Determination of indications for bacteriological, virological, serological studies in deciphering the etiology of oki and evaluation of the results. Virological research What is the meaning of research

Archive of sites Internet Archive Wayback Machine Very often the attack of black PR people comes unexpectedly for you. In this case, for the first time, you are faced with the need to closely study the enemy. If you even assumed such a development of events (for example, in

4.9. Backup with Time Machine

4.9. Backing up with Time Machine Mac OS X Leopard allows you to regularly back up your computer using the Time Machine application. After the appropriate settings, the application will automatically

4.9.2. Create your first backup with Time Machine

4.9.2. Create your first backup using Time Machine Before you start creating your first backup, you must either insert an external drive or have a free hard drive partition set aside for backup only.

4.9.4. Using Time Machine

4.9.4. Using Time Machine Once you've made the necessary Time Machine settings and created a number of backups, you can start searching for and restoring earlier versions of your files. For this: 1. Open a Finder window and select the file you need to restore.2. If a

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

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

In the diagnosis of viral infections, in the cultivation, isolation and identification of viruses, as well as in the preparation of vaccine preparations, the method of tissue and cell culture 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 (more often kidneys) of human and animal embryos. A suspension of cells in a nutrient medium is placed in the 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 introduced in certain dilutions, and after contact with the cells, fresh nutrient medium is added, usually without serum.

Cells from most primary cultures can be subcultured and are referred to as secondary cultures. With further passaging 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 the so-called diploid cells. In serial cultivation of 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 monolayer and suspension. Monolayer cultures grow in the form of a continuous layer on the glass surface, suspension cultures grow in the form of suspensions in various vessels using agitators. There are over 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 cultivated 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 a change in cell morphology, cytopathic action, 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 titration of viruses in tissue culture, chick embryos or sensitive animals; by detecting individual viral nucleic acids in cells by molecular hybridization or clusters of nucleic acids by cytochemical method using fluorescent microscopy.

Isolation of viruses is a laborious and lengthy process. It is carried out in order to determine the type or variant of the virus circulating among the population (for example, to identify the serovariant of the influenza virus, 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 a virus obtained from a single virion is called a viral clone, and the process of obtaining it is called cloning.

To isolate viruses, infection of susceptible laboratory animals, chicken embryos is used, but tissue culture is most often 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 (in hemagglutinating viruses), etc. These signs can be detected only after 2-3 passages of the virus.

For the isolation of a number of viruses, such as influenza viruses, chicken embryos are used, for the isolation of some Coxsackie viruses and a number of arboviruses, newborn mice are used. Identification of isolated viruses is carried out using serological tests 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 fluid, 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 virus-destroyed cells of a single-layer tissue culture under agar coating. Colony counting allows a quantitative analysis of the infectious activity of viruses on the basis that one infectious virus particle forms one plaque. Plaques are identified by staining the culture with vital dyes, usually neutral red; plaques do not adsorb the dye and therefore are visible as light spots against the background of stained live cells. The titer of the virus is expressed as the number of plaque-forming units in 1 ml.

Purification and concentration of viruses is usually carried out by differential ultracentrifugation followed by centrifugation in concentration or density gradients. To purify viruses, immunological methods, ion-exchange chromatography, immunosorbents, etc. are used.

Laboratory diagnosis of viral infections includes the detection of the pathogen or its components in clinical material; virus isolation from this material; serodiagnosis. The choice of laboratory diagnostic method in each individual case depends on the nature of the disease, the period of the disease and the capabilities of the laboratory. Modern diagnostics of viral infections is based on express methods that allow you to get a response a few 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, the detection of antibodies of the IgM class, etc.

Electron microscopy of negatively stained viruses allows differentiation of viruses and determination of 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 the clinical material is sufficiently high (10 5 in 1 ml and higher). The disadvantage of the method is the inability to distinguish between viruses belonging to the same taxonomic group. This disadvantage is eliminated by using immune electron microscopy. The method is based on the formation of immune complexes when specific serum is added to viral particles, while the simultaneous concentration of viral particles occurs, which makes it possible to identify them. The method is also used to detect antibodies. For the purpose of express diagnostics, an 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 method of molecular hybridization, based on the detection of virus-specific nucleic acids, makes it possible to detect single copies of genes and has no equal in terms of sensitivity. The reaction is based on the hybridization of complementary strands of DNA or RNA (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 variants of molecular hybridization: point hybridization, blot hybridization, sandwich hybridization, in situ hybridization, etc.

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

Serological methods in virology are based on classical immunological reactions (see Immunological methods of research) : 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 being continuously improved. These methods are used to identify viruses using a set of known sera and for serodiagnosis in order 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). Diagnostic value is not less than a fourfold increase in antibodies in the second serum. If the detection of antibodies of the lgM class indicates a recent infection, then the antibodies of the lgC class persist for several years, and sometimes for life.

Immunoblotting is used to identify individual antigens of viruses and antibodies to them in complex mixtures without prior purification of proteins. The method combines protein fractionation using polyacrylamide gel electrophoresis with subsequent immunoassay of proteins by enzyme immunoassay. The separation of proteins 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 immunoassay reactions are due to the presence of antibodies to cell antigens, which are present as a result of insufficient purification of viral proteins. Identification of antibodies in the sera of patients to internal and external viral antigens makes it possible to determine the stage of the disease, and in the analysis of populations - the variability of viral proteins. Immunoblotting in HIV infection is used as a confirmatory test to detect 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, determining their size and the presence of antigenic determinants.

