Culture of Virus in Chick Embryo and Cell Lines

Cultivation of viruses

As virus being an obligate parasite, they cannot be grown in inanimate culture medium; they can be grown in animal inoculation, embryonated eggs, and cell culture. The valuable and widely used medium for the isolation and subsequent passage of many viruses for stock cultures as well as for the production of vaccines is done in the avian embryo, especially the chicken embryo. Chicken embryos are used almost preferred because of:

• availability
• economy
• convenient size
• relative freedom from latent infection and extraneous contamination
• Lack of production of antibodies against the viral inoculums.

The healthy and disease-free flocks should be used from an embryonated egg. Some of the factors influencing the growth of viruses in chicken embryos are:

• Age of the embryo
• Route of inoculation
• Concentration of virus and volume of inoculums
• Temperature of incubation
• Time of incubation following inoculation.

The presence of maternal antibodies in the yolk of egg immunized against viral infections. Different way of which virus can be cultivated are as follows:

Animal inoculations

This is the earliest method of cultivation of viruses that causes human diseases. Generally, the infant (sucking mice) are very susceptible to most if the viruses. The mice may be inoculated by several routes. For example intracerebral, subcutaneous, and intraperitoneal. Beside mice, others are guinea pigs, rabbits, and some rodents are also being used in some conditions. The growth of viruses in inoculated animals may be indicated by death diseases or visible lesson. The animal inoculation for the cultivation of viruses is also used for the study of pathogenesis, immune response as well as epidemiology.

2. Inoculation in embryonated egg

An Embryonated egg was first used for cultivation of viruses by Good Pasture (1981). Embryonated egg cultivation is most commonly used for growth of influenza viruses. However, it offers several sites for cultivation of viruses while cultivation in embryonated eggs. The eggs are first prepared for cultivation by using surface disinfectant like iodine. The egg is placed in front of the light source to locate the non-veined area of the allantoic cavity just below the air sac. This is then marked well with a pencil. A small nick is made in the shell at this position and next a hole is drilled at the top of the egg. If this is not done the pressure in the air sac will simply force out the inoculums.

After all the eggs have been nicked and drilled, they are inoculated with viruses using a tuberculin syringe which is fitted with a half inch, 27 gauze needle. The needle passes through the hole in the shell through the chorioallantoic membrane and the virus is placed in the allantoic cavity (considering influenza as an example). The two holes in the shells are sealed with melted paraffin and the egg is placed at 37C for 48hrs. During the incubation period, the virus replicates in the cell of chorioallantoic the membrane. As the virus particles are produced they are released into an allantoic fluid.

Some viruses may produce visible lesions of pocks. The morphology of pock is different in different viruses, so pocks counting can be used for the assay of viruses.

There are various sites for inoculation in embryonated egg depending upon the virus. They are as follows:

• Chorioallantoic membrane inoculation employs 10 to 12-day old embryos and inoculums of 0.1-0.5 cc. This route is particularly effective for primary isolation and cultivation of the viruses of HSV, variola, vaccinia, fowlpox, and Pox virus
• Amniotic inoculation is done for Influenza and Mumps virus and inoculation in embryos is done from 7 to 15days incubation.
• Yolk sac inoculation is mostly done for HSV and be also used for initial isolation of mumps virus. Inoculation is performed with 5 to8 day old embryos and inoculum of 0.2-1.0 cc.
• Allantoic inoculation is preferred for Influenza, Mumps, and Avian adenovirus. This inoculation is useful for inoculation and collection of specimens when large quantities of virus-infected fluid are to be obtained for use in vaccine production, chemical analysis, and as well as for preparation of antigen for serologic tests.

Advantages

• Embryonated egg is the ideal substrate for growth and multiplication of viruses.
• Cost effective and maintenance is much easier.
• Embryonated eggs are readily available.
• They are free from contaminating bacteria and latent viruses.

Disadvantages

• The site of inoculation varies with different viruses.

Tissue culture

Cultivation of viruses is tissues, organs or cell is carried out to study the physiological and morphological phenomenon of viruses. The tissue culture of viruses is mainly carried out in 3 different forms:

Organ culture

In this case, small bits of organs are used for isolation of highly specialized parasites of certain organs for example tracheal organs culture is employed for the isolation of corora viruses, a respiratory pathogen.

Explant culture

Fragments of tissue can be grown as explants embedded in the plasma clots. However, this method is new seldom use.

Cell culture

It is the process by which cells are grown under the controlled condition. Cells are grown in vitro on glass or treated plastic surfaces in a suitable growth medium. The components in the growth medium include 13 amino acids, 9 vitamins, salts, glucose, a buffering system (generally bicarbonate buffer), fetal path, serum, phenol red, indicator etc.

Based on their origin, cell culture can be divided into 3 types:

• Primary cell culture

This is derived directly from excised normal animal tissue. They are capable of limited growth and cannot be maintained in the culture. For example Rhesus Monkey kidney cell culture, chick embryo, fibroblast cell culture etc.

• Semi-continuous culture

They can be sub-culture up to 50 times by serial transfer of inoculums. They have used for isolation of some fastidious viruses and production of viral vaccines. Such as Human Embryonic Tissue culture cell.

• Continuous cell culture

These are cells of a single type derived from cancer cells that are capable of series cultivation for several times. This cell grows faster and can be used for many different research purposes. Many laboratories have been using a cell line which has undergone several hundred passages. These cell lines may be maintained by several sub-cultivations and stored in cold (-70C) for use when necessary. These are not used for the preparation of a viral vaccine.

Example Hela cells, BHK 21, HEP 1(Human epithelioma)

Hela cell lines

A HeLa cell line is immortal cell line used for virus cultivation. It is oldest and most commonly used cell lines. The cell line was derived from cervical cancer cells and the name. Hela came from initial letters of a patient named Henrietta lacks.

The stable growth of HeLa cell line enabled a research successfully to grow polio virus and enabling the development of a vaccine by Salk for polio using these cells. Hela cells were the first human cell to be cloned. Hela cells have also been used in research of cancer AIDS, the effect of radiation and toxic substance, gene mapping and many other scientific purposes. The cell lines are also used in the study of sex steroid hormone including estrogen and others.

REFERENCE

Cheesbrough, M. Medical Laboratory Manual for Tropical Countries. Vol. Vol 2. ELBS London, 2007.

Tille, P. Diagnostic Microbiology. 13th. Elsevier, 2014.

D, Grenwood, Slack RCB and Peutherer J. Medical Microbiology. Dunclude Livingstone: ELBS, 2001.