virology.
Virology is a section of microbiology that studies viruses, their morphology, physiology, genetics, as well as the evolution of viruses and environmental issues.
Let's be frank with our readers, as always.
Not even Trump, who has the geography and location of some countries of the world, constantly lied on every phrase, didn't look so stupid and ridiculous. The entire progressive world of erudite people is already laughing at this U.S. administration. They managed to turn the whole country into a laughing stock quite quickly.
Apart from squeamishness, contempt and disgust for the White House administration as a whole, from their exorbitant and brazen lies, these clowns 🤡 no longer evoke any other feelings.
"The US declared independence from The UK 245 years ago and will now become independent of the crown," Biden said, congratulating the country on a national holiday.
https://t.me/Tribulelouis/4761
The most patriotic he called on the vaccination points: relax early, COVID is not yet defeated.'
https://telegra.ph/DOCUMENTARY-EVIDENCE-OF-THE-TERRORIST-OPERATION-COVID-19-07-04
https://telegra.ph/On-time-and-time-zones-of-segregation-07-05
It's a disgrace.
In 1976, knew how to treat the virus, and now?
We went to the sources of the USSR and this is what we saw and read:
An article in the 1976 Arsenal newspaper from open sources
The point of the article is that coronavirus is one of the varieties of a common virus, it is treated by conventional means. Time will tell whether or not it will be.
Now almost anyone knows (well, if someone does not know - check) to develop a vaccine against "any" virus should spend 1-2 years on its manufacture.
Coronaviridae is a family of viruses that combine RNA-containing pleomorphic viruses of medium size, on the surface of which there are characteristic fringed lint. Initially, viruses belonging to this family were classified as mixoviruses, but subsequent studies gave rise (1968) to separate them into an independent taxonomic group. The ancestral unit of the Coronavirus family has not been determined.
An electronogram of the coronavirus derived from the cultural fluid of the organ culture of the human embryo's trachea: on the surface of the virions are seen fringe lint - indicated by arrows (K. McIntosh, 1974).
propagation
Coronaviruses are ubiquitous around the globe and are an etiological factor in acute human respiratory diseases, bird bronchitis, mice hepatitis, pneumonia in rats, gastroenteritis and encephalomyelitis in pigs, often fatal in animals.
structure
Coronaviruses contain RNA, which is characterized by a single-weight non-segmented structure, its molecular weight of 9 x 106 daltons. Coronavirus RNA is not infectious. Using electrophoresis in polyacrylamide gel, coronaviruses contain 6 or 8 polypeptides, the molecular weight of which is in a person from 13,000 to 210,000 color blinds, in other Ks - from 14,000 to 180,000 color tones. Some virio K proteins are glycoproteins. RNA-dependent RNA polymerase has been detected in human virions. A number of K strains contain Hemagglutinins.
Coronavirus virions are pleomorphic, with a diameter of 80 to 220 nm. K. is characterized by the presence of a shell with lint (Figure 1), more rare than that of the influenza virus. The lint is attached to the virion by a narrow "stem" and expands to the distal end, resembling a solar corona during an eclipse (hence the name of the family). The lint is 12 to 24 nm long. Viriona K. has a lipid shell. The nucleocapside, which has the appearance of a spiral, is swirled freely, its diam. 55 nm. Floating specific density of virions in sucrose p-rach is 1.18-1.19 g/ml, the ratio of c. human sedimentation is 374-416S.
properties
The characteristic characteristic of K. is their sensitivity to fat-disbursers. Exposure to both ether and chloroform significantly reduces the infection of these viruses. The stability of K. at a certain storage temperature depends on the number of virions in the units and on the presence of ions and colloids in the environment. However, it has been established that the inactivation of viruses at t q 56 degrees is completed in 10 to 15 minutes, at t q 37, their infection persists for several days, and at t q 4 - a few months. K. is resistant to freezing and thawing, and at t-60 and below for several years retain the info. properties without change, the most stable at pH 6.0-6.5.
Electrogram of cytoplasm of W1-38 cells infected with human coronavirus, strain 229 E: synthesized sickle-shaped structures (1) and kidney virions (2) coronaviruses in the area of large vacuoles (K. McIntosh, 1974) are visible.
