Objectives:
To study
the different routes of viral inoculation in the embryonated eggs.
Principle:
General Properties of Virus:
Viruses do not fall in the category of unicellular microorganism.
They lack cellular organizations and contain only one type nucleic acids,
either DNA or RNA.Viruses are obligate intracellular parasites and lack the
enzyme necessary for protein and nucleic acid synthesis. They depend on the
host machinery for their growth and survival. Unlike other microorganism,
complex processes are involved in their multiplication. Outside of the host
cells, viruses are inactive. However, inside living cells, viruses show some of
the characteristics of living things. Viruses are of medical importance because
they have the ability to cause a very large number of human diseases. Virus
diseases range from the minor common cold to sporadic and endemic diseases such
as mumps, hepatitis and so on.
Methods
for Cultivation of Virus:
Since the viruses are obligate intracellular parasites, they cannot be grown on any inanimate culture medium. Viruses can be cultivated within suitable hosts, such as a living cell. Generally three methods are employed for the virus cultivation.
1. Inoculation of virus into animals.
2.
Inoculation of virus into embryonated eggs.
3.
Tissue culture.
noculation
of Virus into Embryonated eggs
Prior to the advent of cell culture, animal viruses could be propagated only on whole animals or embryonated chicken eggs. Good pasture in 1931first 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 which is used. The egg used for cultivation must be sterile and the shell should be intact and healthy. A hole is drilled in the shell of the embryonated egg, and a viral suspension or suspected virus- containing tissue is injected into the fluid of the 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. An embryonated egg offers various sites for the cultivation of viruses. The different sites of viral inoculation in embryonated eggs are:
1. Chorioallantoic membrane(CAM)
2.
Amniotic Cavity
3.
Allantoic Cavity
4. Yolk
sac
Chorioallantoic Membrane(CAM) is mainly employed in the growth of poxvirus. Virus growth and replication in the CAM is indicated by visible lesions (pocks); grey white area in transparent CAM. Herpes simplex virus is also grown. Each pock is derived from a single virion. The morphology of the pocks may vary depending on the nature of the virus. Under optimal conditions, each infectious virus particle can form one pock. Hence this method is suitable for plaque studies. Herpes simplex virus can also be inoculated via CAM.
Allantoic Cavity 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 will provide 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 were 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.
Amniotic Cavity: The amniotic sac is employed inoculated for primary isolation of influenza a virus and the mumps virus. Growth and replication of virus in egg embryo can be detected by haemagglutination assay.
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.
Materials Required:
1. Eggs: 9-day old or 10-day old embryonated
eggs.
2. Egg
shell punch/Carborundum disc
3.
Cotton
4.
Spirit
5. 1ml
disposable syringe
6.
Stationery tape (also called cello or sticky tape)
7.
Viral suspension in Beijenox bottle
8.
Biohazard
Procedure:
Swab the
end of the eggs to be inoculated with 70% ethanol. Allow the alcohol to
evaporate.
Place
used cotton wool in discard tray.
Candle
the egg with high intensity LED torch and mark “X” over the embryo’s eye.
Draw a
line on the shell marking the air space.
With a
sterile egg shell puncher or carborundum disc pierce a hole in the end of the
egg at the marked inoculation site.
Attach a
needle to 1 ml syringe.
Draw
0.1ml of inoculums (Viral suspension) into 1 ml syringe.
Place
the needle through the hole in the eggshell keep the needle and syringe
vertical. The needle will need to penetrate approximately 16 mm into the egg to
reach specific site of inoculation.
Inject
0.1 ml of inoculums into the egg.
Withdraw
the needle from the egg.
Seal the
hole in the shell with stationery tape or melted wax.
Discard
the used needles and syringes in a biohazard box.
Place
the inoculated eggs into a second incubator. Check the temperature and humidity
of incubator.
In the
case of amniotic inoculation, when the needle reaches the embryo a thrust will
strike the embryo and the embryo will move away from the needle. Then inject
the virus suspension.
Expected Results:
The virus suspension are succesfully inoculated into the eggs. The
growth of the virus can be detected by visible pocks in the egg.
Courtesy: http://vlab.amrita.edu/?sub=3&brch=76&sim=1223&cnt=2
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