Cell cultures

Animals

Indication of viruses based on the following phenomena

Developmental delay

death, change

membranes of the embryo, RGA

CPP, plaque formation, RIF, RGads, interference

Illness, death,

histological changes

in tissues, inclusions

Titration of the isolated virus; selection of working dose.

Virus titer- maximum dilution of the virus-containing material, in which the expected effect is still observed (CPE, RGA, death of the animal).

Identification of the isolated virus in neutralization reactions, RTGads, RSC, suppression of plaque formation, etc. with diagnostic sera. Type (type) of virus determined by neutralization of the specific effect of the virus by the appropriate immune serum.

Note: Titration and virus identification are performed using the same phenomenon.

Virus cultivation

Course work

"Methods of clinical virology"

Introduction

Laboratory diagnosis of viral infections is carried out mainly using electron microscopy, sensitive cell cultures and immunological methods. As a rule, one method is chosen to make a diagnosis depending on the stage of the viral infection. For example, all three approaches may be useful in diagnosing varicella, but the successful use of microscopy and cell culture techniques depends on the ability to collect satisfactory samples relatively early in the disease.

Largely a success viral diagnostics depends on the quality of the samples received. For this reason, laboratory staff themselves must be directly involved in collecting the necessary samples. Characteristics of the samples, as well as methods for their delivery to the laboratory, are described by Lennett, Schmidt, Christ, et al.

Most reagents and instruments used in laboratory diagnostics can be purchased from various companies. In most cases, the same reagent is produced simultaneously by several companies. For this reason, we have not indicated individual companies, unless the reagent is supplied by only one company. In all other cases, you should refer to the general list of suppliers indicated in table. 1.

We did not aim to provide a comprehensive description of all currently available methods for diagnosing human viral infections. First of all, we characterized the main methods. As you gain experience independent work these basic techniques can be used to solve more complex problems.

1. Electron microscopy

For electron microscopic diagnosis of viral infections, thin sections of affected tissue can be used. The most common material used for electron microscopy is feces or liquid.

Table 1. List of companies supplying reagents and equipment

vesicles that characterize some diseases, for example chicken pox. When analyzing such material, viruses can be detected using negative staining, which results in electron-dense material delineating the components of the virion. The method is effective when the virus concentration is high in the test samples, such as in feces or vesicular fluid. In cases where the content of viral particles in samples is low, the probability of detecting the virus can be increased by concentrating the virus by ultracentrifugation or aggregating it with specific antibodies. The latter method is also convenient for identifying viruses. Here we describe an electron microscopic diagnostic method rotavirus infection and the method of immunoelectron microscopy using the example of detection of specific antibodies to parvoviruses. Electron microscopy methods are described in more detail by Field.

2.1 Direct electron microscopic examination of feces

1. Dip the tip of a Pasteur pipette into the feces and draw up enough material to obtain a 1 cm smear.

2. Resuspend the fecal smear in electron microscopic negative contrast dye until a translucent suspension is obtained. Negative contrast dye is a 2% solution of phosphotungstic acid in distilled water.

3. To obtain an electron microscopic specimen, a drop of the suspension is placed on an electron microscopy grid coated with a carbon-formvar film. During this operation, the mesh is held with a pair of thin tweezers.

4. The drug is left in air for 30 s.

5. Excess liquid is removed by touching the edge of the glass with filter paper.

6. The drug is dried in air.

7. If necessary, the viable virus is inactivated by irradiating both sides of the grid with ultraviolet light with an intensity of 440,000 μW-s/cm2. In this case, shortwave is used ultraviolet lamp with filter. The lamp should be at a distance of 15 cm from the grid; Irradiation time for each side is 5 minutes.

8. Rotavirus virions can be characterized by transmission electron microscope with an increase from 30,000 to 50,000.

2.2 Immunoelectron microscopy

The immunoelectron microscopy method described below is only one of many similar immunological methods. To study virus-specific antibodies, in addition, a method is used that involves binding to the microscopic network of protein A. The working concentration of antiviral antibodies is determined by trial and error in the range from 1/10 to 1/1000. The concentration we indicate is usually used in routine work. To obtain optimal results for the interaction of antibodies with the virus, serum containing parvovirus is titrated in the same way.