20) The main structural component of virions (complete viral particles) is a nucleocapsid, i.e. the protein sheath (capsid) that encloses the viral genome (DNA or RNA). The nucleocapsid of most virus families is surrounded by a lipoprotein envelope. Between the envelope and the nucleocapsid in some viruses (ortho-, paramyxo-, rhabdo-, filo- and retroviruses) there is a non-glycosylated matrix protein, which gives additional rigidity to the virions. Viruses of most families have an envelope that plays an important role in infectivity. Virions acquire the outer layer of the envelope when the nucleocapsid penetrates the cell membrane by budding. Envelope proteins are encoded by the virus, and lipids are borrowed from the cell membrane. Glycoproteins usually in the form of dimers and trimers form peplomers (protrusions) on the surface of virions (ortho-, paramyxoviruses, rhabdo-, filo-, corona-, bunya-, arena-, retroviruses). Glycosylated fusion proteins are associated with peplomers and play a key role in the entry of the virus into the cell. Capsids and envelopes of virions are formed by many copies of one or more types of protein subunits as a result of a self-assembly process. Interaction in the protein-protein system, due to weak chemical bonds, leads to the unification of symmetrical capsids. Differences in viruses in the shape and size of virions depend on the shape, size and number of structural protein subunits and the nature of the interaction between them. The capsid consists of many morphologically expressed subunits (capsomeres) assembled from viral polypeptides in a strictly defined manner, in accordance with relatively simple geometric principles. Protein subunits, connecting with each other, form capsids of two types of symmetry: isometric and helical. The structure of the nucleocapsid of enveloped viruses is similar to the structure of the nucleocapsid of non-enveloped viruses. On the surface of the envelope of viruses, morphologically expressed glycoprotein structures - peplomers are distinguished. The composition of the supercapsid membrane includes lipids (up to 20-35%) and carbohydrates (up to 7-8%), which are of cellular origin. It consists of a double layer of cellular lipids and virus-specific proteins located outside and inside the lipid biolayer. The outer layer of the supercapsid membrane is represented by one or more types of peplometers (protrusions), consisting of one or more glycoprotein molecules. The nucleocapsid of enveloped viruses is often referred to as the core, while the central part of the virions containing the nucleic acid is called the nucleoid. Capsomeres (peplomers) consist of structural units built from one or several homologous or heterologous polypeptide chains (protein subunits). classification of viruses Isometric capsids are not spheres, but regular polyhedra (icosahedrons). Their linear dimensions are identical along the axes of symmetry. According to Kaspar and Klug (1962), capsomeres in capsids are arranged according to icosahedral symmetry. Such capsids are composed of identical subunits forming an icosahedron. They have 12 vertices (corners), 30 faces and 20 surfaces in the form of isosceles triangles. In accordance with this rule, the capsid of poliovirus and foot-and-mouth disease virus is formed by 60 protein structural units, each of which consists of four polypeptide chains. The icosahedron optimally solves the problem of packing repeating subunits into a strict compact structure with a minimum volume. Only some configurations of structural subunits can form the surfaces, vertices and faces of the viral icosahedron. For example, the structural subunits of adenovirus form hexagonal capsomeres (hexons) on surfaces and faces, and pentagonal capsomeres (peptones) at the tops. In some viruses, both types of capsomeres are formed by the same polypeptides, in others, by different polypeptides. Since the structural subunits of different viruses differ from each other, some viruses seem to be more hexagonal, others more spherical. All known DNA-containing vertebrate viruses, with the exception of smallpox viruses, as well as many RNA-containing viruses (7 families) have a cubic capsid symmetry type. Reoviruses, unlike other vertebrate viruses, have a double capsid (outer and inner), each consisting of morphological units. Viruses with a helical symmetry type have the form of a cylindrical filamentous structure, their genomic RNA has the form of a spiral and is located inside the capsid. All animal viruses of helical symmetry are surrounded by a lipoprotein envelope. Helical nucleocapsids are characterized by length, diameter, helix pitch, and the number of capsomeres per turn of the helix. Thus, in the Sendai virus (paramyxovirus), the nucleocapsid is a helix about 1 μm long, 20 nm in diameter, and a helix pitch of 5 nm. The capsid consists of approximately 2400 structural units, each of which is a protein with a molecular weight of 60 kD. There are 11-13 subunits per turn of the helix. In viruses with helical nucleocapsid symmetry, the folding of protein molecules into a helix ensures maximum interaction between nucleic acid and protein subunits. In icosahedral viruses, the nucleic acid is coiled inside the virions and interacts with one or more polypeptides located inside the capsid.

Antireceptors (receptors) Viral- surface virion proteins, for example, hemagglutinin, which bind in a complementary manner to the corresponding receptor of a susceptible cell.

21) Immunological methods in virological research.

Serological tests vary in their ability to detect individual classes of antibodies. The agglutination test, for example, is good at detecting IgM antibodies, but is less sensitive for detecting IgG antibodies. Complement fixation and hemolysis tests, which require complement, do not detect non-complement-attaching antibodies, such as IgA antibodies and IgE antibodies. Only antibodies directed against antigenic determinants of the virion surface associated with pathogenicity participate in the virus neutralization reaction. Sensitivity I. m. and. surpasses all other methods for the study of antigens and antibodies, in particular, radioimmunoassay and enzyme immunoassay allow capturing the presence of protein in quantities measured in nanograms and even in picograms. With the help of I. m. and. determine the group and check the safety of the blood (hepatitis B and HIV infection). At transplantation of fabrics and bodies And. m. allow determination of tissue compatibility and testing of incompatibility suppression methods. In forensic medicine, the Castellani reaction is used to determine the species specificity of a protein and the agglutination reaction to determine the blood type.

Immunological methods are widely used in laboratory diagnostics of infectious diseases. The etiology of the disease is also established on the basis of the increase in antibodies to the pathogen in the blood serum of the convalescent compared with the sample taken in the first days of the disease. Based on I. m. and. study the immunity of the population in relation to mass infections, such as influenza, and also evaluate the effectiveness of preventive vaccinations.

Depending on their mechanism and the account of results And. m. and. can be divided into reactions based on the phenomenon of agglutination; reactions based on the phenomenon of precipitation; reactions involving complement; neutralization reaction; reactions using chemical and physical methods.

Reactions based on the phenomenon of agglutination. Agglutination is the gluing of cells or individual particles - carriers of an antigen with the help of immune serum to this antigen.

The bacterial agglutination test using an appropriate antibacterial serum is one of the simplest serological tests. A suspension of bacteria is added to various dilutions of the tested blood serum and after a certain contact time at t ° 37 ° it is recorded at which highest dilution of blood serum agglutination occurs. The agglutination reaction of bacteria is used to diagnose many infectious diseases: brucellosis, tularemia, typhoid fever and paratyphoid fever, bacillary dysentery, and typhus.

The reaction of passive, or indirect, hemagglutination (RPGA, RNGA). It uses erythrocytes or neutral synthetic materials (for example, latex particles), on the surface of which antigens (bacterial, viral, tissue) or antibodies are adsorbed. Their agglutination occurs when the appropriate sera or antigens are added.

The passive hemagglutination reaction is used to diagnose diseases caused by bacteria (typhoid and paratyphoid, dysentery, brucellosis, plague, cholera, etc.), protozoa (malaria) and viruses (influenza, adenovirus infections, viral hepatitis B, measles, tick-borne encephalitis, Crimean hemorrhagic fever, etc.), as well as to determine certain hormones, to identify the patient's hypersensitivity to drugs and hormones, such as penicillin and insulin.

The hemagglutination inhibition test (HITA) is based on the phenomenon of prevention (inhibition) of the immune serum of hemagglutination of erythrocytes by viruses, and is used to detect and titrate antiviral antibodies. It serves as the main method of serodiagnosis of influenza, measles, rubella, mumps, tick-borne encephalitis and other viral infections, the causative agents of which have hemagglutinating properties. for example, for serodiagnosis of tick-borne encephalitis, two-fold dilutions of the patient's serum in an alkaline borate buffer solution are poured into the wells of the panel. Then a certain amount, usually 8 AU (agglutinating units), of tick-borne encephalitis antigen is added, and after 18 hours of exposure at t ° 4 °, a suspension of goose erythrocytes prepared in an acidic phosphate buffer solution is added. If there are antibodies to the tick-borne encephalitis virus in the patient's blood serum, then the antigen is neutralized and erythrocyte agglutination does not occur.

