Cultivation of viruses in chicken embryos. Cultivation on chicken embryos Infection in blood vessels CAO

Most known viruses have the ability to multiply in the chicken embryo (Fig. 4). Embryos aged from 8 to 14 days are used, depending on the type of virus, method of infection and research objectives. Influenza viruses are cultivated in 9-10 days, smallpox vaccines - in 12 days, mumps - in 7-day chicken embryos. Reproduction of the virus in chicken embryos occurs in different parts of the embryo, which is due to the characteristics of the tropism of the virus. The technique of growing a virus in a chicken embryo is widely used in industrial cultivation.

The structure of a chicken embryo and methods of its infection: 1 - into the amnion; 2 - into the allantoic cavity; 3 - into the yolk sac. (Microbiology and immunology. Edited by Vorobyov A.A. - M. - 1999).

The outermost extraembryonic membrane, adjacent to the shell or maternal tissues and therefore serving as a place of exchange between the embryo and its environment, is called Chorion. In egg-laying species, the main function of the chorion is to carry out respiratory gas exchange. In mammals, the chorion is involved in respiration and nutrition, excretion, filtration and synthesis of substances. In primitive organisms, the chorion is a secondary membrane, and in advanced organisms it is the membrane of the fetus. The cavity between the chorion and amnion is the chorioamniotic cavity.

Amnion(Greek Amnion), amniotic sac or aqueous membrane - one of the embryonic membranes in the embryos of reptiles, birds, and mammals.

Evolutionarily, the amnion arose to protect embryos from drying out during development outside the aquatic environment. Therefore, vertebrate animals that lay eggs (reptiles and birds), as well as mammals descended from reptiles, are classified as amniotes (“Animals with egg membranes”). The previous classes and superclasses of vertebrates (cephalochordates, cyclostomes, fish, amphibians) lay eggs in the aquatic environment, so they do not require an aquatic shell. These classes of animals are combined into the group anamnia. Unlike anamnia, amniotes do not require an aquatic environment for reproduction and early development, therefore amniotes are not attached to bodies of water. This is the evolutionary role of the amnion.

During the birth of mammals, the water membrane bursts, the water flows out, and the remains of the amnion on the body of the newborn are often called the “shirt,” which has been a sign of good luck and other superstitions everywhere since ancient times (hence, in particular, the Russian proverb about those who were “born in a shirt” ).

Allantois (from the Greek allantoeid?s - sausage-shaped) is the embryonic respiratory organ of higher vertebrates; germinal membrane developing from the hindgut of the embryo. In addition, the allantois is involved in the gas exchange of the embryo with the environment and the release of liquid waste. The allantois, together with other embryonic membranes - amnion and chorion, is a defining feature of higher vertebrates - mammals, birds and reptiles.

In oviparous birds and reptiles, the allantois develops around the embryo along the walls of the shell. In its outer layer, called mesoderm, it creates an extensive network of blood vessels through which it interacts with the external environment. In mammals, the allantois is part of the umbilical cord.

There are several ways to infect a developing chicken embryo: on the chorioallantoic membrane, in the allantoic and amniotic cavities, the yolk sac, and the body of the embryo.

Infection of the chorioallantoic membrane is used to isolate and cultivate viruses that form plaques on the membranes (vaccine viruses, smallpox, herpes simplex). Before infection, the eggs are examined using an ovoscope, and the boundary of the air space and the chorioallantoic membrane is outlined with a pencil. The surface of the egg above the air space and at the site of infection is wiped with alcohol, burned, treated with iodine and a hole is made in the cavity of the air sac.

At the site of infection, the shell is removed so as not to damage the subshell membrane, which is then pierced with a short sterile needle so as not to damage the chorioallantoic membrane. The air from the cavity of the air sac is sucked out. Viral material (0.05 - 0.2 ml) is applied to the chorioallantoic membrane with a tuberculin syringe with a short needle or Pasteur pipette. The hole in the shell is covered with a sterile cover glass or the same sawed-out piece of the shell and the edges are filled with molten paraffin. Infected embryos are placed horizontally on a stand and incubated in a thermostat. The embryos are dissected no earlier than 48 hours of incubation. On the infected membrane, whitish opaque spots of various shapes (plaques) are found.

