Laboratory diagnosis of viral infections

Etiological diagnosis of viral diseases is carried out 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; 4) blood; 5) urine; 6) fluid from serous cavities; 7) smear from the conjunctiva; 8) contents of vesicles; 8) sectional material.

For getting flushing from the oropharynx use 15-20 ml of VTS. The patient carefully rinses the VTS throat for 1 minute and collects the flush in a sterile vial.

A smear from the posterior pharyngeal wall take 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 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.

Vesicle content 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 taken as soon 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).

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.

Virology which infections are being investigated. Methods of virological research

Virological research methods— 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 antigen with antibodies), biological properties of the virus (ability to hemagglutinate, hemolysis, enzymatic activity), 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 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 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; determination of 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 secrets 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 class lgM 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-m antisera (anti-IgM heavy chain sera).

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 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.

To identify individual antigens of viruses and antibodies to them in complex mixtures without prior protein purification, immunoblotting is used. 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, establishing their size and the presence of antigenic determinants.

Research for the diagnosis of diseases with a viral nature. This is necessary in order to identify the virus, to study its biology and ability to affect animal and human cells. Thus, it becomes possible to understand the pathogenesis of viral diseases and, accordingly, to choose the right treatment method.

What is the diagnosis?

in living cells. To investigate it, it is necessary to cultivate it at the level of an experimental organism or For this, virological research methods are carried out in medical practice and microbiology in general, which have the following main approaches:

straight;
indirect;
serological.

The material can be examined directly for the presence of nucleic acids, viral antigen, or, for example, to isolate and identify the virus from clinical material.

In addition to the ability to establish the etiology of the disease, monitoring the therapeutic effect, virological research methods play an important role in anti-epidemic measures. For isolation and use chicken embryos, laboratory animals or cell cultures.

How are they researched?

The fastest is the direct method. It allows you to detect a virus, antigen or NA (nucleic acid) in the clinical material itself. It takes from two hours to a day.

EM - electron microscopy. Detects the virus directly.
IEM - immune electron microscopy. Uses specific antibodies to viruses.
RIF - immunofluorescence reaction. Uses dye-bound antibodies. Such virological research methods are widely used as a quick decoding of the etiology of ARVI (acute respiratory viral infections), when smears are taken from the mucous membrane of the upper respiratory tract.
ELISA - enzyme immunoassay - determination of viral antigens, similar to RIF, but based on enzyme labeling of antibodies.
RIA - radioimmunoassay. Uses radioisotope labeling of antibodies to provide high sensitivity in viral antigen detection.
Molecular - NK hybridization or isolation of virus genomes using PCR (polymerase chain reaction).
Cytology - rarely used, but for certain infections, these virological methods of research are very effective. Biopsy materials, autopsies and smears processed for staining and analysis under a microscope are examined.

What is the point of research?

For successful isolation of viruses, clinical material is taken in accordance with the pathogenesis and as early as possible. Often this process requires several passages before certain virological assays are applied.

Microbiology is the study of microscopic beings. And her field is not only medicine. It is a fundamental science for agriculture, veterinary medicine, space and technical industry, and geology.

But of course, everything is created for man and his development on this beautiful planet. Therefore, it is very important to detect the danger in time and neutralize it. Viruses are different from bacteria. These are structures that enter the body and cause the formation of a new generation. They look like crystals and are aimed at controlling the process of their reproduction, although they themselves do not feed, do not grow, and do not excrete metabolic products.

The virus can cause serious illness in any living organism into which it has entered. Plus, it can evolve. That is why virological research methods in microbiology must be developed and improved, since human civilization as a whole may be under threat.

materials

To detect and identify viruses in medicine, as a rule, they take:

nasopharyngeal lavage (respiratory infections);
flushing and faeces (enterovirus infections);
scrapings, the contents of the vesicles (skin lesions, mucous membranes, like herpes, chicken pox);
flushes (exanthemic infections like measles, rubella);
blood, cerebrospinal fluid (arbovirus infections).