Reactions based on the phenomenon of precipitation. Precipitation occurs as a result of the interaction of antibodies with soluble antigens. The simplest example of a precipitation reaction is the formation of an opaque precipitation band in a test tube at the border of antigen layering on an antibody. Widely used are various types of precipitation reactions in semi-liquid agar or agarose gels (Ouchterlon double immunodiffusion method, radial immunodiffusion method, immunoelectrophoresis), which are both qualitative and quantitative. As a result of free diffusion in the gel of antigens and antibodies in the zone of their optimal ratio, specific complexes are formed - precipitation bands, which are detected visually or by staining. A feature of the method is that each antigen-antibody pair forms an individual precipitation band, and the reaction does not depend on the presence of other antigens and antibodies in the system under study.

Reactions involving complement, which is used as fresh guinea pig serum, are based on the ability of the Clq complement subcomponent and then other complement components to attach to immune complexes.

The complement fixation reaction (CFR) allows titration of antigens or antibodies according to the degree of complement fixation by the antigen-antibody complex. This reaction consists of two phases: the interaction of the antigen with the test blood serum (test system) and the interaction of hemolytic serum with ram erythrocytes (indicator system). With a positive reaction, complement fixation occurs in the system under study, and then, with the addition of erythrocytes sensitized with antibodies, hemolysis is not observed. The reaction is used for serodiagnosis of syphilis (Wassermann reaction), viral and bacterial infections.

The neutralization reaction is based on the ability of antibodies to neutralize certain specific functions of macromolecular or soluble antigens, such as enzyme activity, bacterial toxins, and the pathogenicity of viruses. The reaction of neutralization of toxins can be assessed by the biological effect, for example, anti-tetanus and anti-botulinum sera are titrated. A mixture of toxin and antiserum administered to animals does not cause their death. Various variants of the neutralization reaction are used in virology. When viruses are mixed with an appropriate antiserum and this mixture is administered to animals or cell cultures, the pathogenicity of the viruses is neutralized and the animals do not get sick, and the cells of the cultures do not undergo destruction.

Reactions using chemical and physical labels. Immunofluorescence consists in the use of fluorochrome-labeled antibodies, more precisely, the immunoglobulin fraction of IgG antibodies. The fluorochrome-labeled antibody forms an antigen-antibody complex with the antigen, which becomes available for observation under a microscope in UV rays that excite the luminescence of the fluorochrome. The direct immunofluorescence reaction is used to study cellular antigens, detect virus in infected cells, and detect bacteria and rickettsia in smears.

The method of indirect immunofluorescence is more widely used. based on the detection of an antigen-antibody complex using a luminescent anti-lgG antibody sera and is used to detect not only antigens, but also antibody titration.

Immunoenzyme, or enzyme immunological, methods are based on the use of antibodies conjugated with enzymes, mainly horseradish peroxidase or alkaline phosphatase. Like immunofluorescence, enzyme immunoassay is used to detect antigens in cells or to titrate antibodies on antigen-containing cells.

The radioimmunological method is based on the use of a radioisotope label of antigens or antibodies. It is the most sensitive method for determining antigens and antibodies, used to determine hormones, drugs and antibiotics, to diagnose bacterial, viral, rickettsial, protozoal diseases, to study blood proteins, tissue antigens.

Immunoblotting is used to detect antibodies to individual antigens or "recognize" antigens from known sera. The method consists of 3 stages: separation of biological macromolecules (for example, a virus) into individual proteins using polyacrylamide gel electrophoresis; transferring the separated proteins from the gel onto a solid support (blot) by applying a polyacrylamide gel plate to activated paper or nitrocellulose (electroblotting); detection of the desired proteins on the substrate using direct or indirect enzyme immunoassay. As a diagnostic method, immunoblotting is used for HIV infection. Diagnostic value is the detection of antibodies to one of the proteins of the outer shell of the virus.

22) Symmetry types of viruses (cubic, helical, mixed). Interaction of proteins and nucleic acids in the packaging of virus genomes.

Depending on the interaction of the capsid with the nucleic acid, virus particles can be divided into several types of symmetry:

1). Cubic symmetry type.

Cubic capsids are icosiders with approximately 20 triangular surfaces and 12 vertices. They form a structure resembling a spherical formation, but in fact it is a polyhedron. In some cases, special lipoprotein formations called spikes are attached to the vertices of such icosahedral polyhedra. The role of these spikes is presumably reduced to the interaction of virions or viral particles with the corresponding areas of host cells that are sensitive to them. With cubic symmetry, the viral nucleic acid is packed tightly (rolled into a ball), and the protein molecules surround it, forming a polyhedron (icosahedron). An icosahedron is a polyhedron with twenty triangular faces that has cubic symmetry and an approximately spherical shape. Icosahedral viruses include herpes simplex virus, reoviruses, etc.

2). Spiral symmetry type. Spiral capsids are somewhat simpler. Those. The capsomeres that make up the capsid cover the helical NK and also form a fairly stable protein shell of these viruses. And when using high-resolution electron microscopes and appropriate preparation methods, one can see spiral structures on viruses. With helical symmetry of the capsid, the viral nucleic acid forms a helical (or helical) figure, hollow inside, and the protein subunits (capsomeres) are also stacked around it in a spiral (tubular capsid). An example of a virus with a helical symmetry of the capsid is the tobacco mosaic virus, which is rod-shaped, and its length is 300 nm with a diameter of 15 nm. The composition of a viral particle includes one RNA molecule about 6000 nucleotides in size. The capsid is made up of 2000 identical protein subunits arranged in a helix.

3). Mixed or complex type of symmetry. As a rule, this type of symmetry is found mainly among bacterial viruses. And classic examples are those phages, E. coli or temperate phages. These are complex formations that have a head with internal nucleic content, various kinds of appendages, a tail process, and a device of varying degrees of complexity. And each component of such particles is endowed with a specific function that is realized in the process of interaction between the virus and the cell. In other words, a complex type of symmetry is a combination of cubic symmetry, the head is an icosider polyhedron, and the rod-shaped formations are the caudal processes. Although among bacterial viruses there are also quite simply organized virions, which are primitive nucleocapsids, spherical or cubic in shape. Bacterial viruses are more complex than plant and animal viruses.

24) Interaction of a phage with a cell. Virulent and temperate phages.

Adsorption.

The interaction begins with the attachment of viral particles to the cell surface. The process becomes possible in the presence of appropriate receptors on the surface of the cell and anti-receptors on the surface of the viral particle.

Viruses use cell receptors designed to transport essential substances: nutrient particles, hormones, growth factors, etc.

Receptors: proteins, carbohydrate component of proteins and lipids, lipids. Specific receptors determine the further fate of the viral particle (transport, delivery to areas of the cytoplasm or nucleus). The virus can attach to nonspecific receptors and even penetrate the cell. However, this process does not cause infection.

Initially, a single bond between the antireceptor and the receptor is formed. This bond is fragile and can break. For the formation of irreversible adsorption, multivalent attachment is necessary. Stable binding occurs due to the free movement of receptor molecules in the membrane. During the interaction of the virus with the cell, an increase in the fluidity of lipids is observed, and the formation of receptor fields in the area of ​​interaction between the virus and the cell. The receptors of a number of viruses can be present only in a limited set of host cells. This determines the sensitivity of the organism to this virus. Thus, viral DNA and RNA have the ability to infect a wider range of host cells.