Coronaviruses multiply in the cytoplasm of infected cells. At the same time, the children's virions appear in 4-6 hours. after infection, and the maximum manifestation of infection associated with cells is observed in 12-36 hours. In the electron microscopic examination of infected K. cells, a fairly characteristic morphology of K. is revealed: near the cytoplasmic vacuoles there is the appearance of sickle-shaped structures, which are synthesized virions, and in the vacuoli itself there are kidney virions (Figure 2). Budding through cytoplasmic vacuoli is a characteristic feature of K., distinguishing them from mixoviruses, budding through the plasma membrane.
The typical type of K. is considered to be the virus of infectious bronchitis of birds. The K. family also includes K. human - respiratory viruses, the hepatitis mouse virus, the vector-borne gastroenteritis virus of pigs, the virus of sialodacrioadenitis rats, the blue scallop virus of turkeys, the virus of neonatal diarrhoea calves, hemagglutinating the virus of pig encephalomyelitis. According to antigenic properties, these viruses are heterogenic. In the immunodiffusion test, they were able to detect one to four lines of pre-pregnancy. However, there are some antigenic relationships between certain human (serotype OS 43) and mouse strains.
All K. has the ability to fix the complement in the presence of hyperimmune serums or serums received from the diseased.
Only two types of Coronaviruses (human coronavirus, OS 38 and OS 43 and Hemagglutinating pig encephalomyelitis) cause hemagglutination; in this case, human agglutinate red blood cells of humans and monkeys only at t q 4, while red blood cells of chickens, rats and mice they agglutinate both at t q 4, and at room temperature and at t q 37. The ability to cause hemagglutination was impaired after processing by ether and trypsin. Moreover, it is shown that as a result of exposure to the named K. bromelain, destroying fringe lint, there was not only a violation of hemagglutination, but also a violation of complementary and inf. K.
Coronaviruses can only reproduce in a limited range of cell cultures, and the use of cells grown as monoslayers can produce a very low yield of the virus. K. of different origins breed, as a rule, only in the cells corresponding to their origin. Thus, primaryly allocated bird K. reproduce only in the cells of birds, mouse K. reproduce only in the tissues of mice; the same can be said for rat and pig strains. Only K. infectious bronchitis of birds reproduce in chicken embryos. Human C. reproduces, showing strainary differences, in human diploid cells, in L-132 cells (cell line derived from a human lung embryo), as well as in HeLa cells, cells of the primary kidney culture of the human embryo and other human cells. Reproduction of some viruses can be detected only in organ cultures (e.g., reproduction of the human virus in the explants of the epithelium of the human embryo). There are no citations in the affected cells. It should be emphasized that the cytopathic action of K does not manifest itself in all cases. Some viruses (e.g., human K. strain, B814) can only be detected by an electron microscope or by interference with other viruses (for the example of K. a person such an interference virus is ECHO virus type 11). When covering cells methylcellulose was able to observe plaque formation in cell cultures L-132 and Wl-38.
The experiment was able to achieve adaptation of Coronaviruses to cells in which they were not primaryly reproduced.
Okay, synthesis of the inf virus. bird bronchitis was observed in the primary culture of monkey kidney cells, and K. human was observed in the culture of monkey kidney cells and the body of sucker mice.
Human coronaviruses
Human coronaviruses are usually the agents of acute diseases of the upper respiratory tract, but cases of bronchitis and pneumonia of coronavirus etiology (in children) are known. The precise role of the virus in comparison with epidemiological data is extremely difficult because of the wide variability of strains and the time-consuming diagnostic method.
K. is usually excreted from the nasal secret, scrapes and washable waters of the nasopharynx, using appropriate cellular or organ cultures. The effectiveness of K. selection is relatively small. Diagnosis is based on virological (electron microscopy of infected cultures, as well as cytopathic action detected in the corresponding monolayer cell culture) and serological (the build-up in the serum of the patient's amount of antibodies to the complementary antigen K. of humans, strain 229 E, and antibodies to hemagglutinin strain OC 43) methods .
In most cases, Coronavirus causes human disease between January and March. The spread of the disease is predominantly intra-domestic.