Phases

All stages of the virological research method include:

collection of material;
selection, obtaining a test system, determining its viability;
infection of the test system;
virus indication;
determining the type of virus.

Basically, pathogenic viruses differ in the presence of tissue and type specificity. Take, for example, poliovirus, which only reproduces in primates (in their cells). Accordingly, a specific tissue culture is used to isolate a particular virus. If we are talking about an unknown pathogen, then it would be advisable to simultaneously infect three, and preferably four cell cultures.

Thus, perhaps one of them will be sensitive. To determine the presence of the virus in infected cultures, look at the development of specific cell degeneration, intracellular inclusions, detection of a specific antigen, positive hemagglutination and hemadsorption tests.

All virological methods of investigation (direct and indirect, serological) should be selected as the most appropriate for a particular case of suspected infection.

Indirect methods are based on the isolation and identification of the virus. They are laborious, lengthy, but accurate.

Serodiagnostics

This diagnosis refers to a method based on the antigen-antibody reaction. Most often, paired blood sera are used, taken at intervals of several weeks. If the increase in antibody titer is 4 or more times, the reaction is considered positive. To determine the type specificity of a virus, a virus neutralization test is used. To determine group specificity, you need to get the complement fixation reaction.

Various variants of enzyme immunoassay, hemagglutination inhibition reaction, passive hemagglutination, reverse passive hemagglutination, RIF are widely used. Even in genetic engineering, a method for obtaining monoclonal antibodies was developed. The narrow specificity of monoclones can be overcome by using several monoclonal antibodies to various viral determinants. Thus, the specificity and sensitivity of the assay with the determination of antigens has been increased.

Some Features

Today, many different test systems have been created for the immunological diagnosis of infections resulting from the entry of a virus into a living organism.

Thus, virological research methods are methods for isolating viruses, studying their properties and establishing their etiological relationship with certain diseases.

Virological studies in the clinic of infectious diseases are becoming increasingly important, which is primarily due to the increase in the proportion of infections of a viral nature, the clinic of which is not always typical. At the same time, fast and reliable methods of virological diagnostics have not been developed for all infectious diseases, many of them are laborious, they require special conditions, experimental animals, nutrient media, and trained personnel. Currently, 3 main types of research are used to diagnose viral infections.
1. Microscopic examination of infectious material in order to detect viral antigen or pathognomonic changes in tissues. For diagnostic purposes, direct microscopic examination of infectious material from patients is used for a limited number of viral infections (rabies, chicken pox, yellow fever, herpes, etc.). A method based on the detection of viral antigen using fluorescent antibodies has gained wider application. The method of immunofluorescence can be reliable only if all technical requirements are strictly met.
2. Virological methods.
3. Serological studies to determine the increase in antibody titer in the course of the disease. Serological research methods are more available in the laboratory.
For these studies, it is necessary to take blood serum in the acute period of the disease and during the period of convalescence (paired sera). Blood samples for serological studies are taken sterile without anticoagulants and preservatives.
The main stages of virological research are the isolation of viruses, their identification, and the characterization of the main biological properties. There is currently no single method for isolating different groups of viruses. This is primarily due to the diversity of their properties and features of cultivation outside the host organism. For the study, biosubstrates are used (washings from the mucous membranes, blood and its components, cerebrospinal fluid, urine and feces, biopsy specimens of organs and tissues or their pieces taken during autopsy), which are subjected to special processing followed by passage of the material. The material taken for research should be stored at a temperature from -20 °C to -70 °C. Depending on the preliminary diagnosis, the processing of the material has its own characteristics, but in all cases it is supposed to obtain a substrate that is maximally purified from impurities of mucus, cells of organs and tissues or their fragments, bacteria. This is achieved by homogenizing the test material in a special apparatus or grinding in a porcelain mortar in the cold with quartz glass (pieces of organs and tissues) with the addition of sterile chilled (+4 C) 0.9 %

