1966).

Cell culture techniques developed significantly in the 1940s and 1950s in connection with research in the field of virology. The cultivation of viruses in cell cultures made it possible to obtain pure viral material for the production of vaccines. The polio vaccine was one of the first drugs to be mass-produced using cell culture technology. In 1954, Enders, Weller and Robbins received the Nobel Prize "for their discovery of the ability of the polio virus to grow in cultures of various tissues." In 1952, the well-known human cancer cell line HeLa was developed.

Basic principles of cultivation[ | ]

Cell isolation[ | ]

For cultivation outside the body, living cells can be obtained in several ways. Cells can be isolated from blood, but only leukocytes can grow in culture. Mononuclear cells can be isolated from soft tissues using enzymes such as collagenase, trypsin, and pronase that degrade the extracellular matrix. In addition, pieces of tissues and materials can be placed in the nutrient medium.

Cultures of cells taken directly from the object (ex vivo) are called primary. Most primary cells, with the exception of tumor cells, have a limited lifespan. After a certain number of cell divisions, such cells grow old and stop dividing, although they can still remain viable.

There are immortalized ("immortal") cell lines that can multiply indefinitely. In most tumor cells, this ability is the result of a random mutation, but in some laboratory cell lines it is acquired artificially, by activating the telomerase gene.

Cell culture[ | ]

Cells are grown in special nutrient media at a constant temperature. Variable lighting is used for plant cell cultures, while mammalian cells usually also require a special atmosphere maintained in a cell culture incubator. As a rule, the concentration of carbon dioxide and water vapor in the air is regulated, but sometimes also oxygen. Nutrient media for different cell cultures differ in composition, glucose concentration, composition of growth factors, etc. Growth factors used in mammalian cell culture media are most commonly added along with blood serum. One of the risk factors in this case is the possibility of infection of the cell culture with prions or viruses. In cultivation, one of the important tasks is to avoid or minimize the use of contaminated ingredients. However, in practice this is not always achieved. The best, but also the most expensive way is to supplement with purified growth factors instead of serum.

Cross-contamination of cell lines[ | ]

When working with cell cultures, scientists can face the problem of cross-contamination.

Features of growing cells[ | ]

When growing cells, due to constant division, their overabundance in culture may occur, and, as a result, the following problems arise:

To maintain the normal functioning of cell cultures, as well as to prevent negative phenomena, the replacement of the nutrient medium, cell aging and transfection are periodically carried out. To avoid contamination of cultures with bacteria, yeasts, or other cell lines, all manipulations are usually carried out under aseptic conditions in a sterile box. Antibiotics (penicillin, streptomycin) and antifungals (amphotericin B) can be added to the culture medium to suppress the microflora.

The cultivation of human cells is somewhat against the rules of bioethics, as cells grown in isolation can outlive the parent organism and then be used to conduct experiments or to develop new treatments and profit from it. The first court ruling in this area came in the California Supreme Court in John Moore v. University of California, whereby patients have no ownership of cell lines derived from organs removed with their consent.

hybridoma [ | ]

Use of cell cultures[ | ]

Mass cell culture is the basis for the industrial production of viral vaccines and a variety of biotechnology products.

Biotechnology products[ | ]

An industrial method from cell cultures produces products such as enzymes, synthetic hormones, monoclonal antibodies, interleukins, lymphokines, antitumor drugs. Although many simple proteins can be obtained relatively easily using bacterial cultures, more complex proteins such as glycoproteins can currently only be obtained from animal cells. One of these important proteins is the hormone erythropoietin. The cost of growing mammalian cell cultures is quite high, so research is currently being done into the possibility of producing complex proteins in insect or higher plant cell cultures.

tissue cultures[ | ]

Cell culture is an integral part of tissue culture technology and tissue engineering, since it defines the basis for growing cells and maintaining them in a viable state ex vivo.

Vaccines [ | ]

Using cell culture techniques, vaccines against poliomyelitis, measles, mumps, rubella, and chickenpox are currently being produced. Due to the threat of an H5N1 influenza pandemic, the United States government is currently funding research into an avian influenza vaccine using cell cultures.

Non-mammalian cell cultures[ | ]

Plant cell cultures[ | ]

Plant cell cultures are usually grown either as a suspension in a liquid nutrient medium or as a callus culture on a solid nutrient base. Cultivation of undifferentiated cells and callus requires maintaining a certain balance of plant growth hormones auxins and cytokinins.

Bacterial, yeast cultures[ | ]

Main article:

For the cultivation of a small number of bacterial and yeast cells, the cells are plated on a solid nutrient medium based on gelatin or agar-agar. For mass production, cultivation in liquid nutrient media (broths) is used.

virus cultures[ | ]

K.K. - These are cells of a multicellular organism that live and multiply in artificial conditions outside the body.

Cells or tissues living outside the body are characterized by a whole complex of metabolic, morphological and genetic features that are sharply different from the properties of cells of organs and tissues in vivo.

There are two main types of single-layer cell cultures: primary and transplanted.

Primarily trypsinized. The term "primary" refers to a cell culture obtained directly from human or animal tissues in the embryonic or postnatal period. The life span of such crops is limited. After a certain time, phenomena of nonspecific degeneration appear in them, which is expressed in granulation and vacuolization of the cytoplasm, rounding of cells, loss of communication between the cells and the solid substrate on which they were grown. Periodic change of the medium, changes in the composition of the latter, and other procedures can only slightly increase the lifetime of the primary cell culture, but cannot prevent its final destruction and death. In all likelihood, this process is associated with the natural extinction of the metabolic activity of cells that are out of control of neurohumoral factors acting in the whole organism.

Only individual cells or groups of cells in the population against the background of degeneration of most of the cell layer can retain the ability to grow and reproduce. These cells, having found the potency of endless reproduction in vitro, give rise to transplanted cell cultures.

The main advantage of transplantable cell lines, in comparison with any primary culture, is the potential for unlimited reproduction outside the body and the relative autonomy that brings them closer to bacteria and unicellular protozoa.

Suspension cultures- individual cells or groups of cells grown in suspension in a liquid medium. They are a relatively homogeneous population of cells that are easily exposed to chemicals.

Suspension cultures are widely used as model systems for studying secondary metabolism pathways, enzyme induction and gene expression, degradation of foreign compounds, cytological studies, etc.

A sign of a "good" line is the ability of cells to rearrange metabolism and a high rate of reproduction under specific cultivation conditions. Morphological characteristics of such a line:

high degree of disaggregation (5-10 cells per group);

morphological uniformity of cells (small size, spherical or oval shape, dense cytoplasm);

Absence of tracheid-like elements.

Diploid cell strains. These are cells of the same type that are capable of undergoing up to 100 divisions in vitro, while retaining the failure of the original diploid set of chromosomes (Hayflick, 1965). Diploid strains of fibroblasts derived from human embryos are widely used in diagnostic virology and vaccine production, as well as in experimental studies. It should be borne in mind that some features of the viral genome are realized only in cells that retain a normal level of differentiation.

130. Bacteriophages. Morphology and chemical composition

Bacteriophages (phages) (from other Greek φᾰγω - “I devour”) are viruses that selectively infect bacterial cells. Most often, bacteriophages multiply inside bacteria and cause their lysis. As a rule, a bacteriophage consists of a protein shell and the genetic material of a single-stranded or double-stranded nucleic acid (DNA or, less commonly, RNA). The particle size is approximately 20 to 200 nm.

The structure of particles - virions - of different bacteriophages is different. Unlike eukaryotic viruses, bacteriophages often have a specialized attachment organ to the surface of a bacterial cell, or a tail process, arranged with varying degrees of complexity, but some phages do not have a tail process. The capsid contains the genetic material of the phage, its genome. The genetic material of different phages can be represented by different nucleic acids. Some phages contain DNA as their genetic material, others contain RNA. The genome of most phages is double-stranded DNA, and the genome of some relatively rare phages is single-stranded DNA. At the ends of the DNA molecules of some phages there are "sticky areas" (single-stranded complementary nucleotide sequences), in other phages there are no sticky areas. Some phages have unique gene sequences in DNA molecules, while other phages have gene permutations. In some phages, DNA is linear, in others it is closed in a ring. Some phages have terminal repeats of several genes at the ends of the DNA molecule, while in other phages this terminal redundancy is ensured by the presence of relatively short repeats. Finally, in some phages, the genome is represented by a set of several nucleic acid fragments.

From an evolutionary point of view, bacteriophages that use such different types of genetic material differ from each other to a much greater extent than any other representatives of eukaryotic organisms. At the same time, despite such fundamental differences in the structure and properties of carriers of genetic information - nucleic acids, different bacteriophages show commonality in many respects, primarily in the nature of their intervention in cellular metabolism after infection of susceptible bacteria.

Bacteriophages capable of causing a productive infection of cells, i.e. an infection resulting in viable offspring is defined as non-defective. All non-defective phages have two states: the state of an extracellular, or free, phage (sometimes also called a mature phage) and the state of a vegetative phage. For some so-called temperate phages, the state of a prophage is also possible.

Extracellular phage are particles that have a structure characteristic of this type of phage, which ensures the preservation of the phage genome between infections and its introduction into the next sensitive cell. The extracellular phage is biochemically inert, while the vegetative phage, the active (“live”) state of the phage, occurs after infection of sensitive bacteria or after induction of a prophage.

Sometimes infection of sensitive cells with a non-defective phage does not result in the formation of viable progeny. This can be in two cases: during an abortive infection or due to the lysogenic state of the cell during infection with a temperate phage.

The reason for the abortive nature of the infection may be the active interference of certain cell systems in the course of infection, for example, the destruction of the phage genome introduced into the bacterium, or the absence in the cell of some product necessary for the development of the phage, etc.

Phages are usually classified into three types. The type is determined by the nature of the influence of a productive phage infection on the fate of the infected cell.

First type are truly virulent phages. Infection of a cell with a virulent phage inevitably leads to the death of the infected cell, its destruction, and the release of the progeny phage (excluding cases of abortive infection). Such phages are called truly virulent to distinguish them from virulent temperate phage mutants.

Second type- temperate phages. In the course of a productive infection of a cell with a temperate phage, two fundamentally different ways of its development are possible: lytic, in general (in its outcome) similar to the lytic cycle of virulent phages, and lysogenic, when the genome of a moderate phage passes into a special state - a prophage. A cell carrying a prophage is called a lysogenic or simply a lysogen (because it can undergo phage lytic development under certain conditions). Temperate phages that respond in the prophage state to the application of an inducing factor by the onset of lytic development are called inducible, and phages that do not react in this way are called non-inducible. Virulent mutants can occur in temperate phages. Virulence mutations lead to such a change in the sequence of nucleotides in the operator regions, which is reflected in the loss of affinity for the repressor.

The third type of phages are phages, the productive infection of which does not lead to the death of bacteria. These phages are able to leave the infected bacterium without causing its physical destruction. A cell infected with such a phage is in a state of constant (permanent) productive infection. The development of the phage results in some slowing down of the rate of bacterial divisions.

Bacteriophages differ in chemical structure, type of nucleic acid, morphology, and interaction with bacteria. Bacterial viruses are hundreds and thousands of times smaller than microbial cells.

A typical phage particle (virion) consists of a head and a tail. The length of the tail is usually 2-4 times the diameter of the head. The head contains genetic material - single-stranded or double-stranded RNA or DNA with the transcriptase enzyme in an inactive state, surrounded by a protein or lipoprotein shell - a capsid that preserves the genome outside the cell.

Nucleic acid and capsid together make up the nucleocapsid. Bacteriophages may have an icosahedral capsid assembled from multiple copies of one or two specific proteins. Usually the corners are made up of pentamers of the protein, and the support of each side is made up of hexamers of the same or a similar protein. Moreover, phages can be spherical, lemon-shaped, or pleomorphic in shape. The tail is a protein tube - a continuation of the protein shell of the head, at the base of the tail there is an ATPase that regenerates energy for the injection of genetic material. There are also bacteriophages with a short process, without a process, and filamentous.

The main components of phages are proteins and nucleic acids. It is important to note that phages, like other viruses, contain only one type of nucleic acid, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In this property, viruses differ from microorganisms that contain both types of nucleic acids in their cells.

The nucleic acid is located in the head. A small amount of protein (about 3%) was also found inside the phage head.

