Virus Culture in Embryonated Egg

Embryonated Egg culture Technique for Viruses

Goodpasture and Burnet in 1931 first used the embryonated hen’s 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

Terms most often refer to eggs:

•      Embryonated: having an embryo,

•      Unembryonated: not having an embryo

•      De-embryonated: having lost an embryo

Embryonated egg are refered 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.

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

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 increase in size as the embryo grows. 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.

An embryonated egg offers various sites for the cultivation of viruses;

1.      Chorioallantoic membrane(CAM)

2.      Amniotic Cavity

3.      Allantoic Cavity

4.      Yolk sac

Figure 1a & 1b: An Embryonated egg showing sites of 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: It 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.

Figure 2a & 2b: Candling of egg to observe embryo

1. 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

2. Amniotic Cavity: 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. It 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.

3. Allantoic Cavity: 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 (8–15 ml) of virus-infected egg fluids.

4. Yolk sac: 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.