Infection in the allantoic cavity. The virus introduced into the allantois multiplies in the endodermal cells, then passing into the allantoic fluid. Infection is carried out in the following way: a puncture is made in the shell above the air chamber with the tip of a scalpel or scissors, after which a needle with a syringe is inserted through the hole in a vertical direction, which passes through the chorioallantoic membrane and enters the allantoic cavity, the material is injected in a volume of 0.1 ml and the hole filled with paraffin.

Infection in the yolk sac. For this purpose, embryos 5 to 10 days old are used. The most common are two methods of infection. According to the first, the material is introduced through the air space. A hole is made in the center of the egg, it is placed on a stand with the blunt end to the right, and a needle attached to a syringe is inserted through the hole in a vertical direction; the needle passes through the chorioallantoic membrane, the allantoic cavity into the yolk. From 0.1 to 0.5 ml of virus-containing material can be injected into the yolk sac. After infection, the hole in the shell is filled with paraffin and the embryo is placed in a thermostat. According to the second method, at the border of the air space on the side where the yolk lies (the side opposite from the embryo), a puncture is made in the shell, through which infectious material is introduced. The direction of the needle should be towards the center of the egg.

A chicken embryo infected with viral material is placed in an incubator for 2-3 days, depending on the nature of the introduced virus. For the development of viruses from samples taken from a patient (for example, nasopharyngeal swabs used to diagnose influenza), the chicken embryo provides a good environment. By the type of changes, say, in the tissues of the chorioallantoic membrane, we can directly determine what kind of virus we are dealing with. Smallpox and herpes viruses produce very characteristic changes. Some viruses multiply very intensively in various tissues of the chick embryo and provide the starting material for the preparation of viral antigens necessary for the laboratory diagnosis of viral diseases. Viruses propagated in chicken embryos were used as starting material to produce eighteen types of vaccination vaccines.

Despite the positive aspects of cell cultures, which have become so widespread in modern virology laboratories, the chicken embryo in many cases has retained its primacy and continues to serve as a classic material for work. Indication of the virus in a chicken embryo is made by the death of the embryo, a positive hemagglutination reaction on glass with allantoic or amniotic fluid, by the formation of focal lesions (“plaques”) on the chorion-allantoic membrane, as well as in the RHA.

Many viruses that infect humans and animals can multiply to a greater or lesser extent in the chick embryo. The presence of a dense shell protects the embryo from microorganisms from the external environment.

The method of cultivating viruses in chicken embryos is used in the laboratory diagnosis of viral infections, as well as for the production of viral vaccines and diagnostic drugs. But this method has disadvantages: 1) it is impossible to observe in dynamics the pathological changes occurring in the embryo after infection with the virus; 2) when opening an embryo infected with a virus, often no visible changes are detected and it is necessary to detect the presence of the virus in the tissues and fluids of the embryo using other virological methods (for example, the hemagglutination reaction); 3) the method of cultivation in chicken embryos is not suitable for all viruses. Despite the existing disadvantages, the method is relatively simple, convenient and cheap and is widely used in virological studies. It is most important when working with orthomyxoviruses, herpesviruses, and poxviruses.

1.3.1. The structure of a chicken embryo

The chicken embryo is covered with a calcareous shell - a shell, to which the shell membrane is adjacent from the inside. At the blunt end of the egg it bifurcates and contains an air space. Under the shell membrane there is a chorioallantoic membrane; at the blunt end of the egg it runs along the inner side of the shell membrane, enclosing the air space; this membrane is rich in blood vessels and serves as a respiratory organ for the embryo. Adjacent to it from the inside is the allantoic cavity, which is an excretory organ and protects the embryo from drying out and injury. The allantoic cavity surrounds the embryo located in the amnion cavity, which is filled with amniotic fluid. Through the yolk cord, the embryo is connected to the yolk sac, the main source of nutrients.