Thus, according to the chemical composition, phages are nucleoproteins. Depending on the type of their nucleic acid, phages are divided into DNA and RNA. The amount of protein and nucleic acid in different phages is different. In some phages, their content is almost the same, and each of these components is about 50%. In other phages, the ratio between these main components may be different.

In addition to these main components, phages contain small amounts of carbohydrates and some predominantly neutral fats.

Figure 1: Diagram of the structure of a phage particle.

All known phages of the second morphological type are RNA. Among phages of the third morphological type, both RNA and DNA forms are found. Phages of other morphological types are DNA-type.

131. Interferon. What it is?

Interfer O n(from lat. inter - mutually, among themselves and ferio - hit, hit), a protective protein produced by cells in the body of mammals and birds, as well as cell cultures in response to their infection with viruses; inhibits the reproduction (replication) of viruses in the cell. I. was discovered in 1957 by the English scientists A. Isaacs and J. Lindenman in the cells of infected chickens; later it turned out that bacteria, rickettsia, toxins, nucleic acids, synthetic polynucleotides also cause the formation of I.. I. is not an individual substance, but a group of low molecular weight proteins (molecular weight 25,000–110,000) that are stable in a wide pH zone, resistant to nucleases, and degraded by proteolytic enzymes. Formation in I.'s cells is associated with the development of a virus in them, that is, it is a reaction of the cell to the penetration of a foreign nucleic acid. After disappearance from a cell of the infecting virus and in normal cells And. it is not found. According to the mechanism of action, I. is fundamentally different from antibodies: it is not specific to viral infections (it acts against different viruses), does not neutralize the infectivity of the virus, but inhibits its reproduction in the body, suppressing the synthesis of viral nucleic acids. When it enters the cells after the development of a viral infection in them, I. is not effective. Besides, And., as a rule, is specific to the cells forming it; for example, I. of chicken cells is active only in these cells, but does not suppress the reproduction of the virus in rabbit or human cells. It is believed that not I. itself acts on viruses, but another protein produced under its influence. Encouraging results have been obtained in testing I. for the prevention and treatment of viral diseases (herpetic eye infection, influenza, cytomegaly). However, the widespread clinical use of I. is limited by the difficulty of obtaining the drug, the need for repeated administration to the body, and its species specificity.

132. Disjunctive way. What it is?

1.A productive viral infection occurs in 3 periods:

· initial period includes the stages of adsorption of the virus on the cell, penetration into the cell, disintegration (deproteinization) or "undressing" of the virus. The viral nucleic acid was delivered to the appropriate cell structures and, under the action of lysosomal cell enzymes, is released from protective protein coats. As a result, a unique biological structure is formed: an infected cell contains 2 genomes (own and viral) and 1 synthetic apparatus (cellular);

After that it starts second group virus reproduction processes, including average and final periods, during which repression of the cellular and expression of the viral genome occur. Repression of the cellular genome is provided by low molecular weight regulatory proteins such as histones, which are synthesized in any cell. With a viral infection, this process is enhanced, now the cell is a structure in which the genetic apparatus is represented by the viral genome, and the synthetic apparatus is represented by the synthetic systems of the cell.

2. The further course of events in the cell is directedfor viral nucleic acid replication(synthesis of genetic material for new virions) and implementation of the genetic information contained in it(synthesis of protein components for new virions). In DNA-containing viruses, both in prokaryotic and eukaryotic cells, viral DNA replication occurs with the participation of the cellular DNA-dependent DNA polymerase. In this case, single-stranded DNA-containing viruses first form complementary strand - the so-called replicative form, which serves as a template for daughter DNA molecules.

3. The implementation of the genetic information of the virus contained in the DNA occurs as follows: with the participation of DNA-dependent RNA polymerase, mRNAs are synthesized, which enter the ribosomes of the cell, where virus-specific proteins are synthesized. In double-stranded DNA-containing viruses, the genome of which is transcribed in the cytoplasm of the host cell, this is its own genomic protein. Viruses whose genomes are transcribed in the cell nucleus use the cellular DNA-dependent RNA polymerase contained there.

At RNA viruses processes replication their genome, transcription and translation of genetic information are carried out in other ways. Replication of viral RNA, both minus and plus strands, is carried out through the replicative form of RNA (complementary to the original), the synthesis of which is provided by RNA-dependent RNA polymerase, a genomic protein that all RNA-containing viruses have. The replicative form of RNA of minus-strand viruses (plus-strand) serves not only as a template for the synthesis of daughter viral RNA molecules (minus-strands), but also performs the functions of mRNA, i.e. goes to ribosomes and ensures the synthesis of viral proteins (broadcast).

At plus-filament RNA-containing viruses perform the translation function of its copies, the synthesis of which is carried out through the replicative form (negative strand) with the participation of viral RNA-dependent RNA polymerases.

Some RNA viruses (reoviruses) have a completely unique transcription mechanism. It is provided by a specific viral enzyme - reverse transcriptase (reverse transcriptase) and is called reverse transcription. Its essence lies in the fact that at first a transcript is formed on the viral RNA matrix with the participation of reverse transcription, which is a single strand of DNA. On it, with the help of cellular DNA-dependent DNA polymerase, the second strand is synthesized and a double-stranded DNA transcript is formed. From it, in the usual way, through the formation of i-RNA, the information of the viral genome is realized.

The result of the described processes of replication, transcription and translation is the formation daughter molecules viral nucleic acid and viral proteins encoded in the virus genome.

After that comes third, final period interaction between virus and cell. New virions are assembled from the structural components (nucleic acids and proteins) on the membranes of the cytoplasmic reticulum of the cell. A cell whose genome has been repressed (suppressed) usually dies. newly formed virions passively(due to cell death) or actively(by budding) leave the cell and find themselves in its environment.

In this way, synthesis of viral nucleic acids and proteins and assembly of new virions occur in a certain sequence (separated in time) and in different cell structures (separated in space), in connection with which the method of reproduction of viruses was named disjunctive(disjointed). With an abortive viral infection, the process of interaction of the virus with the cell is interrupted for one reason or another before the suppression of the cellular genome has occurred. Obviously, in this case, the genetic information of the virus will not be realized and the reproduction of the virus does not occur, and the cell retains its functions unchanged.

During a latent viral infection, both genomes function simultaneously in the cell, while during virus-induced transformations, the viral genome becomes part of the cellular one, functions and is inherited along with it.

133. Camelpox virus

Smallpox (Variola)- an infectious contagious disease characterized by fever and a papular-pustular rash on the skin and mucous membranes.
The causative agents of the disease belong to various genera and types of viruses of the smallpox family (Poxviridae). Independent species are viruses: natural cow yuspa, vaccinia (genus Orthopoxvirus), natural sheep pox, goats (genus Carpipoxvirus), pigs (genus Suipoxvirus), birds (genus Avipoxvirus) with three main species (causative agents of smallpox of chickens, pigeons and canaries).
Smallpox pathogens different animal species are morphologically similar. These are DNA-containing viruses characterized by relatively large sizes (170 - 350 nm), epitheliotropy and the ability to form elementary rounded inclusions in cells (Paschen, Guarnieli, Bollinger bodies), visible under a light microscope after Morozov staining. Although there is a phylogenetic There is a strong relationship between the causative agents of smallpox in different animal species, the spectrum of pathogenicity is not the same, and immunogenic relationships are not preserved in all cases. Variola viruses of sheep, goats, pigs and birds are pathogenic only for the corresponding species, and under natural conditions each of them causes an independent (original) smallpox. Variola cowpox and vaccinia viruses have a wide spectrum of pathogenicity, including cattle, buffalo, lo-boats, donkeys, mules, camels, rabbits, monkeys and humans.

Camel pox VARIOLA CAMELINA a contagious disease that occurs with the formation of a characteristic nodular-pustular smallpox rash on the skin and mucous membranes. The name of smallpox Variola comes from the Latin word Varus, which means crooked (pockmarked).

Epizootology of the disease. Camels of all ages are susceptible to smallpox, but young animals are more often and more severely ill. In stationary areas with smallpox problems, adult camels rarely get sick due to the fact that almost all of them get smallpox at a young age. In pregnant camels, smallpox can cause abortions.

Animals of other species are not susceptible to the original camelpox virus in natural conditions. In addition to cows and camels, buffaloes, horses, donkeys, pigs, rabbits and people who are not immune to smallpox are susceptible to the cowpox virus and vaccinia. Of the laboratory animals, guinea pigs are sensitive to cowpox and vaccinia viruses after the virus has been applied to the scarified cornea of ​​​​the eyes (FA Petunii, 1958).

The main sources of smallpox viruses are smallpox animals and people with vaccinia and recovering from hypersensitivity after immunization with vaccinia virus in smallpox calf detritus. Sick animals and people disseminate the virus in the external environment, mainly with the rejected epithelium of the skin and mucous membrane containing the virus. The virus is also released into the external environment with aborted fetuses (K. N. Buchnev and R. G. Sadykov, 1967). The causative agent of smallpox can be mechanically carried by domestic and wild animals immune to smallpox, including birds, as well as people immune to smallpox from children vaccinated with vaccinia.

Under natural conditions, healthy camels become infected through contact with sick animals in a virus-contaminated area through infected water, feed, premises and care items, as well as aerogenically by spraying virus-containing outflows by sick animals. More often, camels become infected when the virus enters the body through the skin and mucous membranes, especially when their integrity is violated or when vitamin A deficiency occurs.

In the form of an epizootic, smallpox in camels occurs approximately every 20-25 years. At this time, young animals are especially seriously ill. In the period between epizootics in zones that are stationary in terms of smallpox, among camels, smallpox occurs in the form of enzootic and sporadic cases that occur more or less regularly every 3-6 years, mainly among animals aged 2-4 years. In such cases, animals get sick relatively easily, especially in the warm season. In cold weather, smallpox is more severe, longer and is accompanied by complications, especially in young animals. In small farms, almost all susceptible camels fall ill within 2-4 weeks. It should be borne in mind that smallpox outbreaks among camels can be caused by both the original camelpox virus and cowpox virus, which do not create immunity against each other. Therefore, outbreaks caused by different smallpox viruses can follow one another or occur simultaneously.

Pathogenesis determined by the pronounced epitheliotropism of the pathogen. Once in the body of an animal, the virus multiplies and penetrates into the blood (viremia), lymph nodes, internal organs, into the epithelial layer of the skin and mucous membranes and causes the formation of specific exanthemas and enanthems in them, the severity of which depends on the reactivity of the organism and the virulence of the virus, pathways its penetration into the body and the state of the epithelial layer. Pocks develop sequentially in stages: from roseola with a nodule to a pustule with a crust and scar formation.

Symptoms. The incubation period, depending on the age of the camels, the properties of the virus and how it enters the body, ranges from 3 to 15 days: in young animals 4-7, in adults 6-15 days. Camels from non-immune camels may become ill 2-5 days after birth. The shortest incubation period (2-3 days) occurs in camels after they are infected with the vaccinia virus.

In the prodromal period, in sick camels, the body temperature rises to 40-41 ° C, lethargy and refusal to feed appear, the conjunctiva and mucous membranes of the mouth and nose are hyperemic. However, these signs are often seen, especially at the beginning of the onset of the disease on the farm.

The course of smallpox in camels, depending on their age, is also different: in young animals, especially in a newborn, it is more often acute (up to 9 days); in adults - subacute and chronic, sometimes latent, more often in pregnant camels. The most characteristic form of smallpox in camels is cutaneous with a subacute course of the disease (Fig. 1).

In the subacute course of the disease, clear, later cloudy, grayish-dirty mucus is released from the mouth and nose. Animals shake their heads, sniff and snort, throwing out the epithelium affected by the virus along with the virus-containing mucus. Soon, puffiness forms in the area of ​​the lips, nostrils and eyelids, sometimes spreading to the intermaxillary region, neck, and even to the dewlap area. Submandibular and lower cervical lymph nodes are enlarged. Animals have reduced appetite, they lie more often and longer than usual and get up with great difficulty. By this time, reddish-gray spots appear on the skin of the lips, nose and eyelids, on the mucous membrane of the mouth and nose; under them dense nodules are formed, which, increasing, turn into gray papules, and then into pustules the size of a pea and a bean with a sinking center and a roller-like thickening along the edges.