Bibliography: L. J. And Shcheboldov A.V. Coronaviruses of man and animals, M., 1977, bibliogre.; Mc Intosh K. Coronaviruses, Curr. Top. Microbiol. Immunol., v. 63, p. 86, 1974; Tyrrell D. A. a. o. Coronaviruses, Nature (Lond.), v. 220, p. 650, 1968; Tyrrell D. A. a. o. Coronaviridae, Intervirology, y. 5, p. 76, 1975; Viral infections of humans, ed. by A. S. Evans, N. Y., 1976.
Volume 11
Source: Great Medical Encyclopedia (BME), edited by Petrovsky B.V., 3rd edition
🔬'S VIROLOGICAL RESEARCH
VIRUSAL RESEARCH - Studies conducted to diagnose viral infections, study the relevant pathogens, their spread in nature, as well as the production of viral drugs. In virological laboratories (see) honey. Profiles are studied as human viruses, and in some cases and animal viruses (e.g., conduct a diagnosis of rabies in dogs, examination of animals used for the production of viral drugs). The methods of research of both are similar.
One of the main stages of I. is the release of viruses. When secreting viruses from people use blood, various secrets and excretions, pieces of organs. The blood is most often examined in arbovirus diseases. Whole defibrinated or hemolyzed blood, individual elements or clots (in the later stages of the disease) are used. Viruses of rabies, epid, mumps, herpes simplex can be detected in saliva. Nosophiled flushes serve to secrete pathogens of influenza, measles, psittacosis, rhinoviruses, respiratory syncytial virus, adenoviruses. Adenoviruses are also found in the conjunctiva flushes. Rinse is taken by rinsing the nose and throat (separately) and washing the conjunctiva with isotonic solution sodium chloride. You can wipe the nasal passages and the back wall of the throat with tampons soaked in broth. Unsterile material is treated with antibiotics (1000 ED penicillin and streptomycin per 1 ml) for 30 minutes. Samples are bred with 1:10 phosphate buffer, centrifuge twice for 20 minutes at 8000 about I min. Antibiotics add, as indicated above. Less often for VA take the contents of pustules (in culcies, chickenpox, herpes) and organs points (with venereal lymphogranule). Sectional material should be taken as soon as possible after the death of the body. It is stored until the time of study at t-20 and below. For the conduct of B. and. fabric is crushed (rubd) and prepared 10-20% weight on isotonic solution sodium chloride or nutrient medium for cell cultures. It is centrifuged for 20 minutes at 1500 rpm; the liquid is used for further research.
In order to eliminate viruses infect laboratory animals, embryos of birds, cellular and tissue cultures. Animals are usable if the virus causes clear clinical symptoms or pathological changes (e.g., paralysis, pneumonia, etc.). The effectiveness of one way of introducing the material depends on the tropism of the virus. Widespread infestation under the skin, intra-abdominal and intravenous. Neurotropic viruses are detected when infecting animals in the hemisphere of the brain (arboviruses, rabies virus, etc.), visual bump (polio virus in experiments on monkeys), spinal cord. Smallpox and herpes viruses can be detected by applying the material to rabbits on the scarified cornea. Some viruses are easy to detect when inoculation in the front eye chamber (e.g., the hepatitis dog virus in the puppy experience). Intrenasal infection of animals (burying the material in the nose of an astrateed animal or injecting it in the form of aerosol in a special chamber) is usually used to study the pathogens of respiratory infections. The material is injected into the digestive tract with food or through the mouth with a blunt needle. In the study of some oncogenic viruses use the method of infection of golden hamsters in the mucous membrane of the sac wounds.
To many viruses newborn animals and suckers are more susceptible to mature individuals. Suckling mice are widely used to secrete arboviruses and Coxsackie viruses (after infection in the brain). Some adenoviruses are able to induce tumors in the subcutaneous infection of newborn golden hamsters. The study of a number of viruses birds spend on chickens the first days of life.
Figure 1. Ospina virus vaccine on the chorion-allantois membrane of a chicken embryo.
The use of chicken embryos has a number of advantages.