For the successful cultivation of viruses in the body of developing chicken embryos, a certain temperature regime (36 0 -38 0), humidity (50-70%), as well as sufficient ventilation are required. Chicken embryos of a certain age are infected, incubated for 6 to 13 days, depending on the type of virus and method of infection. It is necessary to prepare: an egg stand, vials of alcohol and iodine, a test tube with sterile paraffin, cover slips, bags of sterile cotton wool and gauze, sterile containers wrapped in paper, sterile syringes, needles, tweezers, dissecting needles. The instruments are placed in a glass of alcohol, where they remain throughout the entire work; before each manipulation, they are additionally sterilized by firing in a burner flame. Hands are washed thoroughly before work; it is recommended to wear a gauze mask.

Viable embryos are selected for work by examining the incubated eggs in an ovoscope. A viable embryo is mobile, the blood vessels of the membrane are filled with blood. Selected eggs are thoroughly disinfected at the blunt end (or on the side of the egg): the shell is wiped with alcohol, lubricated with iodine, re-treated with alcohol and fired.

1.3.2. Infection of the chick embryo on the chorioallantoic membrane

Chicken embryos 10-12 days old are used for infection. Main stages of infection:

1. The egg is placed on a stand in a vertical position so that the air bag is at the top, and the shell is sterilized at the blunt end of the egg.

2. A puncture of the shell is made above the center of the air sac using a dissecting needle.

3. The jaw of the scissors is inserted into the resulting hole and a window about 1.5 cm in diameter is cut out in the shell.

4. Through the hole, carefully tear the inner leaf of the shell with a needle and peel off in a small area (0.5-1 cm 2).

5. The chorioallantoic membrane is infected by applying 0.1-0.2 ml of virus-containing material to it using a Pasteur pipette or syringe.

6. The window in the shell is covered with a special elastic film or a sterile cover glass and attached with molten paraffin.

Infected embryos are placed in a thermostat in a vertical position and incubated for 2-3 days, after which an autopsy is performed according to the following rules:

1. The egg is placed on a stand so that the air space is at the top, and the opening site is sterilized.

2. Using sterile scissors, cut off the shell along the border of the air space.

3. Using tweezers, remove the shell along the border. The exposed chorioallantoic membrane is trimmed along the edge of the shell. Through the resulting hole, pour the entire contents of the egg into a cup or tray.

4. The chorioallantoic membrane remaining inside the shell is carefully removed with tweezers and placed in a sterile cup with saline solution. Here they straighten it and study the changes by placing the cup on a dark background.

To obtain material containing the virus from the chorioallantoic membrane, it must be crushed with scissors and then ground in a mortar with quartz glass, adding a saline solution. The resulting suspension is centrifuged at 2000 rpm for 10-15 minutes and the supernatant is used as a virus-containing material.

Rice. 3. Infection of chicken embryos

1.3.3. Infection in the allantoic cavity

Embryos of 10-11 days of age are taken for infection. Main stages of infection:

1. The egg is placed on a stand with the blunt end up and the shell is sterilized over the air space.

2. A puncture of the shell is made in the center of the blunt end using a dissecting needle.

3. The needle of a syringe containing a dilution of the virus is inserted into the hole. The needle is advanced in a vertical direction 2-3 mm below the level of the air sac and then 0.1-0.2 ml of material is injected.

4. The hole in the shell is sealed with melted sterile paraffin.

Infected embryos are kept in a thermostat usually for 2 days. Before opening, the eggs are placed in the refrigerator overnight at 4 0 C. Opening is carried out in the following order:

1. The egg is placed on a stand so that the air bag is at the top, the shell above it is sterilized.

2. Using scissors, the shell is cut off slightly above the border of the air space.

3. Carefully remove the shell membrane with tweezers, after which the chorioallantoic membrane is pierced with a Pasteur pipette in the place where there are no vessels, and the allantoic fluid is sucked in.

4. The allantoic fluid is transferred to a sterile test tube, and part of it is inoculated into the broth to test for bacteriological sterility.

Embryos are used at the age of 8 to 14 days, depending on the type of virus and method of infection. Reproduction in chicken embryos occurs in different parts of the embryo. Methods of infection: on the chorioallantoic membrane, in the allotonic and amniotic membrane, yolk sac, embryo body.