Antireceptors can be found in unique viral organelles: outgrowth structures in T-bacteriophages, fibers in adenoviruses, spikes on the surface of viral membranes, corona in coronaviruses.

Penetration.

2 mechanisms - receptor endocytosis and membrane fusion.

Receptor endocytosis:

The usual mechanism for the entry of nutrients and regulatory substances into the cell. Occurs in specialized areas - where there are special pits covered with clathrin, specific receptors are located at the bottom of the pit. The pits provide rapid invagination and the formation of vacuoles covered with clathrin (no more than 10 minutes pass from the moment of adsorption, up to 2000 vacuoles can form in one minute). Vacuoles fuse with larger cytoplasmic vacuoles to form receptorosomes (no longer containing clathrin), which in turn fuse with lysosomes.

Fusion of viral and cell membranes:

In enveloped viruses, fusion is mediated by point interactions of the viral protein with cell membrane lipids, resulting in the integration of the viral lipoprotein envelope with the cell membrane. In non-enveloped viruses, one of the surface proteins also interacts with cell membrane lipids and the internal component passes through the membrane (in paramyxoviruses, the F-protein; in orthomyxoviruses, the HA2 hemagglutinating subunit). The conformation of surface proteins is affected by pH.

Strip.

In this process, infectious activity disappears, sensitivity to nucleases often appears, and resistance to antibodies arises. The end product of undressing is nucleic acids bound to an internal viral protein. The undressing stage also limits the possibility of infection (viruses are not able to undress in every cell). Undressing occurs in specialized areas of the cell: lysosomes, the Golgi apparatus, and the perinuclear space.

Undressing takes place as a result of a series of reactions. For example, in picornaviruses, undressing proceeds with the formation of intermediate subviral particles with sizes from 156 to 12S. In adenoviruses in the cytoplasm and nuclear pores and has at least 3 stages:

Formation of subviral particles with a higher density than virions;

The formation of cores in which 3 viral proteins are missing;

Formation of a DNA-protein complex in which DNA is covalently linked to a terminal protein.

Characterization of virulent and temperate phages.

When a bacterium is infected with a phage, the so-called lytic infection occurs, i.e., the infection ends with the lysis of the host cell, but this is characteristic only of the so-called virulent phages, the interaction of which with the cell leads to cell death and the formation of phage progeny.

At the same time, the following stages are distinguished according to the interactions of the phage with the cell: mixing the phage with the cell culture (the multiplicity of infection is 1 phage per 10 cells), and the concentration must be high enough so that the phages can contact the cells. In order to avoid re-infection - after infection for a maximum of 5 minutes, when the phages are adsorbed - this mixture of cells with phage is diluted. There is a latent period during which the number of phages does not increase, then a very short exit period, when the number of phage particles increases sharply, when the cell lyses and phage progeny is released, and then the number of phages remains at the same level, because re-infection does not occur. Based on this curve, these phases can be distinguished: the vegetative period of "growth" (latent period), the exit period and calculate the phage yield per 1 infected cell. During the latent period, nothing resembling phage particles can be found in bacteria, and it is not possible to isolate the infectious principle from such cells in the latent period. Only mature phage particles can infect bacteria. Thus, virulent phages always cause the death of bacteria and produce infection, which is revealed in the production of new viral particles capable of infecting the next and other sensitive cells.

In contrast to virulent ones, infection with temperate phages does not lead to lysis of bacterial cells, but the formation of a special state of coexistence of a phage with a bacterial cell is realized. This coexistence is expressed in the fact that a certain beginning of the phage is present in the bacterial cell without any unfavorable conditions for it and is preserved from generation to generation. At certain stages of such coexistence, the phage is activated in the cell and enters the state of the lytic cycle of development, causing cell lysis and release of phage progeny. Such phages are called lysogenic or temperate phages, and the state of moderate existence with the phage is lysogeny, and the bacteria that contain such a latent phage are called lysogenic bacteria. The term lysogenic bacteria came from the fact that cultures were once discovered in which a phage spontaneously appeared, and this bacteriophage began to be considered as contamination of the culture, that is, a bacterial virus enters the culture, and such cultures were called lysogenic, that is, they generate lysis .

Viruses, unlike bacteria, reproduce only in living cells. In this regard, the cultivation of viruses can be carried out at the level of the body of an experimental animal (chicken embryo as a developing organism is referred to as experimental animals) or a living cell grown outside the body, i.e. at the cell culture level.

Use of laboratory animals. One of the methods for isolating and cultivating viruses is to infect laboratory animals. They are used to isolate viruses that do not cause the development of cytopathic changes in cell cultures and do not multiply in chicken embryos. The use of laboratory animals also makes it possible to identify the nature of the viral infection by the clinical symptom complex. White mice, hamsters, guinea pigs, and rabbits are most often used as laboratory animals, depending on the purpose of the work and the type of viruses being studied. Of the larger animals, monkeys of various species and some other animals are used. From birds use chickens, geese, ducks. In recent years, newborn animals (more susceptible to viruses), "sterile animals" (extracted from the uterus and kept under sterile conditions using sterile air and sterilized food) and animals of pure lines with known heredity (inbred or linear animals) have been more often used.

Only healthy animals are taken into the experiment, preferably from one nursery and one batch. Body temperature is measured at the same time, since there are daily fluctuations in it. The test material is administered taking into account the tropism of viruses to certain tissues. So, for the isolation of neutrotropic viruses, the material is injected into the brain, for the isolation of pneumotropic viruses - through the nose (under light ether anesthesia).

In laboratory animals, after infection with a virus-containing material, it is important to take the material for further research in a timely and correct manner, and aseptically. Virus isolation results are considered positive if the animal develops symptoms of infection after an appropriate incubation period.

Use of chick embryos. In the tissues of the embryo, its membranes, the yolk sac, many pathogenic human and animal viruses are able to multiply. In this case, the selectivity of viruses to a particular tissue is important: smallpox viruses reproduce well and accumulate in the cells of the chorion-allantoic membrane, the mumps virus in the amnion, influenza viruses in the amnion and allantois, and the rabies virus in the yolk sac.

The cultivation of viruses in developing embryos has a number of advantages over other methods: a dense shell quite reliably protects the internal contents from microbes; when infecting chicken embryos, a greater yield of virus-containing material is obtained than with other methods of cultivation; the method of infection of chicken embryos is simple and accessible to any virological laboratories; embryos have sufficient viability and resistance to pathogens of external factors. However, chicken embryos are not always free from latent viral and bacterial infections. It is difficult to observe the dynamics of pathological changes occurring in the embryo after infection with a virus. Autopsy of infected embryos often reveals no visible changes and detects the virus using a hemagglutination test and other methods. In infected embryos, it is impossible to follow the increase in antibody titer. The method is not suitable for all viruses.

For virological studies, embryos of 7-12 days of age are used, which are obtained from poultry farms. You can grow embryos in a conventional thermostat, at the bottom of which trays with water are placed to humidify the air. The temperature in the thermostat should be 37 ° C, and the humidity should be 60-65%. Select large, clean (but unwashed), fertilized eggs from white chickens, stored for no more than 10 days at a temperature of 5-10°C. Fertilized eggs are recognized by the presence of a germinal disc, which, when translucent with an ovoscope, looks like a dark spot.