sodium chloride solution to obtain a 10-30% suspension and subsequent centrifugation at 1500-3000 rpm for 10-15 minutes. The supernatant thus obtained is used for further studies.
Before the intensive development and introduction into wide practice of the method of tissue and cell culture, infection of experimental animals or chicken embryos was used. These methods are still in use today. The detection of viruses using animals is most appropriate in cases where it is possible to reproduce in the experiment a typical picture of an infectious disease or its individual manifestations. Thus, pathogens of the arboviruses and Coxsackie groups can be detected by infection in the brain of suckling mice, influenza - by infection of chicken embryos or intranasal administration of the test material to mice. In recent years, virological laboratories have most widely used the method of cell and tissue culture, which makes it possible to isolate adenoviruses, herpes viruses, respiratory syncytial virus, myxoviruses, and others, and to carry out etiological diagnosis of the disease already at the first stages of the study. The basis for this is the well-studied cytological features of the interaction of most viruses and cells. Thus, infection of HeLa, Hep-2 cells with a material containing type 2 adenovirus leads already on the 3rd day to a change in the growth pattern of the cell monolayer and to the appearance of typical cells in the form of grape bunches, etc., which are well defined in the usual light microscope at low magnification.
Of exceptional importance for the etiological diagnosis of an infectious disease is the standardization of virus isolation from the test material, which at this stage of the work involves the use of genetically pure linear animals, taking into account their phenotypic (primarily age) characteristics. This is primarily due to the fact that experimental animals of various genetic lines and ages are susceptible to viruses to varying degrees. Thus, during intracerebral infection of mice with a neurotropic WSN strain of the influenza virus, the BALB/c, A, CBA and outbred animal lines showed the highest sensitivity, the same pattern was established in cases of intranasal administration of the test material. Essential for the final results of virus isolation is a preliminary examination of animals, chicken embryos, cell and tissue cultures for latent virus carriers. Very widely used in laboratory practice, chicken embryos can be infected with avian leukemia viruses, avian encephalomyelitis, infectious sinusitis, psittacosis, Newcastle disease, pathogens of some bacterial infections (paratyphoid, etc.), as well as mycoplasma. An even greater number of bacterial and especially viral agents are capable of spontaneously infecting cell and tissue cultures and surviving in them. Their presence significantly affects the assessment of the material under study. Some types of mycoplasmas in cell culture
can cause hemagglutination and hemadsorption and even form plaques under the agar coating, similar to the viruses formed. Of no small importance is also the contamination of cell cultures of one type by others, most often this is associated with HeLa cells and is observed when working with different types of cultures in the same room, with poor processing of laboratory glassware, etc. The presence of contamination of cell cultures or infection of animals with bacterial agents, such as as a rule, it manifests itself quite clearly (changes in the growth pattern of the cell monolayer, the properties of the culture medium, the death of chicken embryos or animals with certain symptoms, etc.) and presents no particular difficulties in evaluating the results. The situation is more complicated with latent forms of infection, where the use of serological and other methods is required. These instructions should be taken into account in the work of a virologist, especially when conducting an etiological diagnosis of unclear cases of the disease.

There are several ways to infect a chicken embryo: in the cavity of the amnion and allantois, on the chorion-allantoic membrane, in the yolk sac (Fig. 1).

Infection in the allantois cavity. The chicken egg is placed vertically, with the air bag up. In the center of the blunt pole of the egg above the air sac, the shell is pierced, a needle for intramuscular injections is inserted 2-3 mm below the border of the air sac, and the test material is injected with a tuberculin syringe. The puncture in the shell is closed with molten paraffin or adhesive tape.

Infection in the amnion cavity. A 1x1 cm window is cut above the air sac of a vertically located egg and a part of the chorion-allantoic membrane above the body of the embryo is carefully removed. The test material is injected into it with tweezers using a tuberculin syringe. Amnion is brought to its original position by releasing the tweezers. The hole in the shell is closed with adhesive tape.

Infection on the chorion-allantoic membrane. Above the air chamber of a vertically located egg, a piece of shell is cut out, creating a window. Then the shell is peeled off under the shell, exposing the area of the chorion-allantoic shell, on which the test material is applied. The hole in the shell is sealed with adhesive tape.

