VIROLOGIC RESEARCHES — the researches conducted for the purpose of diagnosis of viral infections, studying of the corresponding activators, their distribution in the nature and also by production of virus drugs. In virologic laboratories (see) a medical profile study as viruses of the person, so in some cases and viruses of animals (e.g., carry out diagnosis of rage at dogs, inspection of the animals used for production of virus drugs). Methods of a research of that and others are similar.
One of the main stages B. and. allocation of viruses is. At allocation of viruses from people use blood, various secrets and excretes, pieces of bodies. Most often blood is investigated at arboviral diseases. Blood, its separate elements or clots is used integral defibrinated or Gemolizirovannaya (at late stages of a disease). Rhabdoviruss, epid, parotitis, a herpes simplex can be found in saliva. Nasopharyngeal washouts serve for allocation of causative agents of flu, measles, a psittacosis, rhinoviruses, a respiratory and syncytial virus, adenoviruses. In washouts from a conjunctiva adenoviruses are also found. Washout is taken by rinsing of a nose and a throat (separately) also by washings of a conjunctiva isotonic solution of sodium chloride. It is possible to wipe the nasal courses and a back wall of a throat with the tampons moistened with broth. Unsterile material is processed antibiotics (on 1000 PIECES of penicillin and streptomycin on 1 ml) within 30 min. From excrements allocate various enteroviruses, adeno-and reoviruses. Tests part with 1:10 phosphatic buffer, centrifuge twice 20 min. at 8000 about the I min. Antibiotics add as it is stated above. More rare for V. and. take contents of pustules (at smallpox, chicken pox, herpes) and punctates of bodies (at a venereal lymphogranuloma). Section material should be taken as soon as possible after death of an organism. It is stored until a research at t ° — 20 ° below. For V.'s carrying out and. fabric is crushed (pound) and prepare 10 — 20% a suspension on isotonic solution of sodium chloride or nutrient medium for cellular cultures. It is centrifuged by 20 min. at 1500 rpm; nadosadochny liquid is used for a further research.
For the purpose of allocation of viruses infect laboratory animals, embryos of birds, cellular and fabric cultures. Animals are suitable if the virus causes in them accurate clinical symptoms of a disease or pathoanatomical changes (e.g., paralyzes, pneumonia, etc.). Efficiency of this or that way of administration of material depends on tropism of a virus. Widely apply infection under skin, intraperitoneally and intravenously. Neurotropic viruses reveal at infection of animals in cerebral hemispheres (an arbovirus, a rhabdovirus, etc.), a visual hillock (a virus of poliomyelitis in experiences on monkeys), a spinal cord. Rabbits can find viruses of smallpox and herpes by putting material on the scarified cornea. It is easy to reveal some viruses at inoculation in an anterior chamber of an eye (e.g., a virus of hepatitis of dogs in experience on puppies). Usually apply intranasal infection of animals to studying of causative agents of respiratory infections (an instillation of material in a nose a narkotizirovanny animal or his introduction in the form of an aerosol in the special camera). Into a digestive tract material is entered with food or through a mouth a stupid needle. During the studying of some oncogenous viruses apply a method of infection of golden hamsters in a mucous membrane of zashchechny bags.
Newborn animals and suckers are more susceptible than puberal individuals to many viruses. Mice suckers are widely used for allocation of an arbovirus and Koksaki's viruses (after infection in a brain). Some adenoviruses are capable to induce tumors at hypodermic infection of newborn golden hamsters. Studying of a number of viruses of birds is carried out on chickens of the first days of life.
Use of chicken embryos has a number of advantages. Their indifferent tissues possess a wide range of sensitivity concerning many viruses. About existence of an infection judge by death of embryos, emergence of changes (pockmarks) on chorion-allantoisnoy to a cover (fig. 1), accumulation in embryonal liquids of hemagglutinins and a complement-linked viral antigen. Infect embryos on a horionallantoisny cover (at the age of 11 — 12 days with viruses of group of smallpox), in allantoisny and amniotic cavities (10 — 11-day myxoviruses), a vitellicle (at the age of 5 — 6 days activators of a psittakozaornitoz, etc.). Inoculation of material to embryos in a brain and intravenously (in vessels of covers) is made seldom. At any way of infection embryos can be injured therefore the dead in the first 24 — 48 hours from the account are excluded.