Their undifferentiated tissues have a wide range of sensitivity to many viruses. The presence of infection is judged by the death of embryos, the appearance of changes (ospin) on the chorion-allantois membrane (Figure 1), accumulation in embryonic fluids of hemagglutinins and complementary-binding viral antigen. The embryos are infected with chorionanlantos (aged 11 to 12 days with smallpox viruses), allantois and amniotic cavities (10-11-day mixoviruses), yolk sac (aged 5-6 days by the pathogens of psitozaornitosis, etc.). Inoculation of the material to embryos in the brain and intravenously (in the vessels of the shells) is rarely produced. In any way of infection, embryos can be injured, so they died in the first 24 to 48 hours. excluded from the accounting.
To study the action on chemical viruses. The substances are very convenient deembrionized eggs in which the embryo is removed, but preserved chorionallantois shell. Inside are placed the virus and the studied substance in 20 ml of isotonic solution sodium chloride. The hole in the shell is covered with a cap with a tube, through the k-ray you can take samples for analysis.
When assessing experiments on animals and embryos of birds should be kept in mind the possibility of provoking latent infections in them or secreting a latent virus.
The cultures of cells and tissues are used exceptionally widely for the release and accumulation of viruses (see). These methods can cultivate most known viruses (see Virus Cultivation). Some of them accumulate intensively already during the primary contamination of crops, to adapt others requires several passages. The reproduction of most viruses in cell cultures is accompanied by the development of cytopathic effect. By its nature to a certain extent it is possible to judge the belonging of viruses to a particular genus: picornaviruses cause rounding and shrivelling of cells, adenoviruses - the formation of rounded cluster cells in the form of grape clusters, mixoviruses and herpetic viruses - the formation of multi-core syncyts. A number of viruses cannot be cultivated outside the body.
Reproduction of some viruses (small group, mixo- and arboviruses) can be detected by the reaction of hemadsorbion, as the affected cells acquire the ability to absorb red blood cells. Appropriate red blood cells (humans, monkeys, guinea pigs, chickens) in a concentration of 0.4-0.5% are placed on a monolayer at t q 4 degrees or at room temperature for 20-30 minutes. Erytrocytes are adsorbed diffusively throughout the culture (e.g., paragrippous viruses) or form islets (influenza viruses).
The reproduction of the virus is sometimes judged by the study of cultural fluid in animals (tick-borne encephalitis) or in the RSC. The presence of a virus that does not have cytopathic activity is sometimes determined by its ability to interact with the cytopathogenic virus. Thus, in the cell cultures of the embryos of chickens infected with the leukemia viruses of birds, the reproduction of the Rausa sarcoma virus is suppressed. The END (exaltation of Newcastle disease virus) method, superinfection of crops by the Newcastle disease virus, has been proposed to detect non-citopatogenic strains of cattle diarrhoea and swine cholera. When the two viruses are jointly, cell destruction occurs.
When there are cytopatic changes or other signs of virus reproduction, the cultural fluid is used to identify the virus or passage. A number of viruses remain associated with cells even when culture degeneration (adenoviruses, smallpox viruses) are frozen and thawed before the liquid is collected. Some herpetic viruses, such as the Marek disease virus in chickens, need to be transplanted along with intact cells.
To study the respiratory corona-viruses of man and some others use the method of tissue cultures, i.e. infection of in vitro-cultivated tissue fragments. Most often use the fabric of the rabbit's trachea. The breeding virus affects the cells of the endothelial mucosa, which is determined by stopping the movement of cilia.
It is necessary to take into account the possibility of the presence of foreign viruses in the cultures of tissues and cells. They can be made with cells if the latter are taken from an infected organism, getting from trypsin or serum used to cultivate cells.
In addition to sowing biopsy or sectional material on already grown crops, direct cultivation of cells of the studied organ after its thripsinization is used, which is often more effective in the release of the virus (e.g., detection of adenoviruses in the tonsils). They also use the method of mixed cultures, when the cells of the studied organ are grown together with any cells sensitive to the virus (e.g., sowing brain cells of patients with subacute sclerosing panencephalitis together with apion cells or hela cells to secrete the measles virus). The mixed-culture method is often the only way to separate the virus from animal-induced tumors that do not produce an active virus but contain a viral gene.
Figure 2. The plaques of the polio virus on the culture of monkey kidney cells.