Infection in the yolk sac. The egg is laid horizontally so that the body of the embryo is at the bottom and the yolk is above it. A needle for intramuscular injections is inserted through the shell puncture in the area of the air sac along the central axis of the egg to a depth of 2/3 of the needle length, and the test material is injected with a syringe. The hole in the shell is sealed with adhesive tape.

After infection, the embryos are incubated in a thermostat, with the blunt end up. The temperature and duration of incubation depend on the biological properties of the isolated virus. At the end of the incubation, the embryos are cooled at +4°C for 16-18 hours. After that, the chicken embryo is sterilely opened by cutting a hole in the shell above the air sac above the designated border. Allantoic, then amniotic fluid is sucked off with a Pasteur pipette or syringe, the chorion-allantoic membrane is cut for study, the rest of the contents of the egg are removed into a Petri dish. Allantoic and amniotic fluids are used to indicate viruses.

Organ cultures. These are properly prepared sections of organs that in vitro retain their structure and functions for several days, and sometimes weeks. Organ cultures are grown on the surface of a liquid nutrient medium using a "raft" or "platform". Infection of organ culture is carried out by introducing pieces of an organ or tissue into a test tube with the test material. The adsorption of the virus is carried out for 1-2 hours at room temperature. Then the test material is drained, fragments of the organ or tissue are washed in Hank's solution, placed in a culture vessel, a nutrient medium is added and kept in a thermostat. Sampling of material for the detection of the virus in tissue culture begins on the 2nd day of cultivation.

Cell cultures. Cell culture is a population of the same type of cells of an animal or human organism, which is grown under artificial conditions and is intended for the cultivation of viruses. According to the life span, cell cultures are divided into: 1) primary; 2) semi-transplantable; 3) transplantable.

Primary cell cultures obtained from animal and human tissues by their enzymatic disintegration. Pieces of tissue are placed in a 0.25% trypsin solution at a temperature of 37°C and periodically mixed. As a result, tissue cells separate from each other. Portions of cells are collected as they are separated, centrifuged, the trypsin is drained, the growth medium is added and the cells are suspended in it. Primary cell cultures can undergo up to 10 divisions in vitro, are highly sensitive to many viruses, can be obtained in large quantities, and are oncogenically safe. The disadvantage of primary cultures is the significant laboriousness and duration of production, as well as possible contamination with latent viruses. Primary cell cultures include kidney cells of a human embryo, rhesus monkey, pig embryo, chicken embryo fibroblasts.

Semi-permanent cell cultures are diploid cells of the same type, which are capable of undergoing up to 100 divisions in vitro, while maintaining the original diploid set of chromosomes. Semi-transplantable cell cultures include human embryonic fibroblasts (Fig. 2). These cells are extremely demanding on cultivation conditions, therefore, they are of limited use in the practice of virological laboratories.

Continuous cell cultures are the same type of tumor or normal human and animal cells with an altered karyotype, capable of unlimited growth under in vitro conditions. Continuous cell cultures are easy to cultivate, and therefore are widely used in the laboratory diagnosis of viral diseases in humans. The transplanted cell cultures include HeLa (human cervical carcinoma cells), KB (human oral cavity carcinoma cells), Vero (green monkey kidney cells), SPEV (porcine embryonic kidney cells), etc.

The cultivation of cell cultures, regardless of their type, is carried out under sterile conditions in special flat glass vessels - mattresses, into which a nutrient medium is introduced. At the bottom of the mattress, cells during their reproduction form a monolayer.

For the cultivation of cell cultures, special nutrient media are used, containing physiological amounts of amino acids, carbohydrates, mineral salts, and having pH=7.2-7.4. Along with the nutrients in the media there is an indicator that changes the color of the medium when the pH shifts from the optimal value. The most widely used when working with cell cultures are: medium 199, Needle medium. Medium 199 includes 60 components and is used for culturing continuous and primary trypsinized cells. Medium Needle contains a minimum set of amino acids (13) and vitamins (8). It is used for cultivation of diploid and continuous cell cultures.