Deembrionirovanny eggs in which the embryo is removed are very convenient for studying of action on viruses of chemical substances, but the horionallantoisny cover is kept. Inside place a virus and the studied substance in 20 ml of isotonic solution of sodium chloride. The opening in a shell is closed a cap with a tubule, through to-ruyu it is possible to take samples for the analysis.
At assessment of animal experiments and embryos of birds it must be kept in mind a possibility of provocation at them latent infections or allocation of the virus which is in an abeyance.
Exclusively widely to allocation and accumulation of viruses apply cultures of cells and fabrics (see). By these methods it is possible to cultivate the majority of the known viruses (see Cultivation of viruses). Some of them intensively collect already at primary infection of cultures, adaptation of others requires several passages. Reproduction of the majority of viruses in cellular cultures is followed by development of cytopathic effect. On its character to a certain extent it is possible to judge belonging of viruses to this or that sort: picornaviruses cause rounding and wrinkling of cells, adenoviruses — education by the rounded cells of accumulations in the form of grape clusters, myxoviruses and herpetic viruses — formation of multinuclear sincytia. A number of viruses does not manage to be cultivated out of an organism.
Reproduction of some viruses (smallpox group, mikso-and an arbovirus) can be found by means of a hemadsorption virus test as the struck cells gain ability to adsorb erythrocytes. The corresponding erythrocytes (the person, a monkey, a Guinea pig, chicken) place in concentration of 0,4 — 0,5% for a monolayer at t ° 4 ° or at the room temperature for 20 — 30 min. Erythrocytes are adsorbed diffuzno on all culture (e.g., parainfluenza viruses) or create islands (influenza viruses, parotitis).
Sometimes judge reproduction of a virus by a research of cultural liquid on animals (vernal encephalitis) or in RSK. Existence of the virus which does not have cytopathic activity is determined sometimes by its ability to interfere with a cytopathic virus. So, in cultures of cells of embryos of the hens infected with viruses of leukoses of birds reproduction of a virus of sarcoma of Raus is suppressed. For detection of not cytopathic strains of viruses of diarrhea of cattle and cholera of pigs the END method (exaltation of Newcastle disease virus) — superinfection of cultures with a virus of a disease of Newcastle is offered. At combined action of both viruses there occurs destruction of cells.
At emergence of cytopathic changes or other signs of reproduction of a virus cultural liquid is used for identification of a virus or a passage. A number of viruses remains connected with cells even at a degeneration of culture (adenoviruses, viruses of group of smallpox) owing to what make freezing and thawing of cultures before liquid collection. Some herpetic viruses, napr, a virus of a disease of Marek at hens, it is necessary to oversow together with the unimpaired cells.
For studying of respiratory coronaviruses of the person and some other use a method of fabric cultures, i.e. infection of the cultivated in vitro of fabric fragments. Most often use tissue of a trachea of a rabbit. The breeding virus affects cells of an endothelium of a mucous membrane that determine by the termination of the movement of cilia.
It is necessary to consider a possibility of presence at cultures of fabrics and cells of foreign viruses. They can be brought with cells if the last are taken from the infected organism, to get from trypsin or the serum used for cultivation of cells.
In addition to crops of biopsy or section material on already grown up cultures, apply direct cultivation of cells of the studied body after its tripsinization that is quite often more effective concerning allocation of a virus (e.g., detection of adenoviruses in almonds). Use also a technique of the mixed cultures when cells of the studied body grow up together with any cells, sensitive to this virus (e.g., crops of cells of a brain of patients with a subacute sclerosing panencephalitis together with cells of kidneys of monkeys or Hela-cells for allocation of a virus of measles). The method of the mixed cultures is often the only way of allocation of a virus from induced by it at animal tumors which do not produce an active virus, however contain a virus genome.