When working with viruses, various methods of infecting embryos can be used, but the most practical application is the application of the virus to the chorion-allantoic membrane, the introduction into the allantoic, amniotic cavity and yolk sac

(Fig. 10.5). The choice of method depends on the biological properties of the virus under study.

Rice. 10.5.

Before infection, the viability of the embryo is determined on an ovoscope. Live embryos are mobile, the pulsation of the vessels of the membranes is clearly visible. When candling, mark with a simple pencil on the shell the boundaries of the air sac or the location of the embryo, which is determined by its shadow on the shell.

Chicken embryos are infected in a box under strictly aseptic conditions using boil-sterilized instruments.

When infected on the chorion-allantoic membrane, 12-day-old embryos are most suitable. For infection in the allantoic cavity, embryos of 10-11 days of age are used, in the amniotic cavity - embryos of 7-11 days of age, in the yolk sac - embryos of 7 days of age.

Eggs with infected embryos are placed on stands with the blunt end up. The temperature regime and incubation period depend on the biological properties of the inoculated virus. The viability of the embryos is monitored daily under the ovoscope. Embryos that died on the first day after infection due to injury are not examined.

Before collecting the material, the embryos are cooled at 4 °C for 18-20 hours to constrict the vessels and prevent bleeding at autopsy. Embryos are opened in the box in compliance with the rules of asepsis.

Allantoic fluid is sucked up with a pipette, sterility is controlled by inoculation in sugar or meat-peptone broth, checked for the presence of a virus in the hemagglutination reaction and stored at 4 ° C in a frozen state.

To obtain amniotic fluid, the allantoic fluid is first aspirated, then the amniotic membrane is captured with tweezers, slightly lifted, and the amniotic fluid is sucked off with a Pasteur pipette.

When studying changes in the chorion-allantoic membrane, it is cut with scissors and all the contents are poured through the hole into a Petri dish. The chorion-allantoic membrane remains inside the shell and is removed with tweezers into a Petri dish with saline. Here it is washed, straightened and the nature of focal lesions is studied against a dark background.

To obtain the amniotic membrane, the amniotic sac in which the embryo is enclosed is cut and freed from the embryo, examined for lesions.

To obtain the yolk membrane, the chorion-allantois is cut, the allantoic and amniotic fluids are sucked off, the fetus is removed with tweezers, separated by the umbilical cord, the yolk sac is captured and placed in a Petri dish. Control for sterility, view for the presence of lesions. The yolk, if necessary, can be removed with a syringe without removing the yolk sac.

The presence of the virus in the allantoic and amniotic fluids of the infected embryo is determined in the hemagglutination test. Fluids from hemagglutination-positive embryos after testing for sterility are pooled and titrated in an extended hemagglutination test.

In the presence of a small amount of virus or the impossibility of detecting it in the test material, successive passages are carried out on chicken embryos. If, after three subsequent passages on embryos, the virus is not detected in the test material, the result is considered negative.

Use of cell cultures. Cultivation of cells outside the body requires the fulfillment of a number of conditions. One of them is the strict observance of sterility during work, since the nutrient media used are an excellent nutrient substrate also for bacteria and fungi. Tissue cells are highly sensitive to salts of heavy metals. Therefore, the quality of the various ingredients that make up saline solutions and nutrient media, as well as the methods of processing utensils and rubber stoppers used in cell culture, must be given exceptional importance.

One of the prerequisites for successful work with cells is the high quality of distilled water (checked twice a week). To work with cells, bidistilled or deionized water is used. The best distillers are devices made of glass or alloy steel: heavy metal ions, which are toxic to cells, are not washed out of such equipment. Deionized water is obtained at special installations, where water is purified from salts during its successive passage through columns with anion exchanger and cation exchanger.

When cultivating cells, particularly high requirements are placed on the preparation and sterilization of dishes and stoppers. In many cases, it is their improper washing and sterilization that causes cells not to attach to glass or rapid degeneration of the cell monolayer.

For the growth and reproduction of cells outside the body, a complex set of physico-chemical factors is required: a certain temperature, the concentration of hydrogen ions, inorganic compounds, carbohydrates, amino acids, proteins, vitamins, oxygen and carbon dioxide, therefore, for the cultivation of viruses in cell cultures, complex nutrient media are used. . According to the nature of the components that make up their composition, these media are divided into two groups.

• 1. Media, which are mixtures of saline solutions (Hanks, Earl, etc.) and natural components (animal and human blood serum, albumin hydrolyzate). The amount of each of these components in different formulations of media is different.
• 2. Synthetic and semi-synthetic media consisting of saline solutions (Earl, Hanks, etc.) with the addition of amino acids, vitamins, coenzymes and nucleotides (Eagle media, 199 etc.). In synthetic media, cells can exist in a viable state for a short time (up to 7 days). To maintain them in a viable state for a longer time, as well as to create better conditions for the growth and reproduction of cells, animal blood serum (cows, calves, etc.) is added to synthetic media.

Various methods of culturing cells outside the body can be used to isolate viruses. However, at present, single-layer cultures of primary trypsinized and transplanted cell lines have received the greatest practical application. Single-layer cell cultures are grown in glass flat-walled vessels-mattresses with a capacity of 1 l, 250 and 100 ml or in conventional bacteriological test tubes treated in an appropriate way.

When using primary trypsinized cell cultures, the essence of the method lies in the destruction of intercellular bonds in tissues by proteolytic enzymes and the separation of cells to grow a monolayer on the glass surface. Tissues and organs of human and animal embryos, slaughtered animals and birds, as well as those extracted from humans during surgery can serve as a source for obtaining cells. Use normal and malignant degenerate tissues, epithelial, fibroblastic type and mixed. The ability to reproduce cells extracted from the body is closely related to the degree of tissue differentiation. The less differentiated the tissue, the more intense the ability of its cells to proliferate in vitro. Therefore, cells of embryonic and tumor tissues are much easier to culture outside the body than normal cells of adult animals.

Daily cultures are viewed under a low magnification microscope to determine the nature of their growth. If the cells do not proliferate, look round, grainy, dark, and flake off the glass, the glassware is poorly processed or the ingredients in the culture medium are toxic.

Along with primary trypsinized tissues, transplanted cell cultures are widely used for virus cultivation; cell cultures capable of reproducing outside the body indefinitely. Cell cultures derived from normal and cancerous human tissues are most commonly used. The HeLa cell line obtained from a cervical tumor, Hep-2 from larynx carcinoma, KV from oral cavity cancer tissue has become widely known. Such cell cultures are also prepared from normal animal tissues - the kidneys of a monkey, a rabbit and a pig embryo (Table 10.1).

For reseeding of transplanted cells, the nutrient medium is sucked off with a pipette and poured out. The formed thin layer of cells is destroyed with a trypsin solution, and the cells released in this way are transferred to a new vessel with fresh nutrient solution, where a cell monolayer is again formed.