Single-layer cell cultures provide an opportunity to get colonies of the virus - plaques (Figure 2). As a rule, plaques form viruses that have cytopal activity. At the same time, this method allows the detection of some non-catotopogenic viruses (e.g., a number of strains of the cattle diarrhoea virus). To obtain plaques, the virus is deposited on the cell monolayer in cups or flat vials. The multiplicity of infection, i.e. the number of viral particles per cell, should be small, so that the plaques did not merge. After 30-60 minutes, adsorptions layer a nutrient medium with 1.35 to 1.5% agar and neutral red in final breeding 1 : 40,000. Cultures in petri dishes are incubated in the atmosphere with 5-10% carbon dioxide, and hermetically closed vials - in a normal thermostat. After a few days, unpainted tricks from degenerated cells begin to stand out among the stained cells.
You can place agar on the cells without neutral red, and after a few days apply a second layer of agar with dye; plaques become visible after a few hours. Agar sometimes contains polysaccharide sulfates, which are inhibitors of virus growth; to neutralize them on Wednesday add protamine-sulfate (60 mg per 100 ml). Methylcellulose and other substances can be used to obtain plaques of a number of viruses. Some viruses (smallpox, measles) form plaques and without agar coating. The plaque method allows for clonal analysis of viral strains. To secrete genetically homogeneous clones extract one plaque, to the next infection. Usually cloning is carried out over three passages.
The plaque method is also suitable for determining in an infected culture the number of cells producing the virus (i.e. the number of infectious centers). To do this, the cells are suspension, placed on a single-layered culture of virus-sensitive indicator cells and filled with agar. Plaques are formed around infected cells.
To diagnose viral infections and study the antigenic structure of viruses, a reaction of pregnancy in the gel is used. Most often for this purpose use agar. Antigens and specific antibodies, placed in the agar gel at a certain distance, diffuse and form at the meeting of the precipitate in the form of white stripes. 0.8-1% of agars in isotonic solution of sodium chloride or phosphate buffer are placed in capillaries or layered on glass. Antigens are preferable to have purified and concentrated. The reaction ingredients are made to the agar at opposite ends of the capillary or in the holes, made in a layer of agar on the glass at a distance of 5-6 mm. Incubation lasts 4-20 hours.
A significant number of VA perform with light and electron microscopy. The largest viruses (e.g. smallpox) after appropriate treatment (silver, coloration of Victoriabau, etc.) can be detected in conventional light microscopy. This method is used in smallpox diagnosis by examining the material from the pustules. Characteristic for some infections is the formation of calfs in cells - inclusions. Thus, in the nuclei there are inclusions in herpetic and adenovirus infection, in the cytoplasm - in pox (The body of Guarniri) and rabies (Babesh-Negri's body). The detection of inclusions is important for the diagnosis of rabies, smallpox, cytomegaly, subacute sclerosing panencephalitis, etc.
Microscopy in the dark field (see Dark-floor microscopy) and phase-contrast microscopy (see) are used to study the dynamics of changes in the cells affected by the virus. Fluorescent microscopy is more widely used (see).
The smears, prints and glass-grown single-layer cell cultures are examined. Drugs (native or fixed) are most often colored with acrydine orange. The method allows you to detect large viruses and clusters of viral components. Formations containing DNA glow bright green, and RNA-containing RNA glows in brick-red. Even more often in VA, infected cells are treated with fluorescent antibodies, which can detect clusters of viral antigen. In direct method use immune gamma-globulin, labeled fluorescent dye, for example, fluorescein-isotiocyanate. In an indirect method, the drug is treated with the usual immune serum of any animal, and then labeled antibodies against the gamma-globulin of this animal. Drugs are viewed in ultraviolet light, viral antigen is detected by a light green glow (see Immunofluorescence). The method of nasal smears allows early diagnosis of respiratory viral infections - influenza, parainfluenza, rhino and adenovirus, respiratory syncytial.
Electron microscopy (see) allows us to study the size and structure of viral particles, as well as the subtle changes they cause in cells. Research on viral weight or ultra-thin slices of infected cells. Some viruses (e.g., a number of human and animal oncorn viruses) can only be detected in tissues by this method.
B.I., whose goal is to determine the amount (titer) of the virus or viral antibodies, are very diverse.