Cell cultivation must be carried out under aseptic conditions, and therefore antibiotics (for example, penicillin and streptomycin) are added to nutrient media.

4. Indication of viruses in living systems. Indication of viruses is the detection of viruses in the test material without establishing their belonging to a family, genus, species or serovariant.

Indication of viruses in laboratory animals. The presence of viruses in the body is primarily indicated by the development of symptoms of the disease or the death of the animal. Samples of affected organs and tissues are taken from a dead animal or previously euthanized with ether, placed in a porcelain mortar, salt solution is added and rubbed with sand. The resulting suspension is centrifuged to precipitate tissue detritus. In the supernatant, viruses are indicated by hemagglutinating, complement-fixing or other antigens.

Virus detection in chick embryos. In the amniotic and allantoic fluid, the indication of viruses is carried out in the hemagglutination reaction (RGA). When a chicken embryo is infected, plaques or pockmarks, which are virus-specific lesions, are often found on the chorion-allantoic membrane. The indication of viruses in the chorion-allantoic membrane is carried out in the reactions of hemagglutination or complement fixation (RCC). To do this, the shell is ground in a mortar, a suspension is prepared, which is centrifuged to precipitate tissue detritus, and the supernatant is examined in the RGA or RSK.

Indication of viruses in cultures of organs and cells carried out according to: 1) cytopathic effect of viruses (CPE); 2) the formation of intracellular inclusions; 3) in the hemagglutination reaction; 4) by plaque formation; 5) by color sample; 6) according to the hemadsorption reaction.

JPC- These are morphological changes in the culture of organs and cells that occur in the process of reproduction of viruses in cells. Viruses that cause CPP are called cytopathogenic. The nature of the CPD depends on the biological properties of the viruses, the dose of the virus, the properties of the cells and the conditions for their cultivation. CPD of viruses can be manifested by necrosis, cluster formation, symplasto- and syncytium formation, round cell degeneration, cell proliferation, and focal destruction.

With necrotic CPD of poliomyelitis, Coxsackie, ECHO viruses, most cells are completely destroyed, the remaining cells are wrinkled (pyknosis of the nucleus and cytoplasmic membrane, vacuolization), they are characterized by birefringence - a strong glow under microscopy.

CPE by the type of clustering is typical for adenoviruses, while the cells are rounded, enlarged, partially merge with each other with the formation of clusters (Fig. 3).

Herpes, measles, mumps, parainfluenza, RS viruses cause CPP by the type of symplasto- or syncytium formation (Fig. 4).

Syncytium consists of cells connected by cytoplasmic bridges, while symplast is a large multinucleated cell formed as a result of multiple incomplete mitoses.

CPD of viruses according to the type of round cell degeneration is characterized by rounding of cells and the loss of their intercellular connections. Pycnosis, wrinkling and destruction of cells can also be observed (Fig. 5).

In oncogenic viruses, CPE can manifest itself as the transformation of cells into malignant ones, which is accompanied by intensive cell proliferation and the formation of multilayer cellular structures. CPE of some strains of influenza viruses, vaccinia, smallpox is manifested by focal destruction of the cell culture - against the background of the monolayer preserved as a whole, foci of cell damage (microplaques) appear.

In the absence or mild CPE, new cell cultures are infected with the culture liquid.

Intracellular inclusions in the cytoplasm or nucleus of cells are formed during the reproduction of rabies, smallpox, influenza, herpes, adenoviruses, etc. viruses in them. Intracellular inclusions are crystal-like clusters of virions. Inclusions are detected by light immersion microscopy after staining glasses with a monolayer according to Romanovsky-Giemsa, or by fluorescence microscopy after treatment with acridine orange. When stained according to Romanovsky-Giemsa, viral inclusions acquire a pink or pink-lilac color. When stained with acridine orange, DNA structures give off a green glow, while RNA structures give off a reddish-orange color. Currently, the detection of intracellular inclusions is carried out in the diagnosis of rabies (Babes-Negri bodies) (Fig. 6). Previously, with natural smallpox, the detection of Guarneri bodies was carried out.