The pustules soften, burst, and a sticky liquid of a light gray color is released from them. The swelling of the head by this time disappears. After 3-5 days, the opened pustules become covered with crusts. If they are not injured by roughage, then the disease ends there. Removed or fallen off primary crusts have a reverse crater-like form of pustules. Scars remain in place of pockmarks. All of these lesions on the skin are formed within 8-15 days.

Pocks in sick camels often appear first on the head. At the age of one to four years, camels get sick, as a rule, easily. Lesions are localized on the scalp, mainly in the lips and nose. In camels, the udder is often affected. A few days after the opening of the primary pustules in the head area, smallpox lesions form on the skin and other low-haired areas of the body (in the areas of the breastbone, armpits, perineum and scrotum, around the anus, the inside of the forearm and thigh), and in camels also on the mucosa lining of the vagina. At this time, the body temperature of the camels usually rises again, sometimes up to 41.5 °, and the camels in the last month of pregnancy bring premature and underdeveloped camels, who, as a rule, soon die.

In some animals, the cornea of ​​​​the eyes (thorn) becomes cloudy, which causes temporary blindness in one eye for 5-10 days, and in camels more often in both eyes. Camel calves who fall ill shortly after birth develop diarrhea. In this case, within 3-9 days after the disease, they die.

With a relatively benign subacute course of smallpox and usually after infection with the vaccinia virus, animals recover in 17-22 days.

In adult camels, opening pustules on the oral mucosa often merge and bleed, especially when injured by roughage. This makes it difficult to feed, the animals lose weight, the healing process is delayed up to 30-40 days, and the disease becomes chronic.

With the generalization of the smallpox process, pyemia and complications (pneumonia, gastroenteritis, necrobacteriosis, etc.) sometimes develop. In such cases, the disease drags on for up to 45 days or longer. There are cases of disorders of the functions of the stomach and intestines, accompanied by atony and constipation. In some sick animals, swelling of the extremities is noted.

In camels with a latent course of smallpox (without characteristic clinical signs of the disease, only in the presence of fever), abortions occur 1-2 months before foaling (up to 17-20%).

The prognosis of the disease in adult camels is favorable, in camels with an acute course, especially at the age of 15-20 days and in camels born from non-immune to smallpox, unfavorable. Camels are seriously ill and up to 30-90% of them die. Camels at the age of 1-3 years are ill with smallpox more easily, and at an older age, although they are seriously ill, with signs of a pronounced generalized process, the mortality rate is low (4-7%).

Pathological changes are characterized by the lesions of the skin, mucous membrane and cornea of ​​the eyes described above. Pinpoint hemorrhages are noted on the epicardium and intestinal mucosa. In the chest cavity on the costal pleura, small hemorrhages and nodules ranging in size from millet grain to lentils of gray and gray-red color with curdled contents are sometimes also visible. The mucous membrane of the esophagus is covered with nodules the size of millet, surrounded by ridge-like elevations. The mucous membrane of the scar (sometimes the bladder) has similar hemorrhages and nodules with jagged edges, as well as small ulcers with a sunken pinkish center. In papules, elementary bodies such as Paschen bodies can be detected, which are of diagnostic value when microscopy of a smear preparation under immersion through a conventional light microscope.

The diagnosis is based on the analysis of clinical and epizootic data (taking into account the possibility of infection of camels from humans), pathological changes, positive results of microscopy (when processing smears from fresh papules using the Morozov silvering method) or electronoscopy, as well as bioassays on those susceptible to smallpox animals. It is possible to isolate the virus from the organs of aborted fetuses of camels with smallpox. When diagnosing smallpox, it is also recommended to use the diffusion precipitation reaction in agar gel and the neutralization reaction in the presence of active specific sera or globulins.

Differential diagnosis is carried out in doubtful cases (taking into account clinical and epizootic features). Smallpox must be differentiated from necrobacteriosis by microscopy of smears from pathological material and infection of white mice susceptible to it; from foot-and-mouth disease - infection of guinea pigs with a suspension of pathological material in the plantar surface of the skin of the hind legs; from fungal infections and scabies - by finding the corresponding pathogens in the examined scrapings taken from the affected areas of the skin; from brucellosis during abortions, miscarriages and premature foals - by examining the blood serum of camels RA and RSK and bacteriological examination of fetuses with the isolation of a microbial culture on nutrient media and microscopy (if necessary, use a bioassay on guinea pigs followed by bacteriological and serological tests of blood and sera).

When diagnosing smallpox in camels, it is also necessary to exclude a non-contagious, but sometimes widespread disease that occurs with skin lesions in the lips and nose - yantak-bash (Turkm.), Jantak-bas (Kazakh), which occurs from injuring them when eating shrubs called camel thorn (yantak, jantak, Alhagi). This disease can usually be observed in autumn in young camels, mainly under the age of one year. Adult camels are only slightly affected by camel thorn. With yantak-bash, there are usually no nodules or papular lesions, unlike smallpox, on the oral mucosa. The grayish coating that appears with yantak-bash is relatively easy to remove. However, it should be taken into account that yantak-bash contributes to the disease of smallpox in camels, and often proceeds simultaneously with it.

When isolating the smallpox virus, it is necessary to determine its type (original, cowpox or vaccinia), using the methods specified in the instructions of the Ministry of Health of the USSR of 1968. On the prevention of cowpox in humans, data obtained after infection (in isolated conditions) of camels who had had smallpox vaccinia virus and isolated pathogens.

Treatment of sick camels is mainly symptomatic. The affected areas are treated with a solution of potassium permanganate (1:3000), and after drying, they are lubricated with a mixture of 10% tincture of iodine with glycerin (1:2 or 1:3). After opening the smallpox, it is treated with a 5% emulsion of synthomycin on fortified fish oil, to which tincture of iodine is added in a ratio of 1:15-1:20; ointments - zinc, ichthyol, penicillin, etc. You can use 2% salicylic or boric ointment and 20-30% propolis ointment on petroleum jelly. In hot weather, 3% creolin ointment, tar and hexachlorane dust are indicated. The affected areas are lubricated with swabs soaked in emulsions and ointments 2-3 times a day.

The affected mucous membrane of the oral cavity is washed 2-3 times a day with a 10% solution of potassium permanganate or a 3% solution of hydrogen peroxide or decoctions of sage, chamomile and other disinfectants and astringents. With conjunctivitis, the eyes are washed with a 0.1% solution of zinc sulfate.

To prevent the development of a secondary microbial infection and possible complications, it is recommended to inject penicillin and streptomycin intramuscularly. With general weakness and complications, cardiac remedies are indicated.

From specific means of treatment in severe cases of the disease, you can use the serum or blood of camels who have had smallpox (subcutaneously at the rate of 1-2 ml per 1 kg of animal weight). The injection sites are carefully cut out beforehand and wiped with tincture of iodine.

Sick and convalescent camels are often given clean water, a mash of bran or barley flour, soft bluegrass or fine alfalfa hay, or cotton husks flavored with barley flour. In cold weather, sick animals, especially camels, are kept in a clean, dry and warm room or covered with blankets.

Immunity in naturally ill smallpox camels lasts up to 20-25 years, that is, almost for life. The nature of immunity is skin-humoral, as evidenced by the presence of neutralizing antibodies in the blood serum of recovered animals and the immunity of camels to re-infection with the homologous smallpox virus. Camels born from camels who have had smallpox are not susceptible to the type of smallpox that the camel has had, especially in the first three years, that is, until puberty. Camel calves, who are under the uterus during the epizootic period, as a rule, do not get smallpox or get sick relatively easily and for a short time.

Prevention and control measures are in strict observance of all veterinary, sanitary and quarantine measures, timely diagnosis of the disease and determination of the type of virus. Persons should not be allowed to care for camels during vaccination and in the post-vaccination period until they (or their children) have completely completed their clinically pronounced reaction to vaccination smallpox. All camels, cows and horses entering the farm must be kept in an isolation cell for 30 days.

When smallpox appears among camels, cows and horses, by a special decision of the district executive committee, the area, settlement or district, pasture where this disease is found is declared unfavorable for smallpox and quarantine, restrictive and health measures are taken.

The appearance of smallpox is immediately reported to higher veterinary organizations, neighboring farms and districts for taking appropriate measures to prevent further spread of the disease.

To prevent infection of camels with cowpox, it is recommended to use a medical preparation - smallpox detritus, which is used to immunize all clinically healthy camels, regardless of their age, sex and physiological state (pregnancy and lactating camels) in disadvantaged and threatened cowpox farms. To do this, wool is cut out in the lower third of the camel's neck, treated with alcohol-ether or a 0.5% solution of carbolic acid, wiped dry with cotton wool or dried, the skin is scarified and applied with a thick needle, the end of a scalpel or a scarifier 2-3 shallow parallel scratches 2 in length -4 cm. 3-4 drops of the dissolved vaccine are applied to the freshly scarified skin surface and lightly rubbed with a spatula. Dissolve the vaccine as indicated on the labels of ampoules and ampoules boxes. The diluted and unused vaccine and vaccine ampoules are disinfected by boiling and destroyed. The tools used for vaccinations are washed with a 3% solution of carbolic acid or a 1% solution of formaldehyde and sterilized by boiling.

If the camel was not immune to cowpox, then on the 5-7th day after vaccination, papules should appear at the site of scarification. If they are not present, the vaccination is repeated, but on the opposite side of the neck and with a vaccine of a different series. Persons immune against smallpox and familiar with the rules of personal hygiene are allowed to care for immunized and sick camels. Young animals, especially from the weak group, can sometimes react strongly to vaccination and get sick with pronounced signs of smallpox.

Sick and highly responsive camels are isolated and treated (see above). Livestock buildings and places contaminated with the smallpox virus are recommended to be disinfected with hot 2-4% solutions of caustic soda and caustic potash, a 3% solution of a sulfur-carbolic mixture or 2-3% solutions of sulfuric acid or clarified solutions of bleach, containing 2-6% active chlorine, which inactivate the smallpox virus within 2-3 hours (O. Trabaev, 1970). You can also use 3-5% solutions of chloramine and 2% formaldehyde solution. Manure must be burned or biothermally disinfected. The corpses of camels that have fallen with clinical signs of smallpox must be burned. Milk from camels sick and suspected of having smallpox, if it does not contain impurities of pus and is not contraindicated for any other reason, can be eaten only after boiling for 5 minutes or pasteurization at 85 ° -30 minutes. Wool and skin from camels killed during the period of trouble for smallpox farms are processed according to the instructions for disinfection of raw materials of animal origin.

It is recommended to remove restrictions from households and settlements that are unfavorable for smallpox no earlier than 20 days after the recovery of all animals and people with smallpox and after a thorough final disinfection.

134. Chemical composition and biochemical properties of viruses

1.1 Structure and chemical composition of virions.

The largest viruses (variola viruses) are close in size to small bacteria, the smallest (causative agents of encephalitis, poliomyelitis, foot-and-mouth disease) to large protein molecules directed to blood hemoglobin molecules. In other words, among viruses there are giants and dwarfs. To measure viruses, a conditional value called a nanometer (nm) is used. One nm is one millionth of a millimeter. The sizes of different viruses vary from 20 to several hundreds of 1 nm.

Simple viruses are made up of proteins and nucleic acids. The most important part of a virus particle, the nucleic acid, is the carrier of genetic information. If the cells of humans, animals, plants and bacteria always contain two types of nucleic acids - deoxyribonucleic acid DNA and ribonucleic RNA, then only one type of either DNA or RNA was found in different viruses, which is the basis for their classification. The second mandatory component of the virion, proteins differ in different viruses, which allows them to be recognized using immunological reactions.

More complex in structure, viruses, in addition to proteins and nucleic acids, contain carbohydrates and lipids. Each group of viruses has its own set of proteins, fats, carbohydrates and nucleic acids. Some viruses contain enzymes. Each component of virions has certain functions: the protein shell protects them from adverse effects, the nucleic acid is responsible for hereditary and infectious properties and plays a leading role in the variability of viruses, and enzymes are involved in their reproduction. Usually, the nucleic acid is located in the center of the virion and is surrounded by a protein shell (capsid), as if dressed in it.