29.Tissue cultures.

Depending on the preparation technique, there are 3 types of cells:

Single-layer glass can reproduce on the surface of chemically neutral glass in the form of a monolayer;

Suspension, spreading throughout the entire volume of the nutrient medium;

Organs are whole pieces of organs and tissues that retain their original structure.

The preparation of a primary cell culture consists of several stages: grinding the tissue, separating the cells, and washing the resulting suspension from trypsin.

Indication:

- cytopathic effect - morphological changes in cells visible under a microscope, up to rejection from the glass.

Viral inclusions are an accumulation of viral particles or separation of viral components in the cytoplasm or nucleus of cells.

Plaques are limited areas consisting of degenerative cells. They are visible as light spots against the background of colored cells.

Color test

Hemadsorption is the ability of cell cultures to adsorb red blood cells on their surface.

Interference.

30. Classification, chemical composition of bacteriophages.

Bacteriophages are bacterial viruses that have the ability to penetrate bacterial cells and cause their dissolution. They have a tadpole shape, some are cubic thread-like. They consist of an icosahedral head and a tail. Inside the caudal process there is a cylindrical rod, outside there is a sheath, the process ends in a hexagonal basal plate with short spines. Phages are composed of nucleic acid and protein. In sperm-shaped phages, double-stranded DNA is tightly packed in a helix inside the head. Proteins are part of the shell. In addition to structural proteins, internal proteins associated with nucleic acid and enzyme proteins involved in the interaction of the phage with the cell were discovered.

31. The process of interaction between virulent phages and a bacterial cell sensitive to them

Virulent phages can only increase in number through the lytic cycle. The process of interaction between a virulent bacteriophage and a cell consists of several stages: adsorption of the bacteriophage on the cell, penetration into the cell, biosynthesis of phage components and their assembly, and release of bacteriophages from the cell.

Initially, bacteriophages attach to phage-specific receptors on the surface of the bacterial cell. The phage tail, with the help of enzymes located at its end (mainly lysozyme), locally dissolves the cell membrane, contracts and the DNA contained in the head is injected into the cell, while the protein shell of the bacteriophage remains outside. Injected DNA causes a complete restructuring of the cell's metabolism: the synthesis of bacterial DNA, RNA and proteins stops. The bacteriophage's DNA begins to be transcribed using its own transcriptase enzyme, which is activated after entering the bacterial cell. First, early and then late mRNAs are synthesized, which enter the ribosomes of the host cell, where early (DNA polymerases, nucleases) and late (capsid and tail proteins, enzymes lysozyme, ATPase and transcriptase) bacteriophage proteins are synthesized. Bacteriophage DNA replication occurs according to a semi-conservative mechanism and is carried out with the participation of its own DNA polymerases. After the synthesis of late proteins and the completion of DNA replication, the final process begins - the maturation of phage particles or the combination of phage DNA with the envelope protein and the formation of mature infectious phage particles.

Infection of the chorioallantoic membrane. This method is one of the most commonly used. Its main advantage is that a number of viruses, when multiplying on the chorioallantoic membrane, cause its characteristic changes, forming lesions in the form of whitish spots-plaques of various morphologies.

Embryos 10-12 days old are used for infection. Typically, the chorioallantoic membrane is penetrated by making a hole in the shell above the air sac or on the side of the egg.

The main stages of infection on the chorioallantoic membrane from the air space:

1. The egg is placed on a stand in a vertical position
so that the air bag is at the top)"; carry out
thorough sterilization of the shell at the blunt end of the egg.

2. The shell is punctured above the center of the air sac.
using a dissecting needle.

3. The jaw of the scissors is inserted into the resulting hole and
cut a window in the shell about 1.5 cm in diameter. (Wherein
Do not allow pieces of the shell to get inside the egg).

4. Through the hole made, the inner leaf is visible
shell membrane, it is carefully torn with the eye
tweezers or a needle and peel off in a small area (0.5-1
cm 2), exposing the chorioallantoic membrane.