Single-layer cellular cultures give the chance to receive colonies of a virus — a plaque (fig. 2). As a rule, plaques create the viruses having cytopathic activity. At the same time this method allows to find some not cytopathic viruses (e.g., a number of virus strains of diarrhea of cattle). For receiving plaques the virus is brought on a cellular monolayer in cups or flat bottles. Plurality of infection, i.e. number of virus particles on one cell, shall be small that the formed plaques did not merge. After 30 — 60 min. adsorption layer a medium from 1,35 — 1,5% of an agar and neutral red in end-point dilution 1: 40 000. Cultures in Petri dishes incubate in the atmosphere from 5 — 10% of carbonic acid, and bottletight bottles — in the ordinary thermostat. In several days among it is intravital the painted cells uncolored focuses from the degenerated cells begin to be allocated.
It is possible to place on cells an agar without neutral red, and in several days to put the second layer of an agar with dye; plaques become visible in several hours. The agar sometimes contains sulfates of polysaccharides which are inhibitors of growth of viruses; for their neutralization on Wednesday add protamine sulfate (60 mg on 100 ml). For receiving plaques of a number of viruses it is possible to use methyl cellulose and other substances as a covering. Some viruses (smallpox, measles) create plaques and without agar covering. The method of plaque-forming cells allows to carry out the clonal analysis of virus strains. For allocation of genetically homogeneous clones take one plaque, to-ruyu use for the following infection. Usually the cloning is carried out throughout three passages.
The method of plaque-forming cells is suitable also for definition in the infected culture of quantity of the cells producing a virus (i.e. number of the infectious centers). For this purpose cells suspend, place on single-layer culture of indicator cells, sensitive to a virus, and fill in with an agar. Around the infected cells plaques form.
The precipitation test in gel is applied to diagnosis of viral infections and studying of an antigenic structure of viruses. Most often for this purpose use an agar. The antigens and specific antibodies placed in agar gel at a certain distance diffuse and form at a meeting precipitated calcium superphosphate in the form of white strips. 0,8 — 1% place an agar in isotonic solution of sodium chloride or the phosphatic buffer in capillaries or apply with a layer on slide plates. It is preferable to have antigens cleared and concentrated. Ingredients of reaction bring on an agar in the opposite ends of a capillary or in the holes made in a layer of an agar on glasses at distance of 5 — 6 mm. The incubation proceeds 4 — 20 hour.
Considerable number B. and. carry out by means of light and a submicroscopy. The largest viruses (e.g., smallpox) after the corresponding processing (silvering, coloring to a viktoriyabla, etc.) can be revealed at usual light microscopy. This method is applied at diagnosis of smallpox by inspection of material from pustules. Formation in cells of little bodies — inclusions is characteristic of some infections. So, in kernels inclusions at a herpes and adenoviral infection appear, in cytoplasm — at smallpox (Guarniyeri's little body) and rage (Babesh's little bodies — Negri). Detection of inclusions matters for diagnosis of rage, smallpox, a cytomegaly, subacute sclerosing a panencephalitis, etc.
Microscopy in a dark field (see. Dark field method ) and phase-contrast microscopy (see) use hl. obr. for studying of dynamics of changes in the cells affected with a virus. Apply more widely luminescent microscopy (see).
Investigate smears, prints and single-layer cellular cultures which are grown up on glasses. Drugs (native or fixed) most often paint acridine orange. The method allows to reveal large viruses and accumulations of virus components. The educations containing DNA shine bright green light, and the containing RNA — brick-red. Even more often at V. and. make processing of the infected cells fluorescent antibodies that allows to reveal accumulations of a viral antigen. At a direct method use immune gamma-globulin, marked fluorescent dye, napr, fluorescein thiocyanate. At an indirect method drug is processed usual immune serum of any animal, and then marked antibodies against gamma-globulin of this animal. Drugs are looked through in an ultraviolet light, the viral antigen is found on a light green luminescence (see. Immunofluorescence ). The method of smears from a nasopharynx allows to carry out early diagnosis of respiratory viral infections — influenzal, parainfluenza, rino-and adenoviral, respiratory and syncytial.
Submicroscopy (see) allows to study the sizes and structure of virus particles, and also the thin changes caused by them in cells. Investigate a virus suspension or ultrathin sections of the infected cells. Some viruses (e.g., a number of oncornaviruses of the person and animals) manage to be found in fabrics only by this method.