An indicator of the presence of the virus in infected cell cultures can be:

• a) development of specific cell degeneration;
• b) detection of intracellular inclusions;
• c) detection of a specific antigen by immunofluorescence;
• d) positive hemadsorption reaction;
• e) positive hemagglutination reaction;
• e) formation of plaques.

Table 10.1

List of the most commonly used cell cultures

To detect specific degeneration in infected cultures, cells are examined daily under a low magnification microscope. Many viruses, when multiplying in cells, cause their degeneration, i.e. have a cytopathogenic effect (CPA) (Fig. 10.6).

Rice. 10.6.

The time of development and the nature of cytopathic changes in infected cell cultures are determined by the properties and dose of the inoculated virus, as well as the properties and conditions of cell cultivation. Some viruses cause CPP within the first week after infection (pox, polio, Coxsackie B, etc.), others - after 1-2 weeks. after infection (adenoviruses, parainfluenza viruses, ECHO, etc.).

Viruses cause cytopathic changes of three main types: the formation of multinucleated giant cells and symplasts, which are the result of the fusion of the cytoplasm of many cells; round cell degeneration resulting from the loss of intercellular connections and rounding of cells; development of foci of cell proliferation, consisting of several layers of cells.

During the reproduction of some viruses in cell cultures, intracellular inclusions are formed in the cytoplasm or nucleus of the affected cells. Cell cultures to detect inclusions are grown on glass plates in test tubes, infected with a virus, and after certain incubation periods, preparations are prepared by staining them with conventional dyes.

To detect a specific antigen in infected cell cultures, preparations are prepared in the same way as for the detection of inclusions using MFA.

The plaque method is based on the formation in a monolayer of virus-infected cells under an agar coating of discolored areas consisting of degenerated (dead) cells. These areas, called plaques, are virus colonies, usually formed from a single viral particle.

In the absence of cytopathic changes, intracellular inclusions, plaque formation, negative reactions of hemadsorption and hemagglutination in cell cultures infected with the test material, two subsequent passages are carried out. In the absence of these changes in the final passage, the result of virus isolation is considered negative.

The following methods can be used to detect viruses in infectious material.

Microscopic:

• a) viroscopy;
• b) detection of intracellular inclusions.

Immunological:

• a) immune electron microscopy;
• b) immunofluorescence;
• c) hemagglutination;
• d) hemadsorption.

Identification of viruses is carried out using immunological methods, including the following reactions:

• a) inhibition of hemagglutination;
• b) hemadsorption delays;
• c) complement binding;
• d) neutralization;
• e) precipitation in agar gel.

microscopic methods. With a light microscope, only large viruses larger than 150 nm can be detected. Recognition of smaller viruses is only possible with an electron microscope. Light, phase contrast, and fluorescent microscopy can be used to detect large viruses.

During viral infections, peculiar inclusions develop in infected cells. Some infections are accompanied by the formation of inclusions in the cytoplasm of the affected cells (rabies, smallpox vaccine), others - in the cytoplasm and nucleus (measles, natural and chicken pox, adenovirus diseases). Inclusions have different nature, structure, shape and size from 0.25 to 25 microns. According to modern data, in some infections, inclusions are the site of virus reproduction and represent its accumulations surrounded by cell substances, while in others, they are a product of cell degeneration.

Inclusions can be detected in stained prints of organs and tissues, cell scrapings, histological sections from the affected tissue, and virus-infected cell culture preparations. Coloring is often done according to the Romanovsky-Giemsa method. For staining by this method, the preparations are fixed in a mixture of Dubosque - Brazil - Bouin, consisting of picric acid, formalin, alcohol, acetic acid. Intracellular inclusions in most viral infections are oxyphilic and stain pink or lilac according to the Romanovsky-Giemsa method.

Immunological methods for diagnosing viral infections. In recent years, these methods have become leading in the laboratory diagnosis of viral infections. This is largely due to economic reasons, since classical methods of virological analysis are quite expensive. In addition, the duration of studies using virological methods (weeks), even if they are quite effective, make them retrospective.

Immunological methods are used both for the detection of viral antigens in various biosubstrates and environmental objects, and for serodiagnosis - the detection of antibodies to viral antigens in the blood sera of sick people and laboratory animals. In addition, immunological research methods are indispensable for the identification of virions.

Interacting with the body, viruses cause the formation of antibodies, which, being adsorbed on virions, prevent the penetration of virions into cells and the development of cytopathic action (CPE); neutralize the deadly effect of viruses during their reproduction in chicken embryos and animals; inactivate virion hemagglutinins and neuraminidase, preventing the hemagglutination reaction (RGA) and the hemadsorption reaction (RGads) on virus-affected cells. These virus-neutralizing antibodies also cause agglutination and precipitation of viral particles, and the resulting immune complexes bind complement. Therefore, for the identification of virions, the classical neutralization reaction (PH) on cell cultures, chicken embryos and animals and its modifications are used: the hemagglutination inhibition reaction (HITA); haemadsorption inhibition reaction (RTGads). The same reactions are used in the serodiagnosis of viral infections to detect virus-neutralizing antibodies in the serum of patients according to a known viral antigen (diagnosticum).

Method of immunoelectron microscopy (IEM). Electron microscopy currently plays an important role in the study of viruses. It is the data of electron microscopy that serve as the basis for the modern classification of viruses.

A new stage in the development of electron microscopic study of viruses is the use of immunoelectron microscopy techniques. Using this method, not only direct detection of viruses, but also their identification, as well as rapid serotyping of viral strains and titration of antibodies to them became possible. IEM acquired great importance for determining the localization of viral antigens inside the cells of a macroorganism.

The undoubted advantage of IEM is its high sensitivity in comparison with conventional electron microscopy methods.

When a virus antigen or viral component comes into contact with a homologous antiserum, an antibody-antigen complex is formed. This phenomenon is the basis of the technique used to detect and identify viral antigens or antibodies to them. It is these complexes of antigens with antibodies after negative staining that can be observed in an electron microscope. In clinical diagnostics, antigenic material does not require thorough purification. So, if an influenza virus is detected, the unpurified allantoic fluid can be examined. Currently, it is believed that almost any kind of clinical material is suitable for IEM. For diagnostic purposes, ordinary unfractionated sera, as well as sera of convalescents, can be used. It should be noted that the ratio of the amounts of antigen and antibodies has a great influence on the final results. With an excess of antigen, an abundance of particles is observed; agglomerates in this case will be few. With an excess of antibodies, the viral particles are surrounded by their thick layer, it is almost impossible to reveal the small structural details of the virion; aggregates are also few. With the optimal ratio of antigen and antibodies, the aggregates are enlarged with a good image of the details of the virions. From the above considerations, it is desirable to use immune serum in several dilutions.

A support film made of palladium is applied to the support grid. When using low concentrations of palladium and to improve the adsorption properties of the substrate, it is reinforced with carbon. To do this, coal is sprayed onto the finished dry film-substrate on an electron microscopic grid in a vacuum. The thickness of the film-substrate and the reinforcing carbon layer has a significant effect on the contrast and image of fine details of the object. Each researcher determines the specific thickness of the substrate films and the carbon layer individually, based on the fact that carbon is more electronically transparent than palladium.