1. 10 µl of antiserum to human parvovirus is diluted 100 times with PBS. The solution is heated in a water bath to 56°C.

2. Melt 10 ml of 2% agarose in PBS in the usual manner and cool to 56 °C in a water bath.

3. At 56 °C, mix 1 ml of diluted antiserum with 1 ml of 2% agarose.

4. Transfer 200 µl of the resulting mixture into two wells of a 96-well microtiter plate.

5. The agarose is allowed to set at room temperature. The tablet can be stored at 4°C for several weeks if it is sealed with adhesive tape.

6. Add 10 µl of serum containing parvovirus to a well containing a mixture of agarose and antiserum.

7. An electron microscopy grid with a pre-prepared carbon-formvar coating is placed on the less shiny side on a drop of serum.

8. The mesh is kept for 2 hours at 37 °C in a humid chamber.

9. Using thin tweezers, take out the mesh and apply a drop of 2% phosphotungstic acid to the surface of the mesh that was in contact with the serum.

10. After 30 s, the excess paint is washed off, the preparation is dried and the virus is inactivated.

Aggregated viral particles are examined under a transmission electron microscope at a magnification of 30,000 to 50,000.

3. Identification of viral antigens

Viruses found in tissues or tissue fluids can be identified by virus-specific proteins using the antigen-antibody reaction. The product of the antigen-antibody reaction is tested against a tag that is introduced either directly into antiviral antibodies or into antibodies directed against virus-specific antibodies. Antibodies can be labeled with fluorescein, radioactive iodine, or an enzyme that cleaves the substrate causing a color change. In addition, the hemagglutination reaction is used to identify the virus. In everyday practice, the described methods are used mainly to detect hepatitis B virus antigens in the blood and search for antigens of various viruses that cause various respiratory diseases.

Currently, many companies produce erythrocyte, radioactive and enzymatic diagnostics, including those for detecting the hepatitis B virus. We do not consider it advisable to outline methods for working with these diagnostics: it is enough to follow the attached instructions. Below we will focus on the immunofluorescence method for identifying respiratory syncytial virus in nasopharyngeal secretions.

3.1 Identification of respiratory syncytial virus in nasopharyngeal secretions by immunofluorescence

The method for obtaining preparations of nasopharyngeal secretions is described by Gardner and McQuillin. In laboratory conditions, this operation is performed in two stages. First, a smear of nasopharyngeal mucus is prepared on a glass slide. The resulting smears can be stored fixed at -20°C for many months. At the second stage, smears are stained to detect the respiratory syncytial virus antigen. For this purpose, the method of indirect immunofluorescence is used.

3.1.1 Preparation of preparations of nasopharyngeal secretions

1. Mucus from special forceps is washed off with 1-2 ml of PBS and transferred to a centrifuge tube.

2. Centrifuge for 10 minutes at 1500 rpm in a tabletop centrifuge.

3. The supernatant is drained.

4. The cell pellet is carefully resuspended in 2-3 ml of PBS until a homogeneous suspension is obtained. To do this, use a wide-necked Pasteur pipette.

5. The resulting suspension is transferred to a test tube.

6. Add another 2-4 ml of PBS to the suspension and mix by pipetting. Large clots of mucus are removed.

7. Centrifuge for 10 minutes at 1500 rpm in a tabletop centrifuge.

8. The supernatant is discarded, the sediment is resuspended in such a volume of PBS that the resulting suspension is easily separated from the walls of the tube.

9. The resulting suspension is applied to a marked glass slide.

10. The glass is dried in air.

Fix in acetone for 10 min at 4°C.

