Cultivation and Preparation of the Virus in Chick Embryo Virtual Lab Simulation

General Aim of Cultivation of Viruses in Embryonated Eggs

The primary purpose of virus cultivation in egg is: Identification and isolation of viruses in clinical samples. To do research on viral structure, replication, genetics and effects on host cell. To prepare viruses for vaccine production.

Method of Isolation of Viruses

In preparation and cultivation of viruses in chick embryo experiment: 1.Fertile eggs are cleaned, dried, rubbed with alcohol. 2.The date is recorded on each egg. 3.Eggs are incubated at 37 °C for 7-12 days. 4.Eggs are gently rocked daily. 5.Be sure of the growth of the embryo (beating heart) by examining the egg against a strong light in a dark chamber. 6.Two points are marked on each egg under transillumination; one of them delimiting the air sac and the other opposite the semitransparent area in the neighborhood of the embryo. 7.The inoculum is introduced through an opening made in the shell using the egg shell puncher. 8.There are five main routes of virus inoculation using a syringe containing the virus: a. Into the chorioallantoic membrane. b. Into the amniotic cavity. c. Into the allantoic cavity. d. Into the yolk sac. e. Into the embryo itself. 9.After inoculation, the eggs are reincubated at 37oC for several days and examined daily. 10.Virus growth is recognized by the development of: Plaques on the chorioallantoic membrane. Haemagglutinin in the amniotic or allantoic cavities. Death of the embryo. (heart beats stop) just this will be showed in the experiment.

Virus Cultivation In EggTheory

Isolation of virus is always considered as a gold standard for establishing viral etiology of a disease. Most of the viruses can be cultivated in:

• Experimental animals
• Embryonated eggs 
• Tissue culture

Experimental animals are rarely used for cultivation of viruses but play an essential role in study of pathogenesis of viral infections and that of viral oncogenesis.

Embryonated chick egg was used first for cultivation of viruses by Goodpasture in 1931.

The method further developed by Burnet was used for cultivation of viruses in different sites of the embryonated egg.

Usually, 8–11 days’ old chick eggs are used for culture of viruses.

The viruses are isolated in different sites of the egg, such as yolk sac, amniotic cavity, and allantoic cavity, and chorioallantoic membrane (CAM).

Many of these viruses cause well-defined and characteristic foci, providing a method for identification, quantification, or assessing virus pathogenicity.

The embryonated egg is also used for growing higher titre stocks of some viruses in research laboratories and for vaccine production.

Yolk sac: Yolk sac inoculation is used for cultivation of Japanese encephalitis, Saint Louis encephalitis, and West Nile virus. It is also used for growth of chlamydia and rickettsia.

Amniotic cavity: Inoculation in the amniotic cavity is used mainly for primary isolation of influenza virus.

Allantoic cavity: Inoculation in the allantoic cavity is used for serial passages and for obtaining large quantities of virus, such as influenza virus, yellow fever (17D strain), and rabies (Flury strain) viruses for preparation of vaccines. For production of rabies virus, duck eggs were used due to their bigger size than that of hen’s egg. This helped in production of large quantities of rabies virus, which are used for preparation of the inactivated non-neural rabies vaccine.

Chorioallantoic membrane: Inoculation of some viruses on chorioallantoic membrane produced visible lesions known as pocks. Each infectious virus particle produces one pock. The pox viruses, such as variola or vaccinia are identified by demonstration of typical pocks on the chorioallantoic membrane inoculated with the pox virus. Nowadays, in a virology laboratory, chick embryo inoculation has been replaced by cell cultures for routine isolation of viruses.

 

Virus Isolation Protocol

Viruses are obligate intracellular parasites so they depend on host for their survival. They cannot be grown in non-living culture media or on agar plates alone, they must require living cells to support their replication.
The primary purpose of virus cultivation is:

1. To isolate and identify viruses in clinical samples.
2. To do research on viral structure, replication, genetics and effects on host cell.
3. To prepare viruses for vaccine production.