5. Infects the chorioallantoic membrane
by applying 0.1-0.2 ml of virus-containing material to it
(for example, vaccinia virus) using a Pasteur pipette
or syringe.

6. The window in the shell is closed with a special elastic
film, sterile coverslip or glass
cap. For attaching glass and cap to shell
use molten paraffin.

Infected embryos are placed in a thermostat in a vertical position and incubated for 2-3 days, after which an autopsy is performed according to the following rules:

1. The egg is placed on a stand so that the air
the space was at the top, the place is being sterilized
upcoming opening by appropriate processing
alcohol and iodine.

2. After removing the film, coverslip or cap,
Using sterile scissors, cut off the shell along the border of the air
space.

3. Using tweezers, remove the shell.
The exposed chorioallantoic membrane (which may
be visible changes) are trimmed along the edge of the shell. Through
the hole formed, pour the entire contents of the egg into the cup
or tray.

4. The remaining chorioallantoic tissue inside the shell
the shell is carefully removed with tweezers and placed in
sterile cup with saline solution. Here it is
straighten and study the changes by placing the cup on a dark
background.

To obtain material containing the virus from the chorioallantoic membrane, it must be crushed with scissors and then ground in a mortar with quartz glass, adding a saline solution. The resulting suspension is centrifuged at 2000 rpm for 10-15 minutes and the supernatant is used! as a virus-containing material (mandatory testing for the absence of bacterial contamination is required).

Infection in the allantoic cavity. This method

It is distinguished by its simplicity and is valuable because it contributes to a significant accumulation of the virus. Embryos of 10 days of age are usually taken for infection. The main stages of infection in the allantoic cavity:

}. The egg is placed on a stand with the blunt end up and the shell is sterilized over the air space.

2. In the center of the blunt end, a puncture is made in the shell with
using a dissecting needle.

3. The needle of a syringe containing a dilution is inserted into the hole
virus (such as influenza virus). The needle is advanced into
vertical direction 2-3 mm below the air level
bag and then inject OD-0.2 ml of material.

4. The hole in the shell is sealed using
melted sterile paraffin.

Infected embryos are kept in a thermostat usually for 2 days. Before opening, the eggs are placed in the refrigerator overnight at 4°. The opening is performed in the following order:

1. The egg is placed on a stand so that the air bag is at the top, the shell above it is sterilized;

2. Using scissors, cut the shell a little higher
airspace boundaries.

3. Carefully remove the shell shell with tweezers, then
why the chorioallantoic membrane is pierced with a Pasteur
pipette in the place where there are no vessels, and suck the allantoic
liquid (you can collect 5-6 ml).

4. Allantoic fluid is transferred to a sterile
test tube, and part is inoculated into the broth to check for
bacteriological sterility.

The allantoic cavity infection method is often used to cultivate influenza virus, as well as mumps and vaccinia.

Infection in the amnion cavity

This method is more difficult to perform and is used somewhat less frequently than the previous ones. Its peculiarity is that when infected with some pneumotropic viruses (for example, influenza viruses, mumps), the latter multiply not only in the cells of the amnion membrane, but also in the respiratory tract and lung tissue of the embryo, where the infected fluid penetrates. Embryos 7-12 days old are used for infection. Infection into the amniotic cavity is carried out using the open method, when a relatively large hole is made in the shell to introduce material, or the closed method, when infection is carried out through a puncture of the shell.

The first of them is more traumatic, the second is less reliable, since, acting blindly, it is not always possible to insert a needle into the amnion cavity.

Open infection technique:

1. The egg is placed on a stand and sterilized
shell in the area of ​​the blunt end.

2. Above the center of the airspace in the shell
Using scissors, cut out a window 2 cm in diameter (as
for infection on the chorioallantoic membrane).

3. Carefully remove the inner leaf of the shell
membrane using tweezers, exposing the underlying
chorioallantoic membrane.

Use scissors to cut a small hole in
chorioallantoic membrane in a place where there are no blood vessels, and ocular tweezers are inserted into the cut. The amnion membrane is grasped with tweezers and the amniotic sac is pulled out over the surface of the chorioallantoic membrane.