12. After fixing, the glass is air dried again.

13. The resulting preparations are stained immediately or stored at -20 °C.

3.1.2. Staining technique

1. Print and dilute commercial RSV antiserum in PBS to the recommended working concentration.

2. Using a Pasteur pipette, apply one drop of antiserum to the prepared preparation.

3. The drug is placed in a humid chamber.

4. The drug is incubated for 30 minutes at 37 °C.

5. Samples are carefully washed with PBS to remove excess antibodies in a special reservoir.

6. Samples are washed in three shifts of PBS, 10 minutes each.

7. Dry the samples, remove excess PBS with filter paper and air dry.

Virological research methods— methods for studying the biology of viruses and their identification. In virology, molecular biology methods are widely used, with the help of which it was possible to establish the molecular structure of viral particles, methods of their penetration into the cell and features of viral reproduction, the primary structure of viral nucleic acids and proteins. Methods are being developed to determine the sequence of the constituent elements of viral nucleic acids and protein amino acids. It becomes possible to link the functions of nucleic acids and the proteins they encode with the sequence of nucleotides and to establish the reasons for the intracellular processes that play a role in important role in the pathogenesis of viral infection.

Virological research methods are also based on immunological processes (interaction of antigen with antibodies), biological properties of the virus (ability for hemagglutination, hemolysis, enzymatic activity), features of the interaction of the virus with the host cell (nature of the cytopathic effect, formation of intracellular inclusions, etc.) .

In the diagnosis of viral infections, in the cultivation, isolation and identification of viruses, as well as in the production of vaccine preparations, the tissue and cell culture method is widely used. Primary, secondary, stable continuous and diploid cell cultures are used. Primary cultures are obtained by dispersing tissue with proteolytic enzymes (trypsin, collagenase). The source of cells can be tissues and organs (usually kidneys) of human and animal embryos. A suspension of cells in a nutrient medium is placed in so-called mattresses, bottles or Petri dishes, where, after attaching to the surface of the vessel, the cells begin to multiply. For virus infection, a cell monolayer is usually used. The nutrient liquid is drained, the viral suspension is added in certain dilutions, and after contact with the cells, fresh nutrient medium, usually without serum, is added.

The cells of most primary cultures can be subcultured; such a culture is called a secondary culture. With further passage of cells, a population of fibroblast-like cells is formed, capable of rapid reproduction, most of which retain the original set of chromosomes. These are so-called diploid cells. By serially culturing cells, stable continuous cell cultures are obtained. During passages, rapidly dividing homogeneous cells with a heteroploid set of chromosomes appear. Stable cell lines can be single-layer or suspension. Single-layer cultures grow in the form of a continuous layer on the glass surface, while suspension cultures grow in the form of suspensions in various vessels using mixing devices. There are more than 400 cell lines derived from 40 various types animals (including primates, birds, reptiles, amphibians, fish, insects) and humans.

Pieces can be cultured in artificial nutrient media individual organs and tissues (organ cultures). These types of cultures preserve tissue structure, which is especially important for the isolation and passage of viruses that do not reproduce in undifferentiated tissue cultures (for example, coronaviruses).

In infected cell cultures, viruses can be detected by changes in cell morphology, cytopathic effects, which may be specific, the appearance of inclusions, by determining viral antigens in the cell and in the culture fluid; establishing the biological properties of viral progeny in culture fluid and titrating viruses in tissue culture, chicken embryos or sensitive animals; by identifying individual viral nucleic acids in cells by molecular hybridization or accumulations of nucleic acids by the cytochemical method using fluorescent microscopy.

Isolating viruses is a labor-intensive and time-consuming process. It is carried out to determine the type or variant of the virus circulating among the population (for example, to identify a serovariant of the influenza virus, a wild or vaccine strain of the polio virus, etc.); in cases where it is necessary to carry out urgent epidemiological measures; when new types or variants of viruses appear; if necessary, confirm the preliminary diagnosis; to indicate viruses in objects environment. When isolating viruses, the possibility of their persistence in the human body, as well as the occurrence of a mixed infection caused by two or more viruses, is taken into account. A genetically homogeneous population of the virus obtained from one virion is called a viral clone, and the process of obtaining it is called cloning.

To isolate viruses, infection of susceptible laboratory animals and chicken embryos is used, but most often tissue culture is used. The presence of a virus is usually determined by specific cell degeneration (cytopathic effect), the formation of symplasts and syncytia, the detection of intracellular inclusions, as well as a specific antigen detected using immunofluorescence, hemadsorption, hemagglutination (for hemagglutinating viruses), etc. These signs can be detected only after 2-3 passages of the virus.