Under an electron microscope, you can calculate the number of viral particles in the weight, if it is sufficiently cleaned. The virus is mixed with polystyrene latex, the number of particles to the horn is known. The mixture is sprayed on the grid, counting the number of particles in individual drops. By the ratio of the number of particles of latex and viruses, the number of virions in this volume is detected. This method does not provide an idea of the infectious activity of the virus, as the viral weight usually contains a significant number of noncommunicable particles.
The method of plaques allows to determine the most accurate number of infectious units in the material. Single-layer cell cultures infect a small dose of the virus. The titre expresses the number of plaque-forming units (BOE) in 1 ml. Similarly, it is possible to determine the titre of some oncogenic viruses by the centers of transformation, as well as those pathogens that form smallpox on the chorionallantoid shell of chicken embryos (e.g., smallpox virus).
Less accurate is the definition of the virus's credits by the end-intelligence method. Consistently increasing breeding (usually tenfold) is injected into sensitive animals, in chicken embryos or cell cultures. Taking into account the result of each injection as positive or negative, determine the lowest dose of the virus, which can cause a certain effect (disease or death of the animal, the appearance of cytopatic changes in culture, etc.). Practically this dose is difficult to determine, so calculate the dose, which gives 50% effect (ED50). It is determined depending on the properties of the virus and the method of titratation by the number of dead animals (LD50), the number of cases (ID50), the number of embryos that have developed viral hemagglutinins or complementary antigens, by the number of cell cultures where cytopatic changes or hemadsorating activity have been detected. The caption is expressed by the number of ED50 in a certain amount of material.
Of the existing ways to calculate ED50, the most common method is the Reed-Munch method, based on the principle of cumulation. It is based on the assumption that each test object (mouse, embryo) who responded positively to the introduction of this breeding would respond with a positive reaction to the introduction of a more concentrated virus. Conversely, if this breeding caused a negative reaction, then the introduction of more breeding will also be a negative reaction. Intervals between breeding in this method should be the same, each breeding infects at least 4 animals (cultures). Next, interpolation is made between doses, which caused the effect closest to 50%. The calculation is made according to the formula:
B - the lowest dose that caused the effect of more than 50%, b - the effect of dose B in percent, a - the effect of the highest dose that caused the effect less than 50%, in percent, d - the interval between the logarithms of two adjacent doses.
Viruses with marked cytopathic activity (eg poliomyelitis virus) can be titrated using the color test. It is based on the fact that in the process of cell growth, the pH of the medium decreases, and in infected cultures, where the cells degenerate, there is no change in the pH of the medium. To detect these changes, an indicator is added to the medium - phenol red, which has a red color at pH above 7, orange at pH 7 and yellow at pH below 7. In a nutrient medium having a pH of 7.3-7.4, add phenol red at a final concentration of 1: 40,000. Determine the minimum dose of cells that changes the color of the medium from red to yellow (usually about 25,000 in 0.25 ml). Then prepare 10-fold dilutions of the virus, each of which is poured into 4-6 test tubes, where the cell suspension is added. The tubes are closed with rubber stoppers or 0.6-0.8 ml of petroleum jelly is poured in. The results are taken into account after keeping for 5-7 days at t ° 37 °. The virus titer is taken as its dilution, which prevents the pH from shifting to the acidic side in 50% of the tubes.
The ability of immune sera to neutralize the infectious activity of viruses is determined using a neutralization reaction. Methods for titration of sera are predetermined by titration methods for the respective viruses. They are all based on the preparation of mixtures of virus with serum, which are then tested for the presence of non-neutralized virus. The reaction is put in two versions. According to one of them, the immune and normal serum in one dilution (for example, 1: 10) is mixed with increasing 10-fold dilutions of the virus in an equal volume and the titer of the virus is determined in the presence of both serum. The quotient of dividing the ED50 of a virus in the presence of normal serum by its ED50 in the presence of immune serum will be an index of neutralization of a given dilution of immune serum. This result cannot be interpolated to other serum dilutions (eg undiluted serum activity will be higher than calculated). In another variant of the reaction, a constant dose of virus (usually about 100 ED50) is combined with a series of 2-fold dilutions of the test serum. The serum titer will be dilution, with which the virus caused a 50% effect.