The capsid consists of similar protein molecules (capsomeres) arranged in a certain way, which form symmetrical geometric shapes in place with the nucleic acid of the virus (nucleocapsid). In the case of cubic symmetry of the nucleocapsid, the nucleic acid strand is coiled into a ball, and the capsomeres are tightly packed around it. This is how the viruses of polio, foot-and-mouth disease, etc.

With helical (rod-shaped) symmetry of the nucleocapsid, the virus thread is twisted in the form of a spiral, each of its coils is covered with capsomeres that are darkly adjacent to each other. The structure of capsomeres and the appearance of virions can be observed using electron microscopy.

Most of the viruses that cause infections in humans and animals have a cubic symmetry type. The capsid almost always has the form of an icosahedral regular twenty-sided hexahedron with twelve vertices and with faces of equilateral triangles.

Many viruses have an outer shell in addition to the protein capsid. In addition to viral proteins and glycoproteins, it also contains lipids borrowed from the plasma membrane of the host cell. The influenza virus is an example of a helical enveloped virion with a cubic symmetry type.

The modern classification of viruses is based on the type and shape of their nucleic acid, the type of symmetry, and the presence or absence of an outer shell.

Biochemical properties - see. manual!!!

135. Pieces of organs that retain functional and proliferating activity in vitro

Cell culture

cells of any animal tissue capable of growing in the form of a monolayer under artificial conditions on a glass or plastic surface filled with a special nutrient medium. The source of cells is freshly obtained animal tissue - primary cells, laboratory strains of cells - transplanted to-ry. cells. Embryonic and tumor cells have the best ability to grow under artificial conditions. The diploid to-ra of human and monkey cells is passaged a limited number of times, therefore it is sometimes called semi-transplantable to-swarm of cells. Stages of receiving to-ry of cells: crushing of a source; trypsin treatment; release from detritus; standardization of the number of cells suspended in a nutrient medium with antibiotics; pouring into test tubes or vials, in which the cells settle on the walls or bottom, and begin to multiply; control over the formation of a monolayer. To-ry cells are used to isolate the virus from the study. material, for the accumulation of viral suspension, the study of St. in. Recently, it has been used in bacteriology.

136. Parasthesias. What it is?

PARESTHESIA(from Greek para-near, contrary to and aisthesis-sensation), sometimes also called dysesthesias, not caused by external irritation, sensations of numbness, tingling, goosebumps (myrmeciasis, myrmecismus, formicatio), burning, itching, painful cold (i.e. n. psychroesthesia), movements, etc., sensations in apparently preserved limbs in amputees (pseudomelia paraesthetica). The causes of P. may be different. P. can occur as a result of local changes in blood circulation, with Renaud's disease, with erythromelalgia, with acroparesthesia, with endarteritis, as an initial symptom of spontaneous gangrene. Sometimes they occur with damage to the nervous system, with traumatic neuritis (cf. typical. P. with a bruise of the n. ulnaris in the sulcus olecrani region), with toxic and infectious neuritis, with radiculitis, with spinal pachymeningitis (compression of the roots), with acute and hron. myelitis, especially with compression of the spinal cord (tumors of the spinal cord) and with tabes dorsalis. Their diagnostic value in all these cases is the same as the diagnostic value of pain, anesthesia and hyperesthesia: appearing in certain areas, along the tract of one or another peripheral nerve or in the area of ​​one or another radicular innervation, they can give valuable indications of the location of the pathology. . process. Items are also possible as manifestations of cerebral damage. So, with cortical epilepsy, seizures often begin with P., localized in the limb from which convulsions then begin. Often they are also observed in cerebral arteriosclerosis or in cerebral syphilis and are sometimes harbingers of apoplectic stroke. mental P., i.e. P. of psychogenic, hypochondriacal origin, for which it is especially characteristic that they have not an elementary, like organic, but a complex character - “crawling of worms under the scalp”, “raising a ball from the abdomen to the neck” (Oppenheim), etc. Their diagnostic value is, of course, completely different from that of organic P

137. Rules for working and safety precautions with virus-containing material

138. Infectious bovine rhinotracheitis virus

Infectious rhinotracheitis(lat. - Rhinotracheitis infectiosa bovum; English - Infectious bovine rhinotracheites; IRT, blistering rash, infectious vulvovaginitis, infectious rhinitis, "red nose", infectious catarrh of the upper respiratory tract) is an acute contagious disease of cattle, characterized mainly by catarrhal necrotic lesions of the respiratory tract, fever, general depression and conjunctivitis, as well as pustular vulvovaginitis and abortion.

The causative agent of IRT - Herpesvirus bovis 1, belongs to the family of herpesviruses, DNA-containing, the diameter of the virion is 120 ... 140 nm. 9 structural proteins of this virus have been isolated and characterized.

RTI virus is easily cultivated in a number of cell cultures, causing CPP. The reproduction of the virus is accompanied by the suppression of mitotic cell division and the formation of intranuclear inclusions. It also has hemagglutinating properties and tropism for cells of the respiratory and reproductive organs and can migrate from the mucous membranes to the central nervous system, is able to infect the fetus at the end of the first and second half of pregnancy.

At - 60 ... -70 "C, the virus survives 7 ... 9 months, at 56 ° C it is inactivated after 20 minutes, at 37 ° C - after 4 ... 10 days, at 22 ° C - after 50 days. At 4 " With the activity of the virus decreases slightly. Freezing and thawing reduces its virulence and immunogenic activity.

Formalin solutions 1: 500 inactivate the virus after 24 hours, 1: 4000 - after 46 hours, 1: 5000 - after 96 hours. In an acidic environment, the virus quickly loses its activity, it remains for a long time (up to 9 months) at pH 6.0 ... 9.0 and a temperature of 4 °C. There is information about the survival of the virus in bull semen stored at dry ice temperature for 4 ... 12 months, and in liquid nitrogen - for 1 year. The possibility of virus inactivation in bull semen was shown when it was treated with a 0.3% trypsin solution.

Sources of the causative agent of infection are sick animals and latent virus carriers. After infection with a virulent strain, all animals become latent carriers of the virus. Breeding bulls are very dangerous, because after getting sick they secrete the virus for 6 months and can infect cows during mating. The virus is released into the environment with nasal secretions, discharge from the eyes and genitals, with milk, urine, feces, and semen. Wildebeest are believed to be the reservoir of the RTI virus in African countries. In addition, the virus can replicate in ticks, which play an important role in causing the disease in cattle.

The factors of transmission of the virus are air, feed, semen, vehicles, care items, birds, insects, as well as humans (farm workers). Ways of transmission - contact, airborne, transmissible, alimentary.

Susceptible animals are cattle regardless of sex and age. The disease is most severe in beef cattle. In the experiment, it was possible to infect sheep, goats, pigs, and deer. Animals usually fall ill 10...15 days after entering a dysfunctional farm.

The incidence of RTI is 30...100%, mortality - 1...15%, may be higher if the disease is complicated by other respiratory infections.

In the primary foci, the disease affects almost the entire livestock, while mortality reaches 18%. IRT often occurs in industrial-type farms when completing groups of animals brought from different farms.

When it enters the mucous membranes of the respiratory or genital tract, the virus invades the epithelial cells, where it multiplies, causing their death and desquamation. Then ulcers form on the surface of the mucous membrane of the respiratory tract, and nodules and pustules form in the genital tract. From the primary lesions, the virus enters the bronchi with air, and from the upper respiratory tract it can enter the conjunctiva, where it causes degenerative changes in the affected cells, which provokes an inflammatory response of the body. Then the virus is adsorbed on leukocytes and spreads through the lymph nodes, and from there it enters the blood. Viremia is accompanied by general depression of the animal, fever. In calves, the virus can be carried by blood into the parenchymal organs, where it multiplies, causing degenerative changes. When the virus passes through the blood-brain and placental barriers, pathological changes appear in the brain, placenta, uterus and fetus. The pathological process also largely depends on the complications caused by the microflora.

The incubation period averages 2-4 days, very rarely more. Basically, the disease is acute. There are five forms of IRT: upper respiratory tract infections, vaginitis, encephalitis, conjunctivitis, and arthritis.

With the defeat of the respiratory organs, chronic serous-purulent pneumonia is possible, in which about 20% of calves die. In the genital form, the external genital organs are affected, endometritis sometimes develops in cows, and orchitis in sires, which can cause infertility. In bulls used for artificial insemination, IRT is manifested by recurrent dermatitis in the perineum, buttocks, around the anus, sometimes on the tail, scrotum. Virus-infected semen can cause endometritis and infertility in cows.

Abortions and death of the fetus in the womb are noted 3 weeks after infection, which coincides with an increase in the titer of antibodies in the blood of pregnant convalescent cows, the presence of which does not prevent abortions and fetal death in the womb.

A tendency of IRT to a latent course was noted with genital form. In the epithelium of the mucous membrane of the vagina, its vestibule and vulva, numerous pustules of different sizes are formed (pustular vulvovaginitis). Erosions and sores appear in their place. After healing of ulcerative lesions, hyperemic nodules remain on the mucous membrane for a long time. In sick bulls, the process is localized on the prepuce and penis. The formation of pustules and vesicles is characteristic. In a small proportion of pregnant cows, abortions, resorption of the fetus or premature calving are possible. Aborted animals, as a rule, had previously had rhinotracheitis or conjunctivitis. Among aborted cows, lethal outcomes due to metritis and fetal decomposition are not excluded. However, cases of abortions are not uncommon in the absence of inflammatory processes on the mucous membrane of the cow's uterus. With IRT, there are cases of acute mastitis. The udder is sharply inflamed and enlarged, painful on palpation. The milk yield is sharply reduced.

At meningoencephalitis along with oppression, a disorder of motor functions and an imbalance are noted. The disease is accompanied by muscle tremor, lowing, gnashing of teeth, convulsions, salivation. This form of the disease mainly affects calves 2-6 months of age.

Respiratory form infection is characterized by a sudden increase in body temperature up to 41 ... 42 "C, hyperemia of the nasal mucosa, nasopharynx and trachea, depression, dry painful cough, profuse serous-mucous discharge from the nose (rhinitis) and foamy salivation. As the disease develops, mucus becomes thick, mucous plugs and foci of necrosis are formed in the respiratory tract.In severe disease, signs of asphyxia are noted.Hyperemia extends to the nasal mirror ("red nose").The etiological role of the IRT virus in mass keratoconjunctivitis of young cattle has been proven.In young cattle, the disease sometimes manifests itself as encephalitis. It begins with sudden excitement, riot and aggression, impaired coordination of movements. Body temperature is normal. In young calves, some strains of RTI virus cause acute gastrointestinal disease.

In general, in sick animals, the respiratory form is clinically clearly expressed, the genital form often goes unnoticed.

An autopsy of animals killed or dead in acute respiratory form usually reveals signs of serous conjunctivitis, catarrhal-purulent rhinitis, laryngitis and tracheitis, as well as damage to the mucous membranes of the adnexal cavities. The mucous membrane of the turbinates is edematous and hyperemic, covered with mucopurulent overlays. In places, erosive lesions of various shapes and sizes are revealed. Purulent exudate accumulates in the nasal and adnexal cavities. On the mucous membranes of the larynx and trachea, petechial hemorrhages and erosions. In severe cases, the mucosa of the trachea undergoes focal necrosis; in dead animals, bronchopneumonia is possible. In the lungs there are focal areas of atelectasis. The lumen of the alveoli and bronchi in the affected areas are filled with serous-purulent exudate. Severe swelling of the interstitial tissue. When the eyes are affected, the conjunctiva of the eyelid is hyperemic, with edema, which also extends to the conjunctiva of the eyeball. The conjunctiva is covered with sebaceous plaque. Often, papillary tubercles about 2 mm in size, small erosions and sores are formed on it.

In the genital form, pustules, erosions and sores are visible on the highly inflamed mucous membrane of the vagina and vulva at different stages of development. In addition to vulvovaginitis, sero-catarrhal or purulent cervicitis, endometritis, and much less often proctitis can be detected. In sires, in severe cases, phimosis and paraphimosis join pustular balanoposthitis.

Fresh aborted fetuses are usually edematous, with minor autolytic phenomena. Small hemorrhages on the mucous membranes and serous membranes. After a longer period after the death of the fetus, the changes are more severe; in the intermuscular connective tissue and in the body cavities, a dark red liquid accumulates, in the parenchymal organs - foci of necrosis.