Century and., which purpose — to define quantity (caption) of a virus or virus antibodies, are very different.
Under a supermicroscope it is possible to count number of virus particles in a suspension if it is rather cleared. The virus is mixed with polystyrene latex, the number of particles to-rogo is known. Mix is sprayed on a grid, count number of particles in separate drops. Based on the ratio of number of particles of latex and viruses reveal number of virions in this volume. This method does not give an idea of infectious activity of a virus as the virus suspension usually contains considerable number of noninfectious particles.
Most precisely the method of plaque-forming cells allows to define number of infectious units in material. Single-layer cellular cultures infect with a small dose of a virus. The caption is expressed number of plaque-forming units (FIGHT) in 1 ml. Similarly it is possible to determine a titre of some oncogenous viruses by the centers of transformation, and also those activators which form pockmarks on a horionallantoisny cover of chicken embryos (e.g., a virus of smallpox).
Less exact is definition of a titre of a virus by method of final cultivations. Consistently increasing cultivations (usually tenfold) enter a sensitive animal, into chicken embryos or cellular cultures. Considering result of each introduction as positive or negative, define the smallest dose of a virus capable to cause a certain effect (a disease or death of an animal, emergence of cytopathic changes in culture, etc.). Practically it is difficult to define this dose therefore calculate the dose giving 50% effect (ED 50 ). It is determined depending on properties of a virus and a method of titration by a death toll of animals (LD 50 ), to number of the diseased (ID 50 ), to quantity of embryos which had virus Hemagglutinins or complement-linked antigens, on number of cellular cultures where cytopathic changes or gemadsorbiruyushchy activity were revealed. The caption is expressed number ED50 in a certain volume of material.
From the existing ways of calculation ED50 the most common is Read's method — Myuncha based on the principle of cumulation. The assumption is its cornerstone that each examinee the object (a mouse, an embryo) which answered positively introduction of this cultivation would answer with positive reaction to introduction of more concentrated virus. And, on the contrary, if this cultivation caused negative reaction, then at introduction of bigger cultivation there will be a negative reaction too. Intervals between cultivations at this method shall be identical, infect with each cultivation not less than 4 animal (cultures). Further make interpolating between doses which caused effect, the closest to 50%. Calculation is made on a formula:
where In — the smallest dose which caused effect is more than 50%, b — a dosage effect In as a percentage, and — the effect of the greatest dose which caused effect is less than 50%, as a percentage, of d — an interval between logarithms of two next doses.
Viruses with the expressed cytopathic activity (e.g., a virus of poliomyelitis) can be titrated by method of color test. It is based that in the course of growth of cells pH of the environment goes down, and in the infected cultures where cells degenerate, change of pH of the environment does not happen. For identification of these changes on Wednesday add the indicator — the phenol red having at pH is higher the 7th red color, at pH 7 — orange and at pH lower than 7 — yellow. Bring phenol red in final concentration in the medium having pH 7,3 — 7,4 1: 40 000. Define the minimum dose of cells changing color of the environment with red on yellow (usually it is about 25 000 in 0,25 ml). Further prepare 10-fold cultivations of a virus, pour each of which in 4 — 6 test tubes where add a cellular suspension. Test tubes close rubber bungs or fill in 0,6 — 0,8 ml of a liquid paraffin. Results consider after keeping within 5 — 7 days at t ° 37 °. Take its cultivation interfering shift of pH in the acid party in 50% of test tubes for a titre of a virus.
Ability of immune serums to neutralize infectious activity of viruses is defined by a neutralization test. Methods of titration of serums are predetermined by ways of titration of the corresponding viruses. All of them are based on preparation of mixes of a virus with serum which then test for existence of not neutralized virus. Reaction is put in two options. On one of them immune and normal serum in one cultivation (e.g., 1: 10) mix with the increasing 10-fold cultivations of a virus in equal volume and define a titre of a virus in the presence of that and other serum. Private from division of ED 50 virus in the presence of normal serum on its ED50 in the presence of immune will be an index of neutralization of this cultivation of immune serum. It is impossible to interpolate this result on other cultivations of serum (e.g., activity of not divorced serum will be higher than calculated). By other option of reaction a constant dose of a virus (usually about 100 ED 50 ) connect to a number of 2-fold cultivations of ispytuyemy serum. Cultivation will be a caption of serum, about the Crimea the virus caused 50% effect.