Chicken embryos are used to isolate a number of viruses, such as influenza viruses, and newborn mice are used to isolate some Coxsackie viruses and a number of arboviruses. Identification of isolated viruses is carried out using serological reactions and other methods.

When working with viruses, their titer is determined. Titration of viruses is usually carried out in tissue culture, determining the highest dilution of the virus-containing liquid at which tissue degeneration occurs, inclusions and virus-specific antigens are formed. The plaque method can be used to titrate a number of viruses. Plaques, or negative colonies of viruses, are foci of cells destroyed by the virus in a single-layer tissue culture under an agar coating. Colony counting allows for a quantitative analysis of the infectious activity of viruses on the basis that one infectious virus particle forms one plaque. Plaques are detected by staining the culture with intravital dyes, usually neutral red; plaques do not adsorb the dye and are therefore visible as light spots against the background of stained living cells. The virus titer is expressed as the number of plaque-forming units per 1 ml.

Purification and concentration of viruses is usually accomplished by differential ultracentrifugation followed by concentration or density gradient centrifugation. To purify viruses, immunological methods, ion exchange chromatography, immunosorbents, etc. are used.

Laboratory diagnosis of viral infections includes detection of the pathogen or its components in clinical material; isolating the virus from this material; serodiagnosis. The choice of laboratory diagnostic method in each individual case depends on the nature of the disease, the period of illness and the capabilities of the laboratory. Modern diagnostics viral infections is based on express methods that allow you to get an answer a few hours after taking clinical material in early dates after the disease, These include electron and immune electron microscopy, as well as immunofluorescence, the method of molecular hybridization, detection of antibodies of the IgM class, etc.

Electron microscopy of negatively stained viruses makes it possible to differentiate viruses and determine their concentration. The use of electron microscopy in the diagnosis of viral infections is limited to those cases where the concentration of viral particles in clinical material is quite high (10 5 in 1 ml and higher). The disadvantage of the method is the inability to distinguish viruses belonging to the same taxonomic group. This deficiency is overcome by the use of immune electron microscopy. The method is based on the formation of immune complexes by adding specific serum to viral particles, while simultaneously concentrating the viral particles, allowing them to be identified. The method is also used to detect antibodies. For express diagnostic purposes, electron microscopic examination of tissue extracts, feces, fluid from vesicles, and secretions from the nasopharynx is carried out. Electron microscopy widely used to study the morphogenesis of the virus, its capabilities are expanded with the use of labeled antibodies.

The molecular hybridization method, based on the detection of virus-specific nucleic acids, makes it possible to detect single copies of genes and has no equal in sensitivity. The reaction is based on the hybridization of complementary DNA or RNA strands (probes) and the formation of double-stranded structures. The cheapest probe is cloned recombinant DNA. The probe is labeled with radioactive precursors (usually radioactive phosphorus). The use of colorimetric reactions is promising. There are several options for molecular hybridization: spot hybridization, blot hybridization, sandwich hybridization, in situ hybridization, etc.

Antibodies of the IgM class appear earlier than antibodies of the G class (on the 3rd-5th day of illness) and disappear after a few weeks, so their detection indicates a recent infection. Antibodies of the lgM class are detected by immunofluorescence or by enzyme-linked immunosorbent assay using anti-m antisera (sera against lgM heavy chains).

Serological methods in virology are based on classical immunological reactions (see. Immunological research methods ): complement fixation reactions, hemagglutination inhibition, biological neutralization, immunodiffusion, indirect hemagglutination, radial hemolysis, immunofluorescence, enzyme immunoassay, radioimmunoassay. Micromethods for many reactions have been developed, and their techniques are constantly being improved. These methods are used to identify viruses using a set of known sera and for serodiagnosis to determine the increase in antibodies in the second serum compared to the first (the first serum is taken in the first days after the disease, the second - after 2-3 weeks). A diagnostic value is no less than a fourfold increase in antibodies in the second serum. If the detection of antibodies of the IgM class indicates a recent infection, then antibodies of the IgC class persist for several years, and sometimes for life.