When the udder is affected, serous-purulent diffuse mastitis is detected. The cut surface is edematous, distinctly granulated due to an increase in the affected lobules. When pressed, a cloudy, pus-like secret flows from it. The mucous membrane of the cistern is hyperemic, swollen, with hemorrhages. With encephalitis in the brain, hyperemia of blood vessels, swelling of tissues and small hemorrhages are detected.

IRT is diagnosed on the basis of clinical and epizootological data, pathological changes in organs and tissues with mandatory confirmation by laboratory methods. Latent infection is established only by laboratory tests.

Laboratory diagnostics includes: 1) virus isolation from pathological material in cell culture and its identification in RN or RIF; 2) detection of RTI virus antigens in pathological material using RIF; 3) detection of antigens in the blood serum of sick and recovered animals (retrospective diagnosis) in RN or RIGA.

For virological examination, mucus is taken from sick animals from the nasal cavity, eyes, vagina, prepuce; from the forcedly killed and fallen - pieces of the nasal septum, trachea, lung, liver, spleen, brain, regional lymph nodes, taken no later than 2 hours after death. Blood serum is also taken for retrospective serological diagnosis. For laboratory diagnostics IRT use a set of bovine IRT diagnosticums and a set of erythrocyte diagnosticum for serodiagnosis of infection in RIGA.

Diagnosis of IRT is carried out in parallel with the study of the material for parainfluenza-3, adenovirus infection, respiratory syncytial infection and viral diarrhea.

Preliminary diagnosis for IRT in cattle is made on the basis of positive results of antigen detection in pathological material using REEF taking into account epizootological and clinical data, as well as pathological changes. The final diagnosis is established on the basis of the coincidence of the results of the RIF with the isolation and identification of the virus.

In the differential diagnosis of infectious rhinotracheitis, it is necessary to exclude foot and mouth disease, malignant catarrhal fever, parainfluenza-3, adenovirus and chlamydial infections, viral diarrhea, respiratory syncytial infection, pasteurellosis.

The disease is accompanied by persistent and long-term immunity, which can be transmitted to offspring with colostrum antibodies. The immunity of recovered animals lasts at least 1.5...2 years, however, even pronounced humoral immunity does not prevent the persistence of the virus in convalescent animals, and they should be considered as a potential source of infection for other animals. Therefore, all animals with antibodies to RTI should be considered as carriers of the latent virus.

139. The reservoir of nutrients in developing bird embryos is

Given the complex and rather lengthy process of embryogenesis in birds, it is necessary to form special temporary extra-embryonic - provisional organs. The first of these forms the yolk sac, and subsequently the rest of the provisional organs: the amniotic membrane (amnion), serous membrane, allantois. In evolution before, the yolk sac was found only in sturgeons, which have a sharply telolecithal cell and the process of embryogenesis is complex and lengthy. During the formation of the yolk sac, fouling of the yolk with parts of the leaves, which we call extraembryonic leaves or extraembryonic material, is noted. But the extraembryonic endoderm begins to grow on the edge of the yolk. The extra-embryonic mesoderm is stratified into 2 sheets: visceral and parietal, while the visceral sheet is adjacent to the extra-embryonic endoderm, and the parietal - to the extra-embryonic ectoderm.

The extra-embryonic ectoderm pushes the protein aside and also overgrows the yolk. Gradually, the yolk masses are completely surrounded by a wall consisting of the extra-embryonic endoderm and the visceral sheet of the extra-embryonic mesoderm - the first provisional organ, the yolk sac, is formed.

Functions of the yolk sac. The endoderm cells of the yolk sac begin to secrete hydrolytic enzymes that break down the yolk masses. The cleavage products are absorbed and transported through the blood vessels to the embryo. So the yolk sac provides trophic function. From the visceral mesoderm, the first blood vessels and the first blood cells are formed and, therefore, the yolk sac also performs a hematopoietic function. In birds and mammals, among the cells of the yolk sac, cells of the genital bud, the gonoblast, are found early.

140. Reactivation. What it is?

By changing the genotype, mutations are divided into point (localized in individual genes) and gene (affecting larger parts of the genome).
Virus infection of sensitive cells is multiple in nature, i.e. several virions enter the cell at once. In this case, viral genomes in the process of replication can cooperate or interfere. Cooperative interactions between viruses are represented by genetic recombination, genetic reactivation, complementation, and phenotypic mixing.
Genetic recombination is more common in DNA-containing viruses or RNA-containing viruses with a fragmented genome (influenza virus). During genetic recombination, an exchange occurs between homologous regions of viral genomes.
Genetic reactivation is observed between the genomes of related viruses with mutations in different genes. When the genetic material is redistributed, a full-fledged genome is formed.
Complementation occurs when one of the viruses infecting a cell synthesizes a nonfunctional protein as a result of a mutation. The wild-type virus, synthesizing a complete protein, makes up for the absence of it in the mutant virus.

Depending on the preparation technique, cell cultures are classified into:

- single layer– cells are able to attach and multiply on the surface of chemically neutral glass or plastic.

- suspension- cells multiply in the entire volume of the nutrient medium when it is stirred.

- organ- whole pieces of organs and tissues that retain the original structure outside the body (limited use).

The most widespread are single layer cell cultures, which can be divided depending on the number of viable generations into

1) primary (primarily trypsinized),

2) semi-transplantable (diploid)

3) transplantable.

Origin they are classified into embryonic, neoplastic and from adult organisms.

By morphogenesis- on fibroblastic, epithelial, etc.

Primary cell cultures are cells of any human or animal tissue that have the ability to grow as a monolayer on a plastic or glass surface coated with a special nutrient medium. The life span of such crops is limited. In each case, they are obtained from the tissue after mechanical grinding, treatment with proteolytic enzymes and standardization of the number of cells. Primary cultures derived from monkey kidneys, human embryonic kidneys, human amnion, chicken embryos are widely used for the isolation and accumulation of viruses, as well as for the production of viral vaccines.

semi-transplantable(or diploid ) cell cultures - cells of the same type, capable of withstanding up to 50-100 passages in vitro, while maintaining their original diploid set of chromosomes. Diploid strains of human embryonic fibroblasts are used both for the diagnosis of viral infections and in the production of viral vaccines. The most commonly used cultures are human embryonic fibroblasts (WI-38, MRC-5, IMR-9), cows, pigs, sheep, etc.

transplanted cell lines are characterized by potential immortality and heteroploid karyotype. Primary cell cultures can be the source of continuous lines(for example, SOC - the heart of a cinamobus monkey, PES - the kidneys of a pig embryo, VNK-21 - from the kidneys of day-old Syrian hamsters; PMS - from a kidney of a guinea pig, Vero - a kidney of a green monkey, etc.) individual cells of which show a tendency to endless reproduction in vitro. The set of changes leading to the appearance of such features from cells is called transformation, and the cells of transplanted tissue cultures are called transformed. Another source of transplantable cell lines are malignant neoplasms. In this case, cell transformation occurs in vivo. The following lines of transplanted cells are most often used in virological practice: HeLa - obtained from cervical carcinoma; Ner-2 - from carcinoma of the larynx; Detroit-6 - from lung cancer metastasis to the bone marrow; RH - from human kidney, KB - oral cavity carcinoma, RD - human rhabdomyosarcoma.

Organ cultures- are sections of animal organs prepared under sterile conditions, which for a certain period (days, weeks) retain their vital activity in special cultivation conditions

The most common are single-layer cell cultures, which can be divided into primary (primarily trypsinized), semi-transplanted (diploid), transplantable, transfected.

Origin they are subdivided into embryonic, tumoral and from adult organisms; by morphogenesis- on fibroblastic, epithelial, etc.

Primary cell cultures are cells of any human or animal tissue that can be cultivated as a monolayer on a plastic or glass surface in a special nutrient medium, but are not capable of long-term reproduction. The life span of such crops is limited. In each case, they are obtained from the tissue after mechanical grinding, treatment with proteolytic enzymes and standardization of the number of cells. Primary cultures derived from monkey kidneys, human embryonic kidneys, human amnion, chicken embryos are widely used for the isolation and accumulation of viruses, as well as for the production of viral vaccines.

semi-transplantable (diploid ) cell cultures - cells of the same genotype, capable of withstanding up to 50-100 passages in vitro, while maintaining their original diploid set of chromosomes. Diploid lines of human embryonic fibroblasts are used both for the diagnosis of viral infections and in the production of viral vaccines.

transplanted cell lines are characterized by immortality and a heteroploid karyotype. The source of transplanted lines can be primary cell cultures (for example, SOC - from the heart of a cynomolgus monkey, PES - from the kidneys of a pig embryo, VNK-21 - from the kidneys of day-old Syrian hamsters; PMS - from a kidney of a guinea pig, etc.), individual cells of which are found tendency to multiply endlessly in vitro. The set of changes that lead to the appearance of such properties in cells is called transformation, and the cells of transplanted tissue cultures - transformed.

Another source of continuous cell lines is malignant neoplasms. In this case, cell transformation occurs in vivo. The following transplantable cell lines have been obtained and most widely used in virological practice: HeLa - obtained from cervical carcinoma; Hep-2 - from carcinoma of the larynx; Detroit-6 - from lung cancer metastasis to the bone marrow; RH - from human kidney tumor.

Transfected cell cultures. Experimental lines of cell cultures have been developed by transfection (transfer) of virus genes that control the biosynthesis of surface antigens. Such cell cultures express the surface protein of a certain virus (HBs antigen, gp120, etc.) on the culture cell membrane. Such cell cultures are used to study the immunological mechanisms of the pathogenesis of viral infections, the development of chemotherapeutic and immunobiological drugs.

To ensure the vital activity of cultured cells, it is necessary culture media . By appointment, they are divided into growth and support. V growth Nutrient media should contain more nutrients that ensure active cell reproduction and monolayer formation. Supportive media ensure the survival of cells in an already formed monolayer during the period of reproduction of viruses in them.

Standard synthetic media, such as Synthetic 199 media and Needle media, are widely used. Regardless of the purpose, all nutrient media for cell cultures are designed on the basis of a balanced salt solution. Most often it is Hank's solution. An integral component of most growth media is the blood serum of animals (calf, bull, horse), without the presence of 5-10% of which, cell reproduction and the formation of a monolayer do not occur. Serum is not included in maintenance media. In order to prevent the possible growth of microorganisms, antibiotics are added to the nutrient media.

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cell virus culture

Introduction

1. Types of cell cultures

2. Nutrient media

4.1 Virus detection

Bibliography

Introduction

For the quantitative accumulation of viruses, cell cultures are the most convenient system. The first attempts to cultivate animal cells outside the body date back to the end of the last century. These fragmentary observations indicated the possibility of preserving the viability of tissues and cells under artificial conditions and marked the beginning of deep scientific research on tissue cultures.

A great merit in the development of methods for cultivating tissues belongs to Carrel. He was the first to prove the possibility of reproduction of animal cells in artificial conditions and thereby demonstrated their "immortality" and similarity to unicellular free-living organisms. Significant progress in this direction has been made by a group of researchers led by Earl. They were the first to obtain the growth of a large number of cells on glass and in liquid stirred suspension. The advent of antibiotics and advances in artificial culture media have ushered in a new era in the development of tissue culture techniques.

Tissue cultures have long been used to solve various problems in biology and medicine. However, only the successes in the field of virology achieved with the help of tissue cultures were a powerful stimulus for their development to the present level.

Cultivation of viruses helps to solve a number of theoretical problems associated with studying the features of the "virus-cell" interaction. In addition, the solution of a number of applied problems related to the diagnosis and production of drugs for the prevention of viral infections is impossible without the accumulation of virus-containing raw materials.

1. Types of cell cultures

The transfer of the cells of a whole organism to the conditions of life in vitro ceases their existence as one of the numerous structural elements of the tissue or organ in which they were previously included. At the same time, the cells get out of control of neurohumoral factors and acquire a number of features that depend both on the very fact of cell rejection from these tissues and on the specific conditions of their existence in vitro.

Cells or tissues living outside the body are characterized by a whole complex of metabolic, morphological and genetic features that are sharply different from the properties of cells of organs and tissues in vivo.