Depending on a method of titration of a virus the neutralization test is carried out on laboratory animals, chicken embryos (on their death or accumulation of hemagglutinins), on cellular cultures (on emergence of cytopathic changes, color test, etc.). If the virus forms pockmarks on a horionallantoisny cover of chicken embryos, define the greatest cultivation of serum reducing number of pockmarks by 50 and more percent. Similarly titrate serums on a reduction of number of virus plaques on single-layer cellular cultures.
Titration of viruses on RGA and antibodies on RTGA is based on ability of a number of viruses (myxoviruses, some representatives of a sort of poksvirus, adenoviruses, picornaviruses and togavirus) to stick together erythrocytes. The virus is titrated in double cultivations with an equal capacity of 0,5 — 1% of a suspension of erythrocytes. (1 AE — the agglutinating unit) take its greatest cultivation which caused for a caption hemagglutination (see). The hemagglutinating titre of a virus is not an indicator of its infectious activity as hemagglutinations) can cause noninfectious «incomplete» particles, the inactivated virus and separable from some Hemagglutinating Antigen viruses. Serums on RTGA titrate in double cultivations from 2 — 4 AE of a virus; a caption consider the greatest cultivation which is completely braking hemagglutinations).
Some viruses are adsorbed on erythrocytes, but do not cause their agglutination. For their identification it is possible to use reaction of indirect hemagglutination. It is based on agglutination of the erythrocytes «loaded» with a virus by serum, specific in relation to this virus. This method is used generally for titration of serums.
Reaction of binding complement (see) it is suitable for titration as viruses (more precisely than complement-linked viral antigens), and antibodies. Essentially RSK with viruses does not differ from that with bacterial and other antigens. Distinctions are available only in a way of preparation of antigens. Blood, nasopharyngeal washouts, urine, excrements of patients, suspensions of the infected bodies, separate parts of the infected chicken embryos and the virus which is grown up in cellular cultures can serve as viral antigens. Cultural antigens are most suitable. Suspensions of the infected bodies before use repeatedly freeze and thaw, connect to the equal volume of ether or chloroform, stir up 1 — 2 hour then maintain 18 — 20 hours at t ° 4 °, again freeze, thaw and clarify centrifuging. For receiving serums it is not necessary to immunize animals the virus cultivated in the same substrate as antigen for reaction. Depending on the purposes of titration connect double cultivations of antigen to a certain dose of serum or, on the contrary, double cultivations of serum with one dose of antigen. The contact of antigen, serum and complement is made within 1 hour at t ° 37 ° or 18 hours at t ° 4 — 6 °. After addition of haemo-system material 30 min. at t incubate ° 37 °. Assessment of results is made by the general rules.
There are methods of titration of viruses and antibodies based on indication of a virus in the infected cells of culture by fluorescent antibodies and also some other which apply quite seldom.
The toxic action inherent a nek-eye in viruses, is revealed by introduction by an animal of high doses in the unusual way, at Krom the virus does not pass a complete cycle of a reproduction. So, e.g., the influenza virus is entered to mice into a brain or intravenously. About its toxic action judge by death of animals within the first days.
Antigenic activity of a virus (or vaccines) is established by immunization of people or animals with the subsequent definition of an antiserum capacity in their serum. Immunogene activity of a vaccine, i.e. ability to cause resistance to an infection, define by immunization of animals, sensitive to this virus. Then immunizirovanny animal and control neimmunizirovanny are infected with a number of cultivations of a virus, determining it by ED50 for both groups. The difference in the received indicators characterizes an immunogenicity of the examinee of drug. If the virus has no pathogenicity for animals, the immunogenicity of the corresponding vaccine can be defined only in epidemiol. experience.
