per egg.

Depending on the virus strain, one or two eggs will produce sufficient virus to produce one 15 microgram dose of vaccine.

Amniotic Sac

It is mainly used for the primary isolation of the influenza virus. Viral growth is measured by the detection of hemagglutinin antigens in amniotic fluid.

Chorioallantoic Membrane

It is preferred for the Herpes Simplex virus and the pox virus. Viruses produce visible lesions as pocks on the chorioallantoic membrane. Each pock is derived from a single virion so the number of pocks would represent the number of viruses present in the inoculum.

Embroyonated Egg culture for Viruses
Cutaway view of an embryonated chicken egg

Yolk Sac

It is a preferred method for arboviruses (e.g. Japanese Encephalitis Virus) and some obligate intracellular bacteria such as Rickettsia and Chlamydia. The growth of encephalitis viruses may result in the death of an embryo.

Advantages of Egg Inoculation

  • Egg inoculation is a convenient and easy method to grow various types of viruses (many avian and few mammalian viruses) in massive quantities. In the US alone, 100-150 million eggs are used to make the flu vaccine.
  • Different viruses can be injected into an egg at different sites and the egg can be easily observed for viral replication throughout the development of an embryo.
  • Eggs have no developed immunologic functions.
  • Eggs are free from bacteria and many latent viruses.
  • Other advantages include easy availability, less cost, easier maintenance, and less labor.

References and further readings

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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