Several types of surviving and growing tissue and cell cultures are distinguished depending on the method of primary explantation of the tissue and the technique of its cultivation. Single-layer and suspension cultures of growing cells are most widely used. They form the basis of modern laboratory and industrial virological practice.

There are two main types of single-layer cell cultures: primary and transplanted.

The term "primary" refers to a cell culture obtained directly from human or animal tissues in the embryonic or postnatal period. The life span of such crops is limited. After a certain time, phenomena of nonspecific degeneration appear in them, which is expressed in granulation and vacuolization of the cytoplasm, rounding of cells, loss of communication between the cells and the solid substrate on which they were grown. Periodic change of the medium, changes in the composition of the latter, and other procedures can only slightly increase the lifetime of the primary cell culture, but cannot prevent its final destruction and death. In all likelihood, this process is associated with the natural extinction of the metabolic activity of cells that are out of control of neurohumoral factors acting in the whole organism.

Only individual cells or groups of cells in the population against the background of degeneration of most of the cell layer can retain the ability to grow and reproduce. These cells, having found the potency of endless reproduction in vitro, give rise to transplanted cell cultures after repeated inoculations.

There are lines and strains of transplanted cells. The first term refers to transplantable cells characterized by potential immortality and, as a rule, a heteroploid karyotype; the second term refers to semi-transplantable cells with a diploid set of chromosomes and a limited lifespan in vitro. The appearance of both those and other cells is associated with the process of selection in the cell population of primary cultures, which are thus the source of all lines and strains of transplanted cells.

The main advantage of transplantable cell lines, in comparison with any primary culture, is the potential for unlimited reproduction outside the body and the relative autonomy that brings them closer to bacteria and unicellular protozoa.

The ability of transplanted cells to reproduce endlessly in vitro marks a qualitative leap, as a result of which cells acquire the ability to autonomous existence, similar to microorganisms grown on artificial nutrient media. The totality of changes leading to the appearance of such features in cells is called transformation, and the cells of transplanted tissue cultures are called transformed.

Improvements in cell culture techniques have greatly expanded the possibilities of obtaining transplantable cell lines from a wide variety of animal and human tissues. At the same time, no age limit was found, above which tissues would lose the ability to adapt to unlimited growth in vitro, i.e. to transformation.

Another source of transplanted cell lines are malignant neoplasms. In this case, cell transformation occurs in vivo as a result of the development of a pathological process, the etiology of which remains largely unclear.

Not all malignant neoplasms are capable of giving rise to transplantable cell cultures. So, for example, unsuccessful attempts were made to obtain transplantable cells from cancerous tumors of the human stomach and mammary glands. It is difficult to adapt to life in vitro cells of squamous cell carcinoma of the skin and mucous membranes. On the other hand, lines are relatively easily derived from the tissues of sarcomas and malignant tumors of the nervous system.

2. Nutrient media

In any cell culture, there are cell and liquid phases. The liquid phase ensures the vital activity of culture cells and is a nutrient medium of various composition and properties.

All media according to their purpose are divided into growth and support. The composition of growth media should contain more nutrients to ensure active multiplication of cells to form a monolayer on the glass surface or a sufficiently high concentration of cell elements in suspension (when obtaining suspension cultures). In fact, supporting media should only ensure the survival of cells in an already formed monolayer during reproduction of viral agents in cells.

Growth and support media are multicomponent. They can include both natural products (amniotic fluids, animal sera), and substrates obtained as a result of partial processing of natural products (embryonic extracts, lactalbumin hydrolyzate, hemohydrolyzate, aminopeptide, etc.), as well as synthetic chemically pure substances (amino acids, vitamins, salts).

As an example of a nutrient medium consisting entirely of natural components, Buckley's medium, proposed for growing cell cultures from the renal epithelium of monkeys, can be mentioned. This medium includes bovine amniotic fluid (85%), horse serum (10%), and bovine fetal extract (5%).

All natural products are of low standard, their use is associated with a great danger of microbial and viral contamination of cell cultures. In this regard, they are gradually being replaced by synthetic standard mixtures. Synthetic medium 199 and Needle medium find the greatest application. Media containing strictly defined amounts of salts, amino acids and vitamins are widely used.

Regardless of the intended use, all tissue culture media are designed with some sort of balanced salt solution with adequate buffering capacity. Most often they are solutions of Hanks and Earl. These solutions are an essential component of any nutrient medium. An integral component of most growth media is animal serum (calf, bovine, horse), without the presence of 5--10% of which cell reproduction and monolayer formation does not occur.

The inclusion of serum at the same time prevents the creation of growth media of precise chemical composition, which are very important for the development of fundamental research in cell physiology, since together with serum (or its derivatives) a whole complex of uncontrolled factors is introduced, which vary depending on the series of serum.

In the 1950s, serum-free media of exact chemical composition were proposed by Ewans and Waymouth. However, these media did not provide those indicators of cell proliferative activity, which determine media with the addition of sera. In this regard, the work of Birch and Pirt is of interest. They showed that the inclusion of sulfate salts of Fe, Zn, Cu, and also MnC1 2 is decisive for ensuring intensive cell growth in serum-free media.

Antibiotics are added to the growth media, as well as to the buffer solution for washing tissues. They are introduced into the medium immediately before use at the rate of 1 ml of the stock solution of antibiotics per 500 ml of the medium.

Below is the composition and method of preparation of one of the common culture media.

Wednesday Needle

l-arginine - 17.4

l-cystine - 4.8

l-histidine - 3.1

l-isoleucine - 26.2

l-leucia - 13.1

l-lysine - 14.6

l-methionine - 7.5

l-phenylalanine - 8.3

l-threonine - 11.9

l-tryptophan - 2.0

l-tyrosine - 18.1

l-valine - 11.7

biotin - 0.24

choline - 0.12

choline chloride - 0.14

vitamin B 12 (pteroylglutamic acid) - 0.44

nicotinamide - 0.12

pantothenic acid - 0.22

calcium pantothenate - 0.48

pyridoxal (pyridoxine hydrochloride) - 0.20

thiamine hydrochloride - 0.34

riboflavin - 0.04

sodium chloride - 5850.0

potassium chloride - 373.0

monosubstituted sodium phosphate (NaH 2 PO 4. H 2 O) - 138.0

calcium chloride - 111.0

sodium bicarbonate (NaHCO 3) - 1680.0

magnesium chloride (MgCl 2. 6H 2 O) - 102.0

glucose - 900.0

l-glutamine - 146.2--292.3

penicillin - 50.0

streptomycin - 50.0

phenol red - 5.0

water up to 1000.0

Cooking.

Solution 1. In 500 ml of water heated to approximately 80 °, the above amounts of amino acids are dissolved with stirring, after which l-glutamine and phenol red are added.

Solution 2. In 100.0 ml of water, dissolve the inorganic salts, with the exception of sodium bicarbonate (NaHCO 3 ), mix with the previous solution of inorganic salts, and add biotin and vitamin B 12 .

Solution 3. All vitamins are dissolved in 200.0 ml of water, except for biotin and ptero-ylglutamic acid, and antibiotics - penicillin and streptomycin. All solutions are sterilized by filtration through a glass suction filter (porous glass plate, pore size 0.7--1.5) or through Seitz asbestos cellulose plates after preliminary washing with water, and then with the prepared solution.

Mix 500 ml of solution 1 + 200 ml of solution 2 + 200 ml of solution 3 and dilute to 1000.0 ml with sterile water. You can add 1 mg of inositol per 1 liter.

3. Obtaining cell cultures

3.1 Preparation of primary cell cultures

Primary - is called a culture obtained from tissue and grown in vitro before the start of subculturing, that is, before the first crop. The primary culture is devoid of many of the cells present in the original tissue, since not all cells are able to adhere to the substrate and survive in vitro. In the process of cell cultivation, the culture is relatively depleted of non-dividing or slowly dividing cells.

At the first stage of obtaining a primary culture, a sterile removal of a tissue fragment, an animal organ and its mechanical or enzymatic disaggregation is carried out. The tissue is crushed to pieces with a volume of up to 1-3 mm, the pieces of tissue are washed from erythrocytes with Hank's solution with antibiotics. Trypsin (0.25% crude or 0.01-0.05% purified) or collagenase (200-2000 units/ml, crude) and other proteolytic enzymes are used for tissue disaggregation. This method of obtaining culture provides a high yield of cells.

Primary cultures can also be obtained from tissue pieces, 1 mm in volume, which adhere to the surface of the substrate due to their own adhesiveness or the presence of notches on the dish, or using a plasma clot. In these cases, cells will grow from fragments. Cells migrating from explants can be used for passage. Tissue fragments (explants) are transferred to new plates, migrating cells can be removed by subculture, a mixture of versene and trypsin, and the remaining explants will form new outgrowths.

Such primary cultures as chick embryo fibroblast culture, calf kidney cell culture, and leukocytes are known.

3.2 Obtaining monolayer continuous cell cultures

As noted above, one of the methods for obtaining transplantable cell lines is the selection of cells with increased activity of growth and reproduction from the population of primary cultures. The selection can be carried out by regularly changing the environment, washing the monolayer of the primary trypsinized cell culture. For this, mattresses with a well-formed cell monolayer are selected (the mattresses themselves must have a flat bottom and must not have scratches and dull spots on the walls). The growth medium prepared for a systematic change is poured into small vessels (to exclude bacterial contamination) and stored at t - 4 °.

The medium is changed regularly, at least once a week. During the first 3 weeks, 20-30% of the volume of the growth medium is replaced, during the next 3-4 weeks - 50-60%, later a complete change of the medium is carried out. Fresh environment immediately before work is heated to t - 37 °.

As the media change, cells change their morphology. Some of the cells are rounded and fall off the glass. Most of the cells shrink towards the center, and the monolayer acquires a stellate appearance. The cells themselves are slightly elongated. After 7-10 changes of the environment in mattresses, as a rule, new cellular elements begin to appear, and in different cultures they have different morphology.

In the culture of chick embryo kidney cells, rounded cellular elements appear in the center of the monolayer or between its contracted areas, from which clusters in the form of small colonies gradually form. In the culture of monkey kidney cells, single formations resembling grains appear. The cells are tightly adjacent to each other, forming small transparent colonies. The number of such cells increases slowly, the size of the colonies also almost does not increase. Colonies appear not only on the bottom of the mattress, but also on the side surfaces, at the border of the nutrient medium. Therefore, a prerequisite for changing the nutrient medium is to maintain its constant volume. In the culture of human embryonic kidney cells against the background of mass degeneration of the monolayer constricted into strands, large polygonal cells with long processes are revealed.

Atypical cellular elements that have arisen during the selection process should be separated from the rest of the monolayer and transferred to separate test tubes or mattresses. For this, the versenization method or mechanical cleavage of atypical cells using a bacteriological loop or spatula can be used. The latter method is especially necessary when working with a cell culture of monkey kidneys, where colonies of atypical cells are tightly attached to the glass and do not separate from it themselves.

When changing the nutrient medium in the event of the appearance of atypical cells, it is recommended to carefully remove most of the growth medium, and with a smaller part, vigorously rinse the monolayer and pour this medium into test tubes. In this case, atypical cells may appear in the medium that have the ability to form colonies suitable for further subcultures.

After transferring the culture of atypical cells to a new dish, it is advisable to continue monitoring the main culture, since the process of breeding a new cell line is very complicated, and not always selected atypical elements give rise to a viable line of transplanted cells. It is necessary that all work on obtaining new cell lines, which lasts for many months, be carried out with the same nutrient media, animal sera and series of antibiotics.

Primary cultures such as golden hamster kidney cell culture (BHK), Siberian mountain ibex kidney cell culture (PSGC), green monkey kidney cell culture (CV) are known.

3.3 Roller cell culture

Until recently, tissue cultures were used in virology mainly in the form of single-layer stationary cultures. In many cases, this cell culture method is indispensable. Many years of experience shows that when using single-layer stationary cultures, a number of difficulties are encountered, associated with huge expenditures of working time and materials. From this point of view, roller cultures are more profitable, which are economical, characterized by an optimal ratio of the useful area of ​​cultivation to the volume of the nutrient medium and open up favorable opportunities for the accumulation of cell mass.