Plaque formation. Plaques are foci of destroyed primary virus-infected cells of the monolayer under the agar coating. Plaques are detected by staining the culture with neutral red, which is either included in the agar coating or added immediately before the results are recorded. Since the plaques consist of dead cells that do not perceive the dye, they are therefore visible as light spots against the background of a pink-red monolayer of living cells. Accounting for plaque formation is carried out for a quantitative analysis of the infectious activity of cells.

color sample. Environments 199 and Needle, in which cell cultures are cultivated, have a crimson color, pH=7.2-7.4, and contain an indicator that changes the color of the medium with a change in pH. When cell cultures not infected with the virus are cultivated in these media, the color of the medium changes to orange due to the release of acidic metabolic products by the cells. Virus-infected cells are destroyed as a result of the suppression of metabolism by viral reproduction, as well as as a result of the CPE of viruses, and the alkaline cytoplasm of the cells enters the medium without changing its color (the medium remains red).

Hemagglutination reaction (RHA) is based on the ability of some viruses containing agglutinin on their outer shell to glue (agglutinate) erythrocytes of certain animal species. For RGA, a cell-free virus-containing material is used (allantoic or amniotic fluid, tissue culture supernatant). The virus-containing liquid is mixed with 0.5 ml of isotonic sodium chloride solution and 0.5 ml of 1% suspension of washed erythrocytes, after which it is incubated at 37˚, 20˚ or 4˚C for 30-60 minutes. With a negative control, the development of agglutination in the experiment indicates the presence of the virus in the test liquid. The control is a mixture of 0.5 ml of erythrocytes with an equal volume of isotonic sodium chloride solution that does not contain the virus.

Hemadsorption reaction (RGads) makes it possible to detect hemagglutinin-containing viruses in cell cultures before the development of CPD (Fig. 7). Hemadsorption is observed only if the hemagglutinin of the virus is present on the cytoplasmic membrane of the culture cells. Rgads is carried out by adding 0.2 ml of a 0.5% suspension of erythrocytes to the cell culture, after which the cells are kept for 15-20 minutes at 37˚, 20˚ or 4˚C (depending on the properties of the virus). Then the tubes are shaken to remove non-adsorbed erythrocytes and their accumulation on individual cells or on the entire monolayer is taken into account under a small magnification of the microscope. On cells not infected with viruses, erythrocyte adsorption is not observed.

5. Titration of isolated viruses - this is an obligatory stage of the virological diagnostic method, the purpose of which is to quantify the content of viral particles per unit volume of the test material.

Titration methods for viruses isolated from laboratory animals provide for the determination of the dose (titer) at which the pathogen causes the death of 50% of infected animals or the characteristic symptoms of the disease. The virus titer is expressed in LD 50 - lethal dose or ID 50 - infectious dose.

Titration of viruses isolated from chick embryos and possessing hemagglutinating activity is carried out in a hemagglutination reaction. RGA is carried out in test tubes or in special tablets. Two-fold dilutions in 0.5 ml of isotonic sodium chloride solution are prepared from the virus-containing material. Add 0.5 ml of erythrocyte suspension to all tubes. The control is a mixture of 0.5 ml of erythrocytes with the same volume of isotonic sodium chloride solution that does not contain viruses. Depending on the properties of the studied virus, the mixture is incubated in a thermostat at 37˚, 20˚ and 4˚C. The results of the reaction are taken into account 30-60 minutes after the complete sedimentation of erythrocytes in the control: (++++) - intensive and rapid agglutination of erythrocytes, the sediment has a star-shaped shape with scalloped edges ("umbrella"); (+++) - erythrocyte sediment has gaps; (++) - less pronounced precipitate; (+) - flocculent erythrocyte sediment surrounded by a zone of lumps of agglutinated erythrocytes and (-) - a sharply defined erythrocyte sediment ("coin column"), the same as in the control. The titer of the virus during RGA is its greatest dilution, at which erythrocyte agglutination is still observed. This dilution is considered to contain one hemagglutinating unit of virus (1 HAU). Dilutions that precede 1 HAU will contain 2 times the amount of HAU compared to the subsequent dilution from them. For example, if 1 GAU corresponds to a dilution of 1:64, then a dilution of 1:32 will correspond to 2 GAU, and dilutions of 1:16 and 1:8 will correspond to 4 and 8 GAU, respectively. Virus titer of 4 HAU is usually used to identify viruses.