Depending on the virus titration method, the neutralization reaction is carried out on laboratory animals, chicken embryos (according to their death or accumulation of hemagglutinins), on cell cultures (according to the appearance of cytopathic changes, color test, etc.). If the virus forms pockmarks on the chorionallantoic membrane of chick embryos, the highest serum dilution is determined, which reduces the number of pockmarks by 50 percent or more. In a similar way, sera are titrated to reduce the number of viral plaques in single-layer cell cultures.
Titration of viruses by RHA and antibodies by RTGA is based on the ability of a number of viruses (mixoviruses, some representatives of the genus of poxviruses, adenoviruses, picornaviruses and togaviruses) to glue erythrocytes. The virus is titrated in two-fold dilutions with an equal volume of 0.5-1% erythrocyte suspension. For the titer (1 AE - agglutinating unit), take its greatest dilution, which caused hemagglutination (see). The hemagglutinating titer of a virus is not an indicator of its infectious activity, since hemagglutination) can be caused by non-infectious "incomplete" particles, an inactivated virus and a hemagglutinating antigen separated from some viruses. Serum for RTGA titrated in two-fold dilutions with 2-4 AE of the virus; The titer is considered the highest dilution, which completely inhibits hemagglutination).
Some viruses are adsorbed on erythrocytes, but do not cause them to agglutinate. To detect them, you can use the reaction of indirect hemagglutination. It is based on the agglutination of virus-loaded erythrocytes with serum specific to this virus. This method is mainly used for titration of sera.
The complement binding reaction (see) is suitable for titration of both viruses (more precisely, complement-binding viral antigens) and antibodies. In principle, CSCs with viruses do not differ from those with bacterial and other antigens. Differences are only in the method of preparation of antigens. Blood, nasopharyngeal washings, urine, feces of patients, suspensions of infected organs, individual parts of infected chicken embryos and a virus grown in cell cultures can serve as viral antigens. Cultural antigens are most suitable. Suspensions of infected organs are repeatedly frozen and thawed before use, combined with an equal volume of ether or chloroform, shaken for 1-2 hours, and then kept for 18-20 hours. at t ° 4 °, again freeze, thaw and clarify by centrifugation. To obtain sera, animals should not be immunized with a virus cultured in the same substrate as the antigen for the reaction. Depending on the purpose of titration, two-fold dilutions of the antigen with a certain dose of serum are combined, or, conversely, two-fold dilutions of serum with one dose of antigen. Contact of antigen, serum and complement is carried out within 1 hour at t ° 37 ° or 18 hours. at t ° 4-6 °. After adding the hematosystem, the material is incubated for 30 minutes. at t ° 37 °. The results are assessed according to the general rules.
There are methods of titration of viruses and antibodies based on the indication of the virus in infected cells of the culture with fluorescent antibodies, as well as a number of others that are rarely used.
The toxic effect inherent in certain viruses is revealed by introducing large doses to animals in an unusual way, when the virus does not go through a full reproduction cycle. For example, the influenza virus is injected into the brain or intravenously in mice. Its toxic effect is judged by the death of animals during the first day.
The antigenic activity of the virus (or vaccine) is established by immunizing humans or animals with subsequent determination of the antibody titer in their serum. The immunogenic activity of a vaccine, that is, the ability to induce resistance to infection, is determined by immunizing animals susceptible to this virus. Then the immunized animals and the control non-immunized are infected with a number of dilutions of the virus, determining its ED50 for both groups. The difference in the values obtained characterizes the immunogenicity of the test drug. If the virus is not pathogenic for animals, the immunogenicity of the corresponding vaccine can only be determined in epidemiol. experience.
To study a number of properties of viruses (size, structure, chemical composition, etc.), it is necessary to have a purified material with a high titer. The most commonly used virus is grown in cell cultures or in the form of allantoic fluid of infected chick embryos. Purification usually begins with differential centrifugation: first, at 15–20 thousand g (g is the acceleration of gravity), the material is freed from cellular debris, and at 50–100 thousand g, the virus itself is precipitated. Filtration through asbestos gaskets (Seitz type), glass and cellulose membrane filters (millipore type) is also used to remove large particles. Fragments that are smaller than viruses can be removed by filtering the material through Sephadex gels, which trap particles of a certain size, depending on the pore size. To isolate and concentrate the virus from large volumes, salting out with ammonium and sodium sulfates, precipitation with alcohol, aluminum hydroxide or polyethylene glycol is used. To get rid of ballast proteins, they are also used for their digestion with proteolytic enzymes. Myxoviruses can be purified and concentrated by adsorption on erythrocytes at low temperatures with elution at higher temperatures.