The term "roller culture" refers to a culture method in which a cell monolayer is located over the entire cylindrical surface of horizontally rotating vessels and is periodically washed with a nutrient medium. For certain reasons, a certain number of cells can in some cases be weighed in the culture medium. In this embodiment, the cell culture is called roller-suspension. The "yield" of the cell culture will be greater, the larger the area from which the cells are harvested. Researchers have used various ways to increase the surface area on which cells can attach and multiply. To do this, various bases with a large specific surface were used: hard, spongy, made of wool, collagen, on glass spirals, etc. These methods were not widely used, since they made manipulations with cells much more difficult, but it should be noted described by L. S. Ratner and V. A. Krikun method of multi-tiered cultivation. Cells were cultured in 1.5, 10 and 15 liter bottles fully loaded with oval glass tubes parallel to the longitudinal axis of the vessels. In this case, the useful area increased in comparison with stationary single-layer cultures in vessels of the same volume by 20 times. Cultivation took place in 2 stages. At the first stage, the bottles were rotated around a horizontal axis in order to evenly distribute the cells. Subsequently, when the cells were attached to the glass, cultivation continued in a stationary position. The described culture system has been successfully used for the cultivation of FMDV.

The rotation of the vessels in which the cells multiplied turned out to be more effective. Initially, cells were grown in test tubes, but with the development of technical equipment, the volume of culture vessels increased, and researchers switched from growing cells in rotating test tubes to rotating bottles. Roller cultures began to be used both for the accumulation of cells and for the propagation of viruses.

For roller cultivation, special rack-and-tier apparatus for rotating bottles or drums are used. Most often, 0.5--3-liter bottles are used for cultivation. Under production conditions, their volume can reach 20 liters or more. Roller culture apparatuses are simple, reliable and easy to maintain. Of great importance is the speed of rotation of the bottles. It should not be too high, so as not to prevent cell attachment, but also not too low, since prolonged exposure of the cells to the gas phase worsens their nutritional conditions. In the works of various authors, the speed of rotation of the bottles varied from 8–12 rpm to 1 rpm. The most suitable for various cell cultures was the speed of rotation of the vials in the range of 0.5--1 rpm. Recommended within the first 30 minutes. after seeding the cells, ensure a high rotation speed of the vials (0.5–1.0 rpm) for uniform distribution of materials, then transfer them to a low rotation speed (20–30 rpm).

The seed concentration in 1 ml of the medium is 60-80 thousand for SPEV, VNK-21, HeLa, L cells, 100-200 thousand for diploid cells, 200-300 thousand for primary cultures. The volume of the growth medium should be 1/ 10, 1/20 of the volume of a roller bottle. The roller method of cultivation allows you to get a larger number of cells. Thus, from a vial with a capacity of 3 l, it is possible to obtain a crop of HeLa cells by 8 times, VNK-21 - by 9 times, AO - 11 times greater than with a monolayer stationary culture of a vial with a capacity of 1 l. The multiplicity of media savings when using 3-liter vials is 2.8 for HeLa cells; VNK-21 - 5.0, AO - 4.0. The ability to grow and reproduce under roller conditions is possessed by subcultures, transplanted cultures, and less often primary, as well as diploid cell strains. For better reproduction of cells in roller devices, it is necessary to adapt them to new cultivation conditions. The roller culture method allows obtaining large numbers of cells. The advantage of roller cultures in comparison with traditional stationary cultures is, in particular, more economical use of nutrient media and a higher yield of viral antigen (by 1-2 lg).

3.4 Suspension cell culture

In 1953, Owens and co-workers first demonstrated the ability of cells to multiply in a liquid medium in a freely suspended state. Since then, the suspension culture method has attracted the attention of researchers due to its high efficiency in the accumulation of large amounts of cells. It turned out that cells of transplanted lines, unlike other types of cell cultures, can be cultivated for a long time in a suspended state. Cells under these conditions multiply without attaching to the walls of the culture vessel, being in a suspension state, due to the constant mixing of the medium. Under the optimal growing regime, cells in suspensions multiply rapidly and have a higher “yield” than in stationary cultures. Primary and continuous cell lines have different ability to grow in suspension. Thus, heteroploid and aneuploid permanent lines, in contrast to pseudodiploid lines, adapt faster to growth in suspension.

Suspension cultures are prepared from monolayer cultures. The cells are peeled off the glass with versene and trypsin solutions. The cell pellet after centrifugation (1000 rpm) is resuspended in fresh nutrient medium. The prepared suspension is placed in culture vessels (reactors, fermenters) and grown with constant stirring. In scientific studies, the concentration of cells in the initial suspension ranges from 0.3 to 10x10 per 1 ml. The optimal concentration of cells in the initial suspension should be 2-5x10 per 1 ml. According to Earle and his collaborators, the concentration of cells in the suspended culture should be such that the logarithmic growth phase occurs no later than 16-24 hours after the preparation of the culture. Suspension cell cultures go through characteristic stages: a lag phase, a logarithmic growth phase, a stationary phase, and a logarithmic death phase. In the first stage, as a rule, the number of cells decreases, in the second stage, the cell population increases (logarithmic growth stage), in the third stage, no increase in the number of cells is observed (stationary phase). If the cultivation is continued further, then a period of decrease in the number of cells is found - the phase of logarithmic death (the phase of destruction). The rate of cell reproduction in the logarithmic growth phase is expressed by the generation time. The generation time refers to the period required to double the population of cells in culture. For cell suspension cultivation, an important condition is the mixing of the liquid, which must be continuous and intense enough to keep the cells in suspension, prevent them from settling and attaching to the walls of the vessel, and at the same time not cause mechanical damage.

Mixing of suspension cultures is carried out with bladed magnetic stirrers, as well as circular rocking chairs. Currently, the rotation of vials and bottles around the longitudinal axis (15-40 rpm) is widely used. On magnetic stirrers, the rotation speed is 100--200 rpm. The mixing speed depends on the culture volume; small culture volumes require a low speed, while it must be increased at large volumes. Siliconization is performed to prevent cell settling on the inner surface of the vessel. The silicone coating, due to its hydrophobicity, prevents cells from attaching to the vessel wall.

The inner walls of the culture vessel are moistened with a 5% or 10% silicone solution, and after evaporation of the solvent (benzene, acetone), the vessels are kept at 85°C and 200° (for 30 and 60 minutes, respectively). After cooling, the vessels are filled with hot bidistilled water and left for 2 hours, then they are rinsed 3 times with bidistilled water and dried at 100°C. Vessels are sterilized in an oven at 170°C for 2 hours.

The maximum cell growth in suspension is observed at pH 7.0-7.2. The nutrient media used to grow cells in suspension do not differ from the media used to grow cell lines in monolayer culture. Most often, when cultivating cells in suspensions, Eagle's medium is taken with a double concentration of amino acids and vitamins.

Suspension cultures consume 2-7 times more glucose than monolayer ones. In the process of consuming glucose, cells release lactic acid, toxic to them, into the environment. The consumption of glucose by cells and the production of lactic acid run in parallel and are directly dependent on the density of the cell population. It turned out to be useful to add insulin to the nutrient medium in an amount of 40 to 200 IU per 1 liter. The addition of insulin to the nutrient medium changes the ratio between the amount of glucose absorbed and the amount of lactic acid released. For L cells, this coefficient can be reduced from 74-81 to 37-38%.

Different amino acids are consumed from the nutrient medium by growing cells at different rates. It is noted that the regular addition of arginine (20-40 mg/l) and an increase in the amount of glutamine to 450 mg/l favor the growth of suspended cultures.

The addition of inositol (0.4 mg/l) accelerates the growth of human amniotic cell cultures. It is desirable to add catalase (1 mg/l) and thyroxine (12 mg/l) to the medium.

Of great interest are works devoted to obtaining suspended cell cultures in a synthetic medium that does not contain blood serum. Attempts are being made to increase the viscosity of the nutrient medium at the expense of protein-free ingredients. For this purpose, methylcellulose and hyaluronic acid are used.

Methylcellulose at a concentration of 0.1-0.2% has a maximum protective effect on cells suspended in the medium. The protective effect of methylcellulose is that the molecules form a protective layer around the cell, preventing cell damage when the medium is stirred. A very important indicator of the state of the suspension culture is the partial pressure of oxygen in the liquid phase. The oxygen concentration in the gas phase depends on the density of the cell population and is often below atmospheric. The lack of oxygen leads to the appearance of granulation of the cytoplasm, the cells lose their regular rounded shape. With a slight excess of oxygen, the cells have a well-defined, regular, rounded shape, and become very large under the damaging effect of an excess of oxygen. The optimal oxygen concentration for various cell cultures ranges from 9 to 17% or 293 mm Hg. pillar. At oxygen concentrations above 20%, cell growth is inhibited. Thus, at an oxygen concentration of 24%, the multiplication of rabbit embryonic kidney cells (ERK line) decreased by half, and at 30% it was reduced to zero. An increase in oxygen concentration has a toxic effect on cellular metabolism.

Thus, the reproduction of cells in suspension depends on the concentration of cells in the initial suspension, aeration and pH of the medium, the composition of the nutrient medium, the method of mixing, the volume of the suspension, and other factors.

The homogeneity of the suspension, the possibility of long-term maintenance of cells in the logarithmic phase of growth, the prospects for mathematical modeling of cell growth processes depending on the influence of environmental factors, the convenience of multiple studies of the physiological state of the cell culture in suspension, the high efficiency of the method - this is not a complete list of the advantages of suspension cultures.

Suspension cultures are widely used in virological studies and for the accumulation of large amounts of virus-containing material, in the manufacture of vaccines and diagnostic preparations.

3.5 Cultivation of cells on microcarriers

In 1967, Van Werel proposed a culture method that combines elements of monolayer and suspension cell growth, which he called the "microcarrier" method. Its essence lies in the fact that the cells attach and multiply on the surface of the polymer balls-particles of "microcarriers" (MN), which are contained in suspension using a mixing device, such as a stirrer. On one MN particle with a diameter of 160–230 mm, 350–630 (or an average of 460) cells can fit. In one ml of the medium, several thousand microcarrier particles can be suspended, with their total area ranging from a few to 50 cm 2 /ml.

Cells inoculated into the cultivator attach to the surface of MN particles and multiply to form a continuous monolayer on each individual particle.

The main advantages of this method are:

1) creating uniform conditions throughout the volume of the vessel, which makes it possible to effectively control the necessary parameters (pH, p0 2, etc.); 2) obtaining a high density of the cell population up to 5-6 million cells per 1 ml; 3) cultivation of several hundreds of billions of cells simultaneously; 4) the introduction of constant control over the dynamics of cell growth; 5) a decrease in the growth of contamination due to a reduction in operations associated with depressurization of the culture vessel; 6) significant savings in nutrient media; 7) the ability to keep the grown cells directly on the particles at low temperatures; 8) the ability to artificially create various concentrations of MNs with cells grown on them; 9) the possibility of passing the culture without the use of trypsin by adding fresh portions of the microcarrier.

Microcarriers must have:

A small positive charge in the range of 1.5-1.8 MEKV / g. Due to the fact that most animal cells have a weakly negative charge, they will more easily attach to such an MN:

Density 1.05--1.15 g/cm; the indicated density is optimal for maintaining MN in suspension;

The particle diameter is from 100 to 250 microns, which provides areas for the growth of several hundred cells;

Smooth surface;

Transparency;

Lack of toxicity of components for cells;

Slight absorption of medium components;

Versatility, providing the possibility of using them for primary, diploid and heteroploid cells. Of no small importance are the properties of MH, which allow them to be used repeatedly.

A study of many granular preparations of various chemical nature, including those made from cross-linked (PS) dextran, PS-agarose, PS-polyvinylpyrrolidone, polyacrylnitrite, porous silica gel, polystyrene, capron, nylon, and aluminosilicate, was carried out in order to use them as microcarriers.

Suitable are only a few of them, mainly based on PS-dextran.

Several foreign firms have developed ready-to-use commercial preparations of microcarriers: Cytodex-1, 2, 3 (France, Sweden), Superbit (USA, England), Biosilon (Denmark). The cost of these drugs is quite high, so it is necessary to conduct research on the development and production of domestic MNs.

Cell culture on microcarriers is carried out in conventional suspension culture fermenters. The fermenter should not have any protrusions, pockets, to prevent the accumulation of microcarriers in stagnant zones. Therefore, it is reasonable to use fermenters with a round bottom and smooth walls. The interior of the fermenter must be siliconized to prevent microcarriers from sticking to the glass or stainless steel of the fermenter.