Titration of viruses in cell cultures carried out by CPP, plaque formation and color test.

The titer of the virus when it is determined in cell cultures by CPE is the highest dilution of the virus-containing material in which the virus is able to cause CPE in 50% of infected cell cultures. This value is called the 50% tissue cytopathic dose (TCD 50). Titration of the virus by CPE includes the following steps: 1) inoculation, cultivation and selection of test-tube cell cultures with a formed monolayer; 2) obtaining 10-fold dilutions of virus-containing material; 3) infection of cell cultures with different dilutions of the virus; 4) keeping cell cultures in a thermostat at 37˚; 5) taking into account the results for 5-7 days according to the system of pluses (++++) and statistical processing of the results. To obtain statistically reliable results, a number of rules must be observed: a) the use of at least 4 tube cultures of cells for infection with 1 dilution of the virus; b) inclusion in the titration series of 2 dilutions of the virus - below and above the CPP 50.

Titration of viruses in cell cultures by plaque formation is one of the most sensitive and accurate methods for the quantitative determination of viruses. However, the method is technically complex and is mainly used in scientific research.

Titration of viruses in cell cultures by the color test method is designed to determine the highest dilution of the virus-containing material, at which the color of the medium containing the cell suspension at a concentration of 200,000 cells per 1 ml changes color. After establishing the virus titer, a working dose is prepared - 100 TCD 50, which is used in the identification of viruses.

6. Identification of viruses in immune reactions. Identification or titration of viruses is the establishment of their variant, species, generic and family affiliation. Identification of viruses is carried out according to the principle: the definition of the unknown by the known. A well-known component in the identification of viruses are specific antiviral sera (anti-influenza, anti-measles, etc.), which are used in serological neutralization tests (RN), hemadsorption inhibition (RTGads), hemagglutination inhibition (RTGA), RPHA, RSK, as well as in ELISA and RIA . These sera contain specific antiviral antibodies and are called diagnostic.

Neutralization reaction (RN) can be carried out on cell culture, chicken embryos and animals. Neutralization mixtures are prepared in test tubes, consisting of equal volumes of virus-containing material (usually 100 TCD50 of the virus in 1.0 ml) and diagnostic serum (1.0 ml). After thorough shaking, the prepared mixtures are kept for interaction for 3 hours at 37°C. Then, the neutralization mixtures are introduced into a sensitive cell culture, which is incubated at 37°C for 5-7 days, after which the results of the CPE and the color sample are taken into account (Table 1).

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, any one method is chosen for the diagnosis, depending on the stage of the viral infection. Thus, for example, all three approaches can be useful in the diagnosis of chickenpox, but the successful use of microscopy and cell culture methods depends on the ability to collect satisfactory samples at a relatively early stage of the disease.

To a large extent, the success of viral diagnostics also depends on the quality of the obtained samples. For this reason, the laboratory staff themselves should be directly involved in the collection of the necessary samples. The characteristics of the samples, as well as the methods of their delivery to the laboratory, are described by Lennett, Schmidt, Krist et al.

Most of the reagents and instruments used in laboratory diagnostics are available from various companies. In most cases, the same reagent is produced simultaneously by several companies. For this reason, we did not list individual firms, unless the reagent is supplied by only one firm. In all other cases, you should refer to the general list of suppliers indicated in Table. one.