Viruses and antibodies to them have a low electron density. Therefore, biological objects cannot be detected using an electron microscope without pre-treatment. Viruses are visualized using a negative contrast (or negative stain) technique. Various salts of heavy metals are used for negative staining of viruses and virus-antibody complexes. Contrasting agents (heavy metal atoms) penetrate into the hydrophilic areas of objects and replace water in them. As a result, the electron density of the object increases, and it becomes possible to observe it in an electron microscope.

The direct IEM method has found the greatest application in practice. The virus suspension is mixed with undiluted antiserum. After vigorous stirring, the mixture is incubated for 1 h at

37°C, then overnight at 4°C. The next day, the mixture is centrifuged to precipitate the immune complexes. The precipitate is resuspended in a drop of distilled water and subjected to negative contrast.

When evaluating the results of IEM, the products of interaction between an antigen and an antibody in an electron microscope can have a different appearance (an individual viral particle covered with antibodies in whole or in part; agglomerates of viral particles). Agglomerates can occupy a different area, have a different appearance, and contain a different number of particles. Therefore, along with the experimental ones, it is necessary to study control preparations (with a buffer solution or heterologous antiserum).

The criterion for evaluating the results obtained using IEM is the presence or absence of clusters of viral particles aggregated by immune serum in the preparations. The presence of antigen agglomerates and specific antiserum antibodies is a sign of a positive reaction. Nevertheless, the possibility of non-specific aggregation of antigen particles under the influence of high-speed centrifugation should be taken into account. For this reason, many authors recommend considering results on a conditional scale from 0 to 4+. It is based on an assessment of the degree of coverage of aggregated particles with serum antibodies.

Methods of hemagglutination and hemadsorption. Many viruses have the ability to agglutinate erythrocytes of strictly defined species of mammals and birds. Thus, influenza and mumps viruses agglutinate erythrocytes of chickens, guinea pigs, and humans; tick-borne encephalitis virus - sheep erythrocytes; Japanese encephalitis viruses - erythrocytes of one-day-old chickens and geese; adenoviruses - erythrocytes of rats, mice, monkeys. Allantoic and amniotic fluids, a suspension of chorion-allantoic membranes of chicken embryos, suspensions and extracts from cultures of cells or organs of animals infected with viruses are used as the test material in the hemagglutination reaction (HA). The hemagglutination reaction can be set by the drip method on glass and in an expanded row in test tubes or wells of polystyrene plates. The first method is indicative.

Being group-specific, RGA does not make it possible to determine the species of viruses. They are identified using the hemagglutination inhibition test (HITA). Known immune antiviral sera are used for its formulation. An equal amount of virus-containing liquid is added to each dilution. The control is a suspension of the virus.

The mixture is kept in a thermostat, then a suspension of erythrocytes is added. A few minutes later, the titer of the neutralizing serum is determined, i.e. its maximum dilution, which caused a delay in erythrocyte agglutination.

In the serological diagnosis of viral diseases, RTGA is recommended to be performed with paired sera, one of which is obtained at the beginning of the disease, and the other after 1-2 weeks or more. A fourfold increase in antibody titer in the second serum confirms the proposed diagnosis.

The hemadsorption reaction (RGads) is used to indicate in infected cell cultures a virus with hemagglutinating activity. The essence of the reaction lies in the fact that erythrocytes sensitive to the hemagglutinating action of viruses are adsorbed on the surface of cells infected with viruses. For example, chicken erythrocytes are adsorbed on cells infected with the variola virus; measles virus - erythrocytes of monkeys; adenoviruses - monkeys and rats, etc.

Neutralization reaction (PH). Reproducing in cell cultures, viruses cause various kinds of CPD, expressed in rounding, wrinkling, reduction or, conversely, increase in cell size, their fusion and the formation of symplasts, destruction of the cytoplasm and nucleus. Finally, in a monolayer of cells infected with viruses, as a result of their destruction of certain sections of the cell layer, “sterile spots” or plaques can appear, which are a clone of the viral particle, which makes it possible not only to isolate the virus, but also to determine its titer.

It is very difficult to identify the virus by the nature of the plaques, and therefore they resort to setting the PH of the isolated virus with known virus-neutralizing sera. For this purpose, the virus obtained from the patient is accumulated in cell culture and its various dilutions are mixed with undiluted antiviral serum.

A mixture of viruses and sera can infect chick embryos or sensitive animals. In such cases, the neutralizing activity of antibodies is most often determined by the neutralization of viral hemagglutinins in the fluids of the embryo and the elimination of the lethal effect of the virus on embryos and animals. At the same time, the neutralization index is calculated, which expresses the maximum number of lethal doses of the virus that is neutralized by this serum, compared with the results of the control experiment, taken as one.

Similarly, using PH, viruses isolated from the material of patients are identified when they infect chicken embryos and animals. To do this, virus-containing fluids of embryos and suspensions of the affected organs of animals are added to the virus-neutralizing sera. After a certain incubation time, cell cultures, chick embryos and animals are infected with mixtures.

In the serodiagnosis of viral infections, the dynamics of the increase in the titer of virus-neutralizing antibodies for a known virus is determined. At the same time, PH is set with paired sera taken from patients at the beginning and at the end of the disease. Diagnostic will be a 4-fold increase in the titer of immunoglobulins in the second of them.

PH is based on the ability of specific antibodies to bind strongly enough to a viral particle. As a result of the interaction between the virus and the antibody, the infectious activity of the virus is neutralized due to the blockade of antigenic determinants responsible for the connection of the viral particle with sensitive cells. As a result, the virus loses its ability to multiply in a sensitive biological system in vitro or in vivo.

The results of PH become apparent after a mixture of the virus and its homologous antibodies, after a timed exposure, is introduced into a sensitive biological system (tissue cell culture, chick embryo, susceptible animal body), where the virus can multiply and cause measurable changes that will suppressed partially or completely in the presence of antibodies.

There are three components involved in PH:

• 1) virus;
• 2) serum containing antibodies;
• 3) a biological object (laboratory animals, developing chicken embryos, tissue cultures), the choice of which depends on the type of virus with which it is supposed to conduct research.

PH is used either to identify an isolated pathogen or to detect and titrate antibodies in sera. In the first case, the sera of specially immunized laboratory animals or recovered people are used. In the second case, sera taken at the initial stage of the disease and during the convalescence period are used.

Virus-neutralizing antibodies in the sera of recovered people, unlike antihemagglutinins or complement-fixing antibodies, persist for many years, and in some viral infections (for example, measles) even for life. This makes it possible in some cases to use the pool of sera of many convalescents as a reference drug, which, after filling into ampoules and lyophilization, is suitable for diagnostic work for a long time.

When identifying isolated pathogens, pre-prepared hyperimmune sera of various animals are used: rabbits, white rats and mice, guinea pigs, monkeys, sheep, horses, etc. The activity of hyperimmune sera for PH depends on the method of immunization of animals.