Standard equipment can be used to control crop conditions such as pH and O 2 . The speed of mixing the suspension with the carrier should be 40-60 rpm. Various types of cells are used for cultivation on MH.

Cytodex concentration can vary from 0.5 to 5 mg/ml. However, in the production of prophylactic vaccines, a final cytodex concentration not exceeding 1 mg/ml is usually used. Increasing the concentration to 3 mg/ml and above creates additional difficulties associated with the need for perfusion of the nutrient medium and its partial replacement, which complicates the technological process.

The inoculum concentration of cells, as well as the conditions of cultivation on MN in the first hours, largely determine the optimal parameters for proliferation and maximum accumulation of cells. It was shown that inoculation of 10 cells/ml of a continuous line of monkey kidneys (Vero) and diploid human embryonic fibroblast cells (MRC-5) in a medium volume reduced to 1/3 and with the stirrer switched on periodically (30 rpm) for one minute after every hour for 4 hours, followed by the addition of the nutrient medium to the final volume, leads to an increase in cell proliferation and their number compared to the control (full volume of the nutrient medium at the time of planting the cells and continuous operation of the stirrer from the beginning of cultivation).

Nutrient medium is important for culturing cells on MN. Proper selection of the nutrient medium will also help optimize the process of cell proliferation and their quality. It is necessary to select nutrient media for culturing cells on various types of MNs. It was shown that the transplanted cell line of monkey kidneys (Vero) on cytodex gives the highest yield when using Eagle DME medium (planting cell concentration 10 5 ml) but compared with BME and 199 medium. If the number of cells during planting is reduced to 10 4 , then the best media 199 gives results. All media tested contained 10% fetal serum.

3.6 Growing viruses in cell cultures

Primary cultures, cell strains, and established cell lines are currently used to isolate and propagate animal viruses. In general terms, the procedure is the same for all viruses.

The medium is removed from the cell monolayer and the monolayer is washed with balanced buffered salt solutions (SBSR) or phosphate buffered saline (PBS) to remove inhibitors (antibodies) that may be present in the medium. Virus particles are suspended in a small amount of SBSR or PBS and adsorbed by cells within 30-60 minutes. The saline solutions are then replaced with fresh medium.

Infection of cultivated cells with viruses causes characteristic morphological changes in cells. The final degenerative cellular processes (cytopathogenic effect, CPE) are detected only after a few weeks of growth in the presence of viruses, but in some cases CPE are detected after 12 hours. Details of morphological changes are different in the case of different viruses.

If, instead of a productive infection, the virus causes cellular transformation, then this is also accompanied by characteristic changes in the morphology and characteristics of cell growth.

4. Precautions when handling virus-infected cells

Viruses have a cytopathogenic effect and serve as etiological agents in many human and animal diseases. In addition, many viruses (eg, oncornaviruses, herpesvirus type II, adenoviruses, polyoma virus, and SV40) appear to be tumor causing agents in animals. Due to the ability of viruses to pass through bacterial filters, it can be difficult to exclude viruses from uninfected cell cultures in the presence of virus suspensions, where transmission of the virus through the air of the culture room is possible.

The following precautions are of a general nature and apply only when viruses do not pose any particular risk. When using highly dangerous viruses that pose a health hazard to laboratory personnel or people and animals in the surrounding world, additional precautions should be taken. Viruses of particular concern include Newcastle disease viruses, foot-and-mouth disease viruses, vesicular stomatitis viruses, pox viruses, rabies viruses, herpes type B viruses, and so on. In addition, one cannot be sure that even viruses such as SV40 do not pose a danger to humans.

A special room or group of rooms must be used to infect cells and grow virus-infected cells.

No live viruses should be removed from these rooms unless they are contained in tightly sealed containers. Precautions should be taken to ensure that the outer surfaces of these containers are not contaminated with viral particles.

When working with viruses, special protective clothing (lab coats) should be used. After work, these clothes should be placed in a special tank for autoclaving.

All media and glassware that have been in contact with viruses should be treated with chloros; only after that they can be taken out of the room designed to work with viruses.

All plastic utensils must be placed in a special autoclaving tank.

Equipment for the storage and handling of viral material should be located inside the virus handling facility.

The laboratory should be equipped with two-cycle autoclaves so that laboratory personnel are protected from harmful materials.

4.1 Virus detection

Viruses can be detected and quantified using a number of different tests, such as:

1) Viral activity is measured by determining the amount of virus-containing material required to elicit a specific response in the host. The virus-induced response can be all-or-nothing (i.e. the presence or absence of infection), or it can be quantified, such as the length of time it takes for an infection to appear or the number of lesions in a susceptible cell layer. The quantitative determination of viral activity is called titration.

The smallest amount of virus that can cause an appropriate reaction is called an infectious unit, and the titer of the initial viral suspension is expressed as the number of infectious units per unit volume. For example, if the minimum amount of influenza virus suspension that causes pneumonia in a mouse when intranasally injected with a suspension of infected lung tissue in a dilution of 1:10 6 is 0.1 ml, then this means that the titer of influenza virus in the initial suspension of lung tissue is 10 7 infectious units per 1ml--1/(10~1-10-6)=10 7 .

As a rule, a mandatory component of the virus titration method is a test that allows you to evaluate the result of inoculation as positive or negative. For example, when titrating animal viruses, such a test may be the presence or absence of visible lesions in cell culture, an inflammatory reaction at the site of inoculation of the virus into the skin or cornea, paralysis resulting from the introduction of the virus into the brain of the animal, etc. Sometimes the reproduction of the virus can be detected even in the absence of a visible reaction from the host organism: for example, infection of chicken embryos with myxoviruses can be detected by the appearance of hemagglutinins in the allantoic fluid.

The best results are obtained by titration methods, which are based on counting the number of discrete lesions in a certain area of ​​a layer of cells infected with a known amount of virus-containing material. In cases where the number of virus-susceptible and accessible cells can be considered practically infinitely large, as, for example, when a monolayer culture of bacteria on nutrient agar is infected with a phage or when a continuous monolayer culture of animal cells is infected with a virus, lesions caused by the virus, or plaques formed phage, in this sense, are similar to bacterial colonies on a dense nutrient medium. Just as the number of these colonies is proportional to the number of bacterial cells contained in the titratable preparation, so the number of plaques is directly proportional to the amount of virus added to the monolayer culture, the formation of viral particles by infected cells, which can be identified, for example, by the nature of nucleic acids and particle symmetry.

2) The production of nucleic acids by infected cells, which may have a characteristic density by density gradient centrifugation or a characteristic size by agarose gel electrophoresis or sucrose concentration gradient centrifugation.

3) Formation by infected cells or transformed cells of characteristic antigens, which can be visualized by staining with fluorescent specific antibodies or cause changes in the cell membrane leading to heme adsorption. In the direct method, antibodies generated against viral antigens are combined with a fluorochrome and used to stain infected cells. A positive reaction (yellow-green in a fluorescent microscope) indicates the presence of viral antigens in the cells. This method can therefore be used to detect cellular transformation when antibodies are generated against, for example, early antigens of the SV40 virus, or to detect the formation of mature viruses when antibodies are generated against capsid proteins. In the indirect method, antibodies are not combined with a fluorochrome. Instead, after the interaction of antibodies with antigens in fixed cell preparations, anti-gamma globulin antibodies conjugated with fluorochrome are added to them. This method is more sensitive and avoids conjugation of each individual antibody with a fluorescent dye. Thus, the same fluorescent antibodies to rabbit antibodies (obtained in sheep against rabbit gamma globulins) can be used to stain any antibodies produced in rabbits and interacting with viral antigens in productively infected or transformed cells. An even more indirect, but much more sensitive method that does not require the use of a fluorescent microscope is the peroxidase-antiperoxidase method. According to this method, rabbit antibodies interact with fixed cell antigens and are then coated with goat antibodies to rabbit immunoglobulins conjugated with complexes of horseradish peroxidase and rabbit antiperoxidase. After that, the preparations are treated with diamino-benzidine and hydrogen peroxide; brown color indicates the presence of antigens.

4) The presence of antigens on viral particles can lead to hemagglutination reactions, allowing for quantitative assessment. Many viruses have antigens that can be adsorbed on red blood cells and cause them to stick together. When a large number of viral particles and erythrocytes interact, a network of cells is formed, and the suspension agglutinates, i.e. erythrocytes precipitate. There is some specificity in that certain viruses cause agglutination of the erythrocytes of certain animals, but if an active combination is found, then hemagglutination becomes a rapid test to determine the virus titer. Blood is drawn into a heparinized syringe and the erythrocytes are washed three times with reprecipitation in 0.85% NaCl at 200 g for 10 min. The final erythrocyte sediment is suspended in a 200-fold volume of 0.85% NaCl solution. The most convenient way to test for hemagglutination is in microtiter plates. In a series of microtiter plate wells, add 25 µl of 0.85% NaCl or PBS; 25 μl of virus suspension is added to the first well and the mixture is stirred (1:2 dilution). 25 µl is then transferred from the first well to the second (dilution 1:4) and so on, until the final dilution is 1:2048. After that, 25 µl of erythrocyte suspension is added to each well, the mixture is stirred and left at 4°C, room temperature or 37°C until hemagglutination occurs (1-2 hours). In wells where agglutination has occurred, the erythrocyte sediment has an irregular shape, and in wells where hemagglutination has not occurred, the cell sediment forms a compact dot at the bottom of the well. The dilution in the last well in which haemagglutination occurred is considered the titer, and a value of 1 haemagglutination unit (HAU) is assigned to this dilution. The previous dilution contains 2 HAU respectively, and so on.

5) The formation by infected cells of characteristic enzymes, or enzymes that are easily distinguished by their properties from the corresponding enzymes of the host cells.

4.2 Cultivation of picornaviruses using the example of obtaining poliovirus type 3 in HeLa cell culture

As a specific technique for culturing viruses, a method for growing poliovirus in continuous cells can be cited.

All procedures are carried out under sterile conditions.

Certain sublines of HeLa cells (eg HeLa S3) can grow in low calcium ion media (eg CaS2MEM). For effective infection, it is essential that the cells grow well in a homogeneous suspension. Cells are pelleted by low speed centrifugation (15 min at 900 g) and resuspended in CaS2MEM to a concentration of ~2. 10 7 cells/ml (~1/20 of the original volume).

The virus is added to the cell suspension at a concentration of 10-50 PFU per cell; incubate it on a magnetic stirrer for 1 hour at 35°C.

The suspension is diluted with the same medium to a concentration of 2. 10 6 cells/ml and add 100 μCi of [ 3 H]-uridine.

The cell suspension is incubated for 8 h, after which the cells are pelleted by low speed centrifugation (15 min at 900 g) and washed once with phosphate buffer solution (PBS) free of magnesium and calcium ions.

Cells are resuspended in PBS (5-10 7 cells/ml) and subjected to three freeze-thaw cycles.

Cell debris is removed by centrifugation.

The supernatant is kept for further purification.

Bibliography

1. Adams R. Cell culture methods for biochemists. - M.: Mir, 1983, 263 p.

2. Virology. Methods. / Ed. B. Meikhi. - M.: Mir, 1988, 344 p.

3. Golubev D.B., Sominina A.A., Medvedeva M.N. Guidelines for the use of cell cultures in virology. - L .: Medicine, 1976, 224 p.

4. Dyakonov L.P., Glukhov V.F., Pozdnyakov A.A., Denisenko G.F., Kalmykova T.P. Cultivation of cells and tissues of animals. - Stavropol: Stavrop. Pravda, 1988, part 2, 91 p.

5. Animal cell in culture under. ed. L.P. Dyakonova, V.I. Sitkova. - M.: 2000.

6. Culture of animal cells. Methods. / Ed. R. Freshni. - M.: Mir, 1989, 333 p.

7. Guide to veterinary virology. / Ed. V.N. Shurin. - M.: Kolos, 1965, 687 p.

8. Sergeev V.A. Reproduction and cultivation of animal viruses. - M.: Kolos, 1976, 303 p.

9. Fenner F., McOslen B., Mims S., Sambrook J., White D. Biology of animal viruses. - M.: Mir, 1977, vol. 1, 447 p.

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