Further purification and concentration of the virus is carried out by one or another fractionation method. These methods are also used to separate virus mutants and individual viral components. When a virus is mixed with a phase system formed by aqueous solutions of two polymers, it passes into one of the phases, depending on its size, ionic composition of the medium, pH, etc. To purify large viruses, the dextran sulfate - methylcellulose system is usually used, small ones - dextran sulfate with polyethylene glycol. The polymers remaining after phase separation are often removed during further virus purification. You can also remove dextran sulfate by adding potassium chloride, methyl cellulose using ammonium sulfate, polyethylene glycol chloroform and other methods.
Purification of viruses using column chromatography is based on their ability to adsorb on a number of substances, and elute from them when the pH or salt concentration changes (see Chromatography). For adsorption, calcium phosphate gels, cellulose-based ion exchangers (most often anion exchangers, for example, diethylaminoethyl cellulose), and ion exchange resins are used. Elution of the virus from calcium phosphate is carried out with phosphate buffer solutions of increasing concentration, and from cellulose-based ion exchangers - with sodium chloride solutions.
Highly purified suspensions are obtained by centrifugation in a liquid column with a density distributed in a continuous gradient. Viral particles are collected at the level where the density of the medium is equal to their buoyant density. Zonal sucrose gradient centrifugation separates particles of different weights. The material is applied to a gradient created by layering the appropriate sucrose solutions. At the end of centrifugation, the fractions are collected by piercing the bottom of the tube. In equilibrium or isopycnic centrifugation, viral particles are suspended in a solution of cesium chloride, cesium sulfate or rubidium chloride. After prolonged centrifugation, a stable continuous density gradient of the solution is created. At the site of localization of viral particles, you can determine their density. It should be noted that the obtained values of the density and mass of viral particles depend to a certain extent on the medium used for centrifugation.
At V. and. viruses labeled with various radioactive isotopes are widely used. The most commonly used carbon 14C, tritium 3H, phosphorus 32P, sulfur 35S, iodine 131I. The easiest way to label a virus is when it is cultured in cell cultures. Radioactivity is determined using liquid scintillation counters or by autoradiography (see).
Chem. the composition of viruses is investigated by the generally accepted chem. methods. Nucleic acid is usually obtained by phenol extraction, less often anionic detergents are used - sodium dodecyl or lauryl sulfate.
To identify viruses (see), first of all, you should establish their generic affiliation. To do this, it is necessary to determine the size and structure of viral particles, the type of nucleic acid included in their composition, the presence of a lipoid envelope. The type of nucleic acid is most often determined by indirect methods, for example, using the ability of bromodeoxyuridine to suppress the reproduction of DNA-containing viruses. The presence of a lipoid envelope in a virus is established by its sensitivity to the action of ether and chloroform (viruses with envelopes are inactivated). Further identification is carried out with a set of immune sera to known viruses, using various reactions - neutralization, RSK, RTGA, etc. Less often, animals are immunized with a known virus with their further infection with an unknown one, or vice versa.
Bibliography:
Laboratory diagnostics of viral and rickettsial diseases, ed. E. Lennett and N. Schmidt, trans. from English, M., 1974, bibliogr .;
Luria G. E. and Darnell J. E. General virology, trans. from English, M., 1970, bibliogr .; Methods of Virology and Molecular Biology, trans. from English., M., 1972; Pshenichnov V.A., Semenov B.F. and Zezerov EG Standardization of methods of virological research, M., 1974, bibliogr .; Guidelines for laboratory diagnosis of viral and rickettsial diseases, ed. P.F.Zdrodovsky and M.I.Sokolov, M., 1965; Sokolov MI, Sinitskiy AA and Remezov PI Virological and serological tests for viral infections, L., 1972; Virologische Praxis, hrsg, v. G. Starke, Jena, 1968, Bibliogr.
Category: Volume 4
Source: Great Medical Encyclopedia (BME), edited by Petrovsky B.V., 3rd edition
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