Lesson 3b: Cultivation of viruses in embryonated eggs

• The embryonated egg is a complex structure comprised of an embryo and its supporting membranes (chorioallantoic, amniotic, yolk).
• The developing embryo and its membranes provide the diversity of cell types that are needed for successful replication of a wide variety of different viruses.
• The embryo serves as an incubator for viral replication.
• Location within the embryo is important as many viruses have a tissue tropism, and must therefore be introduced into a specific site for growth.
• Viruses can be cultivated in various parts of the egg like the chorioallantoic membrane, allantoic cavity, amniotic sac, and yolk sac.
• Good pasture in 1931 first used the embryonated hen’s egg for the cultivation of virus.
• Viruses are inoculated into chick embryos of 7-12 days old.
• For inoculation, eggs are first prepared for cultivation, the shell surface is first disinfected with iodine and penetrated with a small sterile drill.
• After inoculation, the opening is sealed with gelatin or paraffin and incubated at 36°c for 2-3 days.
• After incubation, the egg is broken and the virus is isolated from the tissue of the egg.
• Viral growth and multiplication in the egg embryo are indicated by the death of the embryo, embryo cell damage, or by the formation of typical pocks or lesions on the egg membranes
• Viral infection may damage tissue membranes, producing lesions called pox; disrupt embryonic development; or cause the death of the embryo.

Advantages:

1. Widely used method for the isolation of virus and growth.
2. Ideal substrate for the viral growth and replication.
3. Isolation and cultivation of many avian and few mammalian viruses.
4. Cost effective and maintenance is much easier.
5. Less labor is needed.
6. The embryonated eggs are readily available.
7. Sterile  and wide range of tissues and fluids
8. They are free from contaminating bacteria and many latent viruses.
9. Specific and non specific factors of defense are not involved in embryonated eggs.
10. Widely used method to grow virus for some vaccine production.

Disadvantages:

1. The site of inoculation for varies with different virus. That is, each virus have different sites for their growth and replication.

The anatomy of a ten-day-old embryonated egg (left) and the different sites of virus inoculation (right)

Chorioallantoic Membrane (CAM):

• After incubation and incubation, viral growth and replication are indicated by visible lesions called pocks (single virion) are observed, which is a grey-white areas in transparent CAM.
• Herpes simplex virus is also grown.
• The single virus gives single pocks
• This method is suitable for plaque studies.
• Inoculation is mainly for growing poxvirus but other viruses like Herpes simplex virus, Rous sarcoma virus, Canine distemper virus, and Infectious Bronchitis virus.

Amniotic Cavity

• Inoculation is mainly done for primary isolation of influenza virus and the mumps virus.
• Growth and replication of virus in egg embryo can be detected by haemagglutination assay.
• Age of egg at inoculation – 9-10 days.

Allantoic Cavity

• 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 popular and simple method for viral inoculation.
• Allantoic inoculation is employed for the growth and replication of the influenza virus for vaccine production.
• This method provides a rich yield of influenza and some paramyxoviruses.
• Other allantoic vaccines include Yellow fever and rabies vaccines.
• Duck eggs provide a better yield of rabies virus and are used for the preparation of the inactivated non-neural rabies vaccines. But they need a longer incubation period than embryonated hen’s egg.
• Most of avian viruses can be isolated using this method.
• Age of egg at inoculation – 10-12 days
• Viruses that can be inoculate via this method include influenza viruses, Yellow fever virus, Rabies virus, Mumps virus, Newcastle disease virus and Avian adenovirus

Yolk Sac

• Is the simplest method for growth and replication of many viruses.
• Mostly mammalian viruses are isolated using this method.
• This method is also used for the cultivation of some bacteria like Chlamydiae and Rickettsiae.
• Immune interference mechanism can be detected in most of avian viruses.

Routes of virus inoculation and the viruses inoculated

Candling of eggs

• Egg candling is when we shine a bright light into an egg to see inside it.
• Candling eggs is a straight-forward process, you can even use a household torch, but it helps to have some pictures to know what you’re looking for inside the egg.
• The term ‘egg candling’ comes from the past before electric light bulbs when people used candles for light. Poultry breeders would place a candle inside a wooden box and cut a hole big enough to allow light to shine through the egg but small enough to stop the egg from falling into the box.
• We candle eggs before incubation to look for cracks. These allow bacteria to enter during incubation. Cracked eggs are likely to become infected, causing the embryos to die prematurely.
• During incubation, candling an egg allows us to see the developing embryo inside the shell and provides a way to remove non-viable eggs (infertile or early death), which will potentially become rotten.
• A ‘viable egg’ is fertile, and we hope it will develop into an embryo in the incubator and eventually hatch into a chick.
• After virus inoculation, we can use egg candling to monitor the effects of infection to the growing embryo (pocks, haemorrhages or embryo death).

Drilling and virus inoculation into embryonated chicken eggs

Procedure of Allantoic Cavity Inoculation (for propagation of influenza virus)

1. Take pathogen-free fertilized chicken eggs of 11-12 days.
2. 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
3. Make a small nick in the marked area using a jeweler’s scribe.
4. 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.
5. Inoculate the eggs using a tuberculin syringe (a 1 ml syringe fitted with a 1/2 inch, 27 gauge needle).
6. 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.
7. Seal these two holes of the shell with melted paraffin.
8. Incubate the eggs at 37 degrees C for 48 hours.

Methods for examination

Inoculated embryonated egg is examined by candling daily for the presence of the virus by:

• Death of the embryos (Togaviruses)
• Deformities of the embryos (Infectious bronchitis virus-IBV)
• Haemorrhages of the embryos (NDV)
• Oedema and necrotic foci ‘pocks’ on the CAM (poxviruses and herpes B virus)
• Histopathological sections- intranuclear or intracytoplasmic inclusion bodies (Herpes virus)
• Serological tests- presence of viral antigens in infected eggs fluid- HI, VNT
• Pock lesions on CAM
• 

Harvesting the virus in the allantoic cavity

• 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.

Harvesting embryo