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

1. Electron microscopy

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

Table 1. List of companies supplying reagents and equipment

Flow Laboratories: Gibco Europe: Tissue Culture Services: Wellcome Diagnostics: Northumbria Biologicals: Oxoid: Dynatech Laboratories Ltd.: Sterilin Ltd.: Abbott Laboratories Ltd.:

Woodcock Hill, Harefield Road, Rickmansworth, Hertfordshire WD3 1PQ, UK Unit 4, Cowley Mill Trading Estate, Longbridge Way, Uxbridge, Middlesex UB8 2YG, UK 10 Henry Road, Slough, Berkshire SL1 2QL, UK Temple Hill, DartfordT Kent DAI 5BR, UK South Nelson Industrial Estate, Cramlington, Northumberland NE23 9HL, UK Wade Road, Basingstoke, Hampshire RG24 OPW, UK Daux Road, Ballingshurst, Sussex RH14 9SJ, UK 43/45 Broad Street, Teddington, Middlesex TW11 8QZ, UK Brighton Hill Parade, Basingstoke, Hampshire RG22 4EH, UK

vesicles that characterize certain diseases, such as chickenpox. In the analysis of such material, viruses can be detected using negative staining, which leads to delineation of the components of the virion with electron-dense material. The method is effective at high concentrations of virus in test specimens, such as in faeces or vesicular fluid. In cases where the content of viral particles in the 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 the electron microscopic method for diagnosing rotavirus infection and the method of immunoelectron microscopy using the example of detection of specific antibodies to parvoviruses. The methods of electron microscopy are described in more detail by Field.

2.1 Direct electron microscopy of faeces

1. The end of the Pasteur pipette is immersed in the feces and enough material is collected to obtain a 1 cm smear.

2. Resuspend the fecal smear in negative electron microscope stain until a translucent suspension is obtained. The negative contrast stain is a 2% solution of phosphotungstic acid in distilled water.

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

4. The drug is left in the air for 30 seconds.

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 at an intensity of 440,000 μW-s/cm 2 . In this case, a short-wave ultraviolet lamp with a filter is used. The lamp should be at a distance of 15 cm from the grid; irradiation time of each side - 5 min.

8. Rotavirus virions can be characterized under a transmission electron microscope with a magnification of 30,000 to 50,000.

2.2 Immunoelectron microscopy

The method of immunoelectron microscopy described below is only one of many such immunological methods. For the study of virus-specific antibodies, in addition, a method is used that involves binding to a 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 indicated by us, as a rule, is used in routine work. To obtain optimal results of the interaction of antibodies with the virus, serum containing parvovirus is titrated in the same way.

1. 10 µl of human parvovirus antiserum diluted 100-fold 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 to two wells of a 96-well microtiter plate.

5. Allow the agarose to solidify at room temperature. The plate can be stored at 4°C for several weeks if sealed with adhesive tape.

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

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

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

9. With thin tweezers, the mesh is taken out and a drop of 2% phosphotungstic acid is applied 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 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 for a label, which 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 with a color change. In addition, a hemagglutination reaction is used to identify the virus. In everyday practice, the described methods are mainly used to detect hepatitis B virus antigens in the blood and to search for antigens of various viruses that cause various respiratory diseases.

Currently, many companies produce erythrocyte, radioactive and enzymatic diagnosticums, including those for the detection of the hepatitis B virus. We do not consider it appropriate to describe the methods of working with these diagnosticums: it is quite enough to follow the attached instructions. Below we will focus on the immunofluorescent method for the identification of 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 McQuilin. In laboratory conditions, this operation is performed in two stages. First, a smear is prepared from nasopharyngeal mucus on a glass slide. The obtained swabs can be stored in a fixed state at -20°C for many months. At the second stage, smears are stained to detect the antigen of the respiratory syncytial virus. For this purpose, the method of indirect immunofluorescence is used.

3.1.1 Preparation 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 discarded.

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

5. The resulting suspension is transferred into 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 drained, the precipitate is resuspended in such a volume of PBS that the resulting suspension is easily separated from the walls of the test tube.

9. The resulting suspension is applied to the 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 again dried in air.

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

3.1.2. Staining technique

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

2. Apply one drop of antiserum to the prepared preparation with a Pasteur pipette.

3. The drug is placed in a humid chamber.

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

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

6. Samples are washed in three shifts of PBS for 10 min each.