Before setting up each neutralization experiment, the virus is pre-titrated, determining the final dilution that causes tissue culture damage or infection of laboratory animals (or chicken embryos). The virus titer is expressed as a 50% dose (TCID50 - 50% infectious dose for tissue culture).

Molecular genetic diagnostic methods in virological practice. Methods of molecular biology were developed in the 50s. XX century. They became possible due to the fact that in the genome of each virus there are unique species-specific nucleotide sequences, by detecting which any infectious agent can be identified. These methods are most important in identifying microorganisms that are long or difficult to cultivate by conventional methods. In the 1970s, DNA probe detection was used to detect an infectious agent or mutation, based on the hybridization of specific oligonucleotide probes labeled with a radioactive isotope (or fluorochrome) with an isolated DNA sample. Hybridization analysis uses the ability of nucleic acids under certain conditions to form specific complexes with nucleic acids that have sequences complementary to them. The method of detecting infectious pathogens by DNA hybridization turned out to be extremely laborious, time-consuming and expensive. In addition, its sensitivity is insufficient for the identification of microorganisms in clinical materials such as faeces and urine.

DNA hybridization has been replaced by a method that mimics the natural replication of DNA and allows you to detect and repeatedly copy a certain DNA fragment using thermophilic DNA polymerase. Polymerase chain reaction (PCR) is an elegant technique that mimics natural DNA replication and allows a specific piece of DNA to be detected and copied repeatedly using thermophilic DNA polymerase.

Due to its high diagnostic qualities, PCR is a generally recognized addition to the traditional methods used in virology: virus propagation in cell culture, immunological detection of viral antigens, and electron microscopy. A significant advantage of this method is the ability to detect viruses in latent infections (cytomegalovirus, herpes virus) and viruses that are difficult or not yet possible to cultivate (human immunodeficiency virus, Epstein-Barr virus, human papillomavirus, Vidr hepatitis virus). Prospects for studying such diseases as Creutzfeldt-Jakob disease, Alzheimer's disease, and multiple sclerosis are associated with the PCR method.

Etiological diagnosis of viral diseases is carried out by virological, virological, serological and molecular genetic methods. The last three methods can be used as express diagnostic methods.

Virological diagnostic method.

The ultimate goal of the method is to identify viruses to a species or serological variant. The virological method includes several stages:

1) selection of material for research;

2) processing of virus-containing material;

3) contamination of sensitive living systems with material;

4) indication of viruses in living systems;

5) titration of isolated viruses;

6) identification of viruses in immune reactions.

1. Selection of material for research .

It is carried out in the early stages of the disease, subject to the rules that prevent contamination of the material with foreign microflora and infection of medical personnel. To prevent virus inactivation during transport, the material is placed in a viral transport medium (VTS) consisting of balanced salt solution, antibiotics, and serum albumin. The material is transported in a special container with thermal insulation and closed plastic bags containing ice. If necessary, the material is stored at -20˚C. Each sample of material for research should be labeled and labeled with the name of the patient, type of material, date of sampling, detailed clinical diagnosis and other information.

Depending on the nature of the disease, the material for the study may be:

1) swabs from the nasal part of the pharynx and a swab from the pharynx;

2) cerebrospinal fluid;

3) feces and rectal swabs;

6) fluid from serous cavities;

7) smear from the conjunctiva;

8) contents of vesicles;

8) sectional material.

To obtain flushing from the oropharynx, 15-20 ml of VTS are used. The patient carefully rinses the VTS throat for 1 minute and collects the flush in a sterile vial.

A smear from the back of the pharynx is taken with a sterile cotton swab, pressing on the root of the tongue with a spatula. The swab is placed in 2-3 ml of VTS, rinsed and squeezed.

Cerebrospinal fluid is obtained by lumbar puncture. 1-2 ml of cerebrospinal fluid is placed in a sterile container and delivered to the laboratory.

Fecal samples are taken within 2-3 days in sterile vials. A 10% suspension is prepared from the obtained material using Hank's solution. The suspension is centrifuged at 3000 rpm, the supernatant is collected, antibiotics are added to it and placed in a sterile dish.

The blood obtained by venipuncture in a volume of 5-10 ml is defibrinated by adding heparin. Whole blood is not frozen and antibiotics are not added. To obtain serum, blood samples are incubated at 37°C for 60 minutes.

Fluid from the serous cavities is obtained by puncture in the amount of 1-2 ml. The liquid is used immediately or kept frozen.

A smear from the conjunctiva is taken with a sterile swab and placed in the VTS, after which the material is centrifuged and frozen.

The contents of the vesicles are aspirated with a syringe with a thin needle and placed in the VTS. The material is sent to the laboratory in the form of dried smears on glass slides or in sealed sterile capillaries or ampoules.

Sectional material is taken as early as possible, observing the rules of asepsis. Separate sets of sterile instruments are used to collect each sample. The amount of selected tissues is 1-3 g, which are placed in sterile vials. First, samples of extracavitary organs (brain, lymph nodes, etc.) are taken. Tissues of the chest cavity are taken before opening the abdominal cavity. The resulting tissue samples are ground in a mortar with the addition of sterile sand and sterile sodium chloride solution, after which the material is centrifuged. The supernatant is collected in vials, antibiotics are added. Material for virological research is used immediately or stored at -20°C.

2. Processing of virus-containing material.

It is carried out in order to free the material from the accompanying bacterial microflora. For this, physical and chemical methods are used.

Physical methods:

1) filtration through various bacterial filters;

2) centrifugation.

Chemical methods:

1) treatment of the material with ether in cases of isolation of viruses that do not have a supercapsid;

2) adding a mixture of heptane and freon to the material;

3) the introduction of antibiotics (penicillin - 200-300 U / ml; streptomycin - 200-500 μg / ml; nystatin - 100-1000 U / ml).

3. Infection of sensitive living systems with material.

1) laboratory animals;

2) chicken embryos;

3) organ cultures;

4) tissue culture.

laboratory animals. White mice, guinea pigs, hamsters, rabbits, etc. are used. White mice are most sensitive to a large number of types of viruses. The mode of infection of animals is determined by the tropism of the virus to the tissues. Infection in the brain is used in the isolation of neurotropic viruses (rabies viruses, polioviruses, etc.). Intranasal infection is carried out when pathogens of respiratory infections are isolated. Widely used intramuscular, intravenous, intraperitoneal, subcutaneous and other methods of infection. Sick animals are euthanized with ether, opened and material is taken from organs and tissues.

chicken embryos. Widely available and easy to use. Apply chicken embryos aged 5 to 14 days. Before infection, chicken embryos are ovoscoped: their viability is determined, the border of the air sac and the location of the embryo (the “dark eye” of the embryo) are marked on the shell. Work with chicken embryos is carried out in a sterile box with sterile instruments (tweezers, syringes, scissors, spears, etc.). After completing a fragment of the work, the instruments are immersed in 70% ethyl alcohol and burned before the next manipulation. Before infection, the shell of a chicken embryo is wiped with a burning alcohol swab and an alcohol solution of iodine. The volume of the test material injected into the embryo is 0.1-0.2 ml. At least 4 chick embryos are used to isolate viruses from one material.