5. Holding the amniotic membrane in this position,
infect the amnion cavity by injecting 0.1-
0.2 ml of virus dilution.

6. The hole in the shell is closed with a sterile cap.
or cover glass, using to fix them and
sealing the egg with molten paraffin.

Infected embryos are incubated for 2 days, the dead ones are discarded during the first day (observations are made through a window in the shell). After opening, the embryos are kept overnight in a refrigerator at 4°C.

Technique for opening an embryo during infection in the amniotic cavity:

1. Having sterilized the site of the upcoming autopsy, cut it off
shell slightly above the edge of the air sac.

2. The chorioallantoic membrane is punctured
pasteur pipette and remove allantoic fluid.

3. Grabbing the amniotic sac with tweezers, pierce
its membrane with a syringe needle or Pasteur pipette and
amnestic fluid is sucked in (from 0.5 to 1.5 ml). U
infected embryo, the fluid should be cloudy, meanwhile
like a normal embryo it is transparent.

4. The taken liquid is transferred into a sterile test tube, 0.1-
0.3 ml of it is inoculated into the broth to control bacteriological
sterility.

The introduction of methods for cultivating viruses in chicken embryos was of great importance in the development of virology. For the reproduction of the virus, chicken embryos of 7-12 days of age are used, incubated in a thermostat at 37 C. A necessary condition for the proper development of the embryo is maintaining a certain air humidity, which can be created by placing a vessel with water in the thermostat. Cultivation of viruses in chicken embryos is carried out in different places of the embryo, which is infected:

1) on the chorion-allantoic membrane;

2) into the allantoic cavity;

3) into the amniotic cavity;

4) into the yolk sac.

Infection of chicken embryos is carried out in a box using sterile instruments. Before: infection, chicken embryos are wiped twice with a cotton swab moistened with alcohol.

Infection of the chorion-allantoic membrane. After disinfection of the egg, carefully cut off a piece of the shell from the blunt end, remove the subshell membrane - this reveals the chorion-allatioic membrane. Infectious material in an amount of 0.1-0.2 ml is applied to the chorion-allantoic membrane using a syringe or Pasteur pipette. After infection, the hole is closed with a cap and the gap between it and the chicken embryo is filled with paraffin. On the other side of the egg, write the name of the infectious material and the date of infection with a simple pencil.

Infection in the amniotic cavity. The egg is ovoscopic and an area is selected on the lateral side where the chorion-allantois is devoid of large blood vessels. This area is marked with a pencil. The eggs are placed on a stand in a horizontal position, disinfected and a hole is pierced with a special sterile spear. And shell to a depth of 2-3 mm, through which a needle with infectious material is inserted at the same distance directly into the amniotic cavity. To prevent the injected liquid from flowing back, a puncture is first made above the air sac. After which both holes are filled with paraffin.

Infection in the allantoic cavity. Infection is also carried out in a shaded box. The air space is marked, the shell above the air space is disinfected and a syringe needle with material is inserted through a hole in the shell towards the embryo. If the needle enters the allantoic cavity, then a displacement of the embryo's shadow is observed. After infection, the hole is filled with paraffin.

Infection in the yolk sac. The shell is disinfected. The egg is placed on the stand with the blunt end to the right so that the yolk mark is facing up. A hole is pierced in the center above the air chamber. A syringe needle is inserted through a hole in the shell in a horizontal direction to a depth of 2-3 mm, which enters the yolk sac. The material is administered in a volume of 0.2-0.3 ml. After introducing the material, the hole is waxed.

The temperature and duration of incubation depend on the biological properties of the virus. Infected eggs are checked daily and ovoscoped to check the viability of the embryo. If embryos die on the first day, the cause is usually trauma during infection. Such eggs are hatched from experience. The presence of the virus in an infected embryo is determined by characteristic changes in the chorion-allantoic membrane of an infected chicken embryo. Viruses that do not have hemagpotinating activity are detected using RSC. To detect the virus in the allantoic or amniotic fluid of infected embryos, RGA is performed.