To identify individual antigens of viruses and antibodies to them in complex mixtures without preliminary purification of proteins, immunoblotting is used. The method combines protein fractionation using polyacrylamide gel electrophoresis with subsequent immunoindication of proteins using the enzyme immunoassay method. Protein separation reduces protein requirements chemical purity antigen and allows you to identify individual antigen-antibody pairs. This task is relevant, for example, in the serodiagnosis of HIV infection, where false-positive enzyme-linked immunosorbent assay reactions are caused by the presence of antibodies to cellular antigens, which are present as a result of insufficient purification of viral proteins. Identification of antibodies in patient sera to internal and external viral antigens makes it possible to determine the stage of the disease, and when analyzing populations, the variability of viral proteins. Immunoblotting for HIV infection is used as a confirmatory test to identify individual viral antigens and antibodies to them. When analyzing populations, the method is used to determine the variability of viral proteins. The great value of the method lies in the possibility of analyzing antigens synthesized using recombinant DNA technology, establishing their sizes and the presence of antigenic determinants.

Virological research includes two main stages: isolation of viruses and their identification. The material for virological research can be blood, other biological and pathological fluids, biopsies of organs and tissues.

Virological blood tests are often performed to diagnose arboviral infections. Rabies viruses can be found in saliva, mumps, herpes simplex, Nasopharyngeal swabs are used to isolate pathogens of influenza and other acute respiratory viral infections, measles. Adenoviruses are found in conjunctival swabs. Various entero-, adno-, pso-, nora- and rotaviruses are isolated from feces.

To isolate viruses, cell cultures, chicken embryos, and sometimes laboratory animals are used.

Most pathogenic viruses are distinguished by the presence of tissue and type specificity; for example, poliovirus reproduces only in primate cells, so an appropriate tissue culture is used to isolate a specific virus. To isolate an unknown pathogen, it is advisable to simultaneously infect 3-4 cell cultures, assuming that one of them may be sensitive. The presence of the virus in infected cell cultures is determined by the development of specific cell degeneration, i.e. cytopathogen action, detection of intracellular inclusions, as well as based on the detection of a specific antigen by immunofluorescence, positive hemadsorption and hemagglutination reactions.

Bird embryos with their poorly differentiated tissues are suitable for cultivating many viruses. Most of the time, chicken embryos are used. When multiplying in embryos, viruses can cause their death (arboviruses), the appearance of changes in the chorion-allantoic membrane (smallpox viruses) or in the body of the embryo, the accumulation of gluten in in the embryonic fluids (influenza viruses, mumps) and complement binding viral antigen .

Identification of viruses is carried out using immunological methods: hemagglutination inhibition reaction, complement fixation, neutralization, gel precipitation, immunofluorescence.

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Virological research methods- methods for studying the biology of viruses and their identification. In virology, molecular biology methods are widely used, with the help of which it was possible to establish the molecular structure of viral particles, methods of their penetration into the cell and features of viral reproduction, the primary structure of viral nucleic acids and proteins. Methods are being developed to determine the sequence of the constituent elements of viral nucleic acids and protein amino acids. It becomes possible to link the functions of nucleic acids and the proteins they encode with the nucleotide sequence and to establish the causes of intracellular processes that play an important role in the pathogenesis of viral infection.

Virological research methods are also based on immunological processes (interaction of antigen with antibodies), biological properties of the virus (ability for hemagglutination, hemolysis, enzymatic activity), features of the interaction of the virus with the host cell (nature of the cytopathic effect, formation of intracellular inclusions, etc.) .

In the diagnosis of viral infections, in the cultivation, isolation and identification of viruses, as well as in the production of vaccine preparations, the tissue and cell culture method is widely used. Primary, secondary, stable continuous and diploid cell cultures are used. Primary cultures are obtained by dispersing tissue with proteolytic enzymes (trypsin, collagenase). The source of cells can be tissues and organs (usually kidneys) of human and animal embryos. A suspension of cells in a nutrient medium is placed in so-called mattresses, bottles or Petri dishes, where, after attaching to the surface of the vessel, the cells begin to multiply. For virus infection, a cell monolayer is usually used. The nutrient liquid is drained, the viral suspension is added in certain dilutions, and after contact with the cells, fresh nutrient medium, usually without serum, is added.

