CULTIVATION OF VIRUS : Embryonated eggs

CULTIVATION OF VIRUS : Embryonated eggs

1. C ULTIVATION OF VIRUS : Embryonated eggs

2. Introduction 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. In vivo-laboratory bred animal and embryonic bird tissue In vitro-cell or tissue culture method The primary purpose of virus cultivation is: To isolate and identify viruses in clinical samples. To do research on viral structure, replication, genetics and effects on host cell. To prepare viruses for vaccine production. 2

3. Cultivation of viruses Inoculation into embryonated egg Woodruff and Goodpasture (1931)used fertilized chicken egg for viral cultivation. This is a simpler technique than animal inoculation ,are inexpensive and easily available . Eggs usually not interfere with virus multiplication due to absence of immune response. Suitable cells for the growth of viruses are available in embryo and its membrane ,which may facilitate the growth of virus. Tissue Culture Cultivation of tissue or organ for the growth of viruses. Cell culture is most widely used in diagnostic virology for cultivation and assays of viruses. 3 Animal inoculation Susceptible experimental animals like Mice, Monkey, Rabbits, Guinea Pigs etc. are used for the cultivation of viruses. Virus sample to be cultivated should be injected into the experimental animal. Intracerebral, subcutaneous, intraperitoneal, or intranasal routes are various routes of inoculation. After inoculation, the animals are observed for signs of disease or death.

4. Embryonated eggs Woodruff and Goodpasture in 1931 first used the embryonated hen’s egg for the cultivation of virus. The method further developed by Burnet was used for cultivation of viruses in different sites of the egg. 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 stocks of some viruses in research laboratories and for vaccine production. Viruses can be cultivated in various parts of egg like Chorioallantoic membrane, Allantoic cavity, Amniotic sac and Yolk sac. 4

5. Continue…. Viruses are inoculated into chick embryo of 7-12 days old. For inoculation, eggs are first prepared for cultivation, the shell surface is first disinfected with iodine, checked in ovoscope if it is alive 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 virus is isolated from tissue of egg. Viral growth and multiplication in the egg embryo is indicated by the death of the embryo, by embryo cell damage, or by the formation of typical pocks or lesions on the egg membranes 5

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7. Chorioallantoic Membrane (CAM) Inoculation is mainly for growing poxvirus. After incubation, visible lesions called pocks are observed, which is grey white area in transparent CAM. Each infectious virus particle produces one pock. The morphology of the pocks may vary depending on the nature of the virus. The pox viruses, such as variola or vaccinia are identified by demonstration of typical pocks on the CAM inoculated with the pox virus. Herpes simplex virus is also grown. This method is suitable for plaque studies. Nowadays, in a virology laboratory, chick embryo inoculation has been replaced by cell cultures for routine isolation of viruses. 7

8. Allantoic cavity Is the most popular and simple method for viral inoculation. Inoculation in the allantoic cavity is used for serial passages and for obtaining large quantities of virus, such as influenza virus, yellow fever, and rabies 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. But they need a longer incubation period than embryonated hen’s egg. Most of avian viruses can be isolated using this method. 8 Amniotic sac 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.

9. Yolk sac inoculation: It is also a simplest method for growth and multiplication of virus. Mostly mammalian viruses are isolated using this method. Yolk sac inoculation is used for cultivation of Japanese encephalitis, Saint Louis encephalitis, and West Nile virus. It is inoculated for cultivation of some viruses and some bacteria (Chlamydia, Rickettsiae ) Immune interference mechanism can be detected in most of avian viruses. 9

10. Procedure : 1. Candling : For propagation of influenza virus, pathogen-free eggs are used 11-12 days after fertilization. The egg is placed in front of a light source to locate a non-veined area of the allantoic cavity just below the air sac. This is marked with a pencil. After all the eggs have been ‘candled’ in this way, a small nick is made in the shell at this position using a jeweler’s scribe. 10

11. Procedure : 2. Drill the hole : Next, a hole is drilled at the top of the egg with a Dremel motorized tool. If this is not done, when virus is injected, the pressure in the air sac will simply force out the inoculum. 11 Drilling pin- point hole.

12. Procedure : 3. Inject the specimen : After all the eggs have been nicked and drilled, they are inoculated with virus using a tuberculin syringe. The needle passes through the hole in the shell, through the chorioallantoic membrane, and the virus is placed in the allantoic cavity, which is filled with allantoic fluid. 4. Hole sealed with paraffin wax: The two holes in the shell are sealed with melted paraffin, 5. Incubation : The eggs are placed at 37 degrees C for 48 hours. 12

13. Advantages Cost effective and maintenance is much easier. Less labor is needed. The embryonated eggs are readily available. Sterile and wide range of tissues and fluids They are free from contaminating bacteria and many latent viruses. Defense factors are not involved in embryonated eggs. Widely used method to grow virus for some vaccine production Chick embryos are packed in their shells and have natural resistant against bacterial contamination. Chick embryo method is cheaper and easy to handle. 13 Disadvantages The site of inoculation for varies with different virus. That is, each virus have different sites for their growth and replication. Some viruses do not show growth on primary inoculation into the eggs. Slight amount of bacterial contamination in the inoculum may kill the embryo.

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