The cells of most primary cultures can be subcultured; such a culture is called a secondary culture. With further passage of cells, a population of fibroblast-like cells is formed, capable of rapid reproduction, most of which retain the original set of chromosomes. These are so-called diploid cells. By serially culturing cells, stable continuous cell cultures are obtained. During passages, rapidly dividing homogeneous cells with a heteroploid set of chromosomes appear. Stable cell lines can be single-layer or suspension. Single-layer cultures grow in the form of a continuous layer on the glass surface, while suspension cultures grow in the form of suspensions in various vessels using mixing devices. There are more than 400 cell lines derived from 40 different animal species (including primates, birds, reptiles, amphibians, fish, insects) and humans.

Pieces of individual organs and tissues (organ cultures) can be cultured in artificial nutrient media. These types of cultures preserve tissue structure, which is especially important for the isolation and passage of viruses that do not reproduce in undifferentiated tissue cultures (for example, coronaviruses).

In infected cell cultures, viruses can be detected by changes in cell morphology, cytopathic effects, which may be specific, the appearance of inclusions, by determining viral antigens in the cell and in the culture fluid; establishing the biological properties of viral progeny in culture fluid and titrating viruses in tissue culture, chicken embryos or sensitive animals; by identifying individual viral nucleic acids in cells by molecular hybridization or accumulations of nucleic acids by the cytochemical method using fluorescent microscopy.

Isolating viruses is a labor-intensive and time-consuming process. It is carried out to determine the type or variant of the virus circulating among the population (for example, to identify a serovariant of the influenza virus, a wild or vaccine strain of the polio virus, etc.); in cases where it is necessary to carry out urgent epidemiological measures; when new types or variants of viruses appear; if necessary, confirm the preliminary diagnosis; for indication of viruses in environmental objects. When isolating viruses, the possibility of their persistence in the human body, as well as the occurrence of a mixed infection caused by two or more viruses, is taken into account. A genetically homogeneous population of the virus obtained from one virion is called a viral clone, and the process of obtaining it is called cloning.

To isolate viruses, infection of susceptible laboratory animals and chicken embryos is used, but most often tissue culture is used. The presence of a virus is usually determined by specific cell degeneration (cytopathic effect), the formation of symplasts and syncytia, the detection of intracellular inclusions, as well as a specific antigen detected using immunofluorescence, hemadsorption, hemagglutination (for hemagglutinating viruses), etc. These signs can be detected only after 2-3 passages of the virus.

To isolate a number of viruses, such as influenza viruses, chicken embryos are used, and to isolate some Coxsackie viruses and a number of arboviruses, newborn mice are used. Identification of isolated viruses is carried out using serological reactions and other methods.

When working with viruses, their titer is determined. Titration of viruses is usually carried out in tissue culture, determining the highest dilution of the virus-containing liquid at which tissue degeneration occurs, inclusions and virus-specific antigens are formed. The plaque method can be used to titrate a number of viruses. Plaques, or negative colonies of viruses, are foci of cells destroyed by the virus in a single-layer tissue culture under an agar coating. Colony counting allows for a quantitative analysis of the infectious activity of viruses on the basis that one infectious virus particle forms one plaque. Plaques are detected by staining the culture with intravital dyes, usually neutral red; plaques do not adsorb the dye and are therefore visible as light spots against the background of stained living cells. The virus titer is expressed as the number of plaque-forming units per 1 ml.

Purification and concentration of viruses is usually accomplished by differential ultracentrifugation followed by concentration or density gradient centrifugation. To purify viruses, immunological methods, ion exchange chromatography, immunosorbents, etc. are used.

Laboratory diagnosis of viral infections includes detection of the pathogen or its components in clinical material; isolating the virus from this material; serodiagnosis. The choice of laboratory diagnostic method in each individual case depends on the nature of the disease, the period of illness and the capabilities of the laboratory. Modern diagnostics of viral infections is based on express methods that make it possible to obtain an answer several hours after taking clinical material in the early stages after the disease. These include electron and immune electron microscopy, as well as immunofluorescence, the method of molecular hybridization, detection of antibodies of the IgM class, etc.

Electron microscopy of negatively stained viruses makes it possible to differentiate viruses and determine their concentration. The use of electron microscopy in the diagnosis of viral infections is limited to those cases where the concentration of viral particles in clinical material is quite high (10 5 in 1 ml and higher). The disadvantage of the method is the inability to distinguish viruses belonging to the same taxonomic group. This deficiency is overcome by the use of immune electron microscopy. The method is based on the formation of immune complexes by adding specific serum to viral particles, while simultaneously concentrating the viral particles, allowing them to be identified. The method is also used to detect antibodies. For express diagnostic purposes, electron microscopic examination of tissue extracts, feces, fluid from vesicles, and secretions from the nasopharynx is carried out. Electron microscopy is widely used to study the morphogenesis of the virus; its capabilities are expanded with the use of labeled antibodies.

The molecular hybridization method, based on the detection of virus-specific nucleic acids, makes it possible to detect single copies of genes and has no equal in sensitivity. The reaction is based on the hybridization of complementary DNA or RNA strands (probes) and the formation of double-stranded structures. The cheapest probe is cloned recombinant DNA. The probe is labeled with radioactive precursors (usually radioactive phosphorus). The use of colorimetric reactions is promising. There are several options for molecular hybridization: spot hybridization, blot hybridization, sandwich hybridization, in situ hybridization, etc.

Antibodies of the IgM class appear earlier than class G antibodies (on the 3rd-5th day of illness) and disappear after a few weeks, so their detection indicates a recent infection. Antibodies of the lgM class are detected by immunofluorescence or by enzyme-linked immunosorbent assay using anti-m antisera (sera against lgM heavy chains).

Serological methods in virology are based on classical immunological reactions (see. Immunological research methods ): complement fixation reactions, hemagglutination inhibition, biological neutralization, immunodiffusion, indirect hemagglutination, radial hemolysis, immunofluorescence, enzyme immunoassay, radioimmunoassay. Micromethods for many reactions have been developed, and their techniques are constantly being improved. These methods are used to identify viruses using a set of known sera and for serodiagnosis to determine the increase in antibodies in the second serum compared to the first (the first serum is taken in the first days after the disease, the second - after 2-3 weeks). A diagnostic value is no less than a fourfold increase in antibodies in the second serum. If the detection of antibodies of the IgM class indicates a recent infection, then antibodies of the IgC class persist for several years, and sometimes for life.

To identify individual antigens of viruses and antibodies to them in complex mixtures without preliminary purification of proteins, immunoblotting is used. The method combines protein fractionation using polyacrylamide gel electrophoresis with subsequent immunoindication of proteins using the enzyme immunoassay method. Protein separation reduces the requirements for the chemical purity of the antigen and makes it possible to identify individual antigen-antibody pairs. This task is relevant, for example, in the serodiagnosis of HIV infection, where false-positive enzyme-linked immunosorbent assay reactions are caused by the presence of antibodies to cellular antigens, which are present as a result of insufficient purification of viral proteins. Identification of antibodies in patient sera to internal and external viral antigens makes it possible to determine the stage of the disease, and when analyzing populations, the variability of viral proteins. Immunoblotting for HIV infection is used as a confirmatory test to identify individual viral antigens and antibodies to them. When analyzing populations, the method is used to determine the variability of viral proteins. The great value of the method lies in the possibility of analyzing antigens synthesized using recombinant DNA technology, establishing their sizes and the presence of antigenic determinants.

Bibliography: Bukrinskaya A.G. Virology, M., 1986; Virology, Methods, ed. B. Meikhi, trans. from English, M., 1988; Handbook of microbiological and virological research methods, ed. M.O. Birger, M., 1982.