For studying of a number of properties of viruses (the sizes, structure, chemical structure, etc.) it is necessary to have the purified material with a high caption. Most often use the virus which is grown up in cellular cultures or in the form of allantoisny liquid of the infected chicken embryos. Cleaning is usually begun with differential centrifuging: at first at 15 — 20 thousand g (g — acceleration of gravity) exempt material from a cellular detritis, and at 50 — 100 thousand g — besiege a virus. Apply to release from coarse particles also filtering through asbestos gaskets (like Zeytts), glass and cellulose membrane filters (like milliporovy). Fragments which there are less viruses can be removed with filtering of material through sephadex-gels, the particles of a certain size detaining depending on the size of a time. To allocation and concentration of a virus from large volumes apply salting-out by means of ammonium sulfates and sodium, sedimentation by alcohol, aluminum hydroxide or polyethyleneglycol. For release from ballast proteins use also their digestion by proteolytic enzymes. Myxoviruses can be cleared and concentrated by adsorption on erythrocytes at a low temperature with elution at higher.
Further cleaning and concentration of a virus are made by this or that method of fractionation. These methods use also for division of mutants of a virus and separate virus components. During the mixing of a virus with the phase system formed by aqueous solutions of two polymers it passes into one of phases depending on the size, ionic structure of the environment, pH, etc. For cleaning of large viruses usually use system dextransulphate — methyl cellulose, small — dextransulphate with polyethyleneglycol. The polymers which remained after phase separation often are removed in the course of further cleaning of a virus. It is also possible to remove dextransulphate with addition of potassium chloride, methyl cellulose by means of ammonium sulfate, polyethyleneglycol chloroform and by other methods.
Cleaning of viruses by means of a chromatography on a column is based on their ability to be adsorbed on a number of substances, and at change of pH or salt content to eluate from them (see the Chromatography). Apply gels of calcium phosphate, ion exchangers on a cellulose basis to adsorption (most often anion exchangers, napr, diethylaminoethyl cellulose), ion-exchange resins. Elution of a virus from calcium phosphate is carried out phosphatic buffered solutions of the increasing concentration, and from ion exchangers on a cellulose basis — solutions of sodium chloride.
High cleaning suspensions receive centrifuging in a fluid column with a density distributed in a continuous gradient. Virus particles gather on that level where density of the environment is equal to their floating density. Zone centrifuging in a gradient of sucrose allows to divide particles with various weight. Material is applied on the gradient created by a lamination of the corresponding solutions of sucrose. Upon termination of centrifuging bring together fractions, puncturing a bottom of a test tube. At equilibrium or isopycnic centrifuging virus particles suspend in solution of chloride of caesium, sulfate of caesium or rubidium chloride. After long centrifuging the steady continuous gradient of density of solution is created. It is possible to determine their density by the place of localization of virus particles. It should be noted that the received values of density and mass of virus particles to a certain extent depend on the environment used during the centrifuging.
At V. and. widely use viruses, marked various radioisotopes. Most often apply carbon 14 C, hyzone 3 H, phosphorus 32 P, sulfur 35 S, iodine 131 I. The easiest to mark a virus at its cultivation in cellular cultures. Radioactivity is determined by liquid scintillation counters or method autoradiography (see).
The chemical structure of viruses is investigated by the standard chemical methods. Nucleinic to - that is usually received phenolic extraction, apply anionic detergents less often — dodecyl - or sodium lauryl sulfate.
For identifications of viruses (see) first of all it is necessary to establish their patrimonial accessory. For this purpose it is necessary to determine the sizes and structure of virus particles, a look being their part nucleinic to - you, existence of a lipoid cover. The look nucleinic to - you most often is determined by indirect methods, napr, using ability of a bromdezoksiuridin to suppress reproduction of the DNA-containing viruses. Existence of a lipoid cover at a virus is established on its sensitivity to effect of ether and chloroform (vaginate viruses are inactivated). Further identification is carried out with a set of immune serums to the known viruses, using various reactions — neutralizations, RSK, RTGA, etc. Make immunization of animals the known virus with their further infection with the unknown or on the contrary less often.
Bibliography: Laboratory diagnosis of viral and rickettsial diseases, under the editorship of E. Lennet and N. Schmidt, the lane with English, M., 1974, bibliogr.; Luriya G. E. and D and r N of e of l of l of Dzh. E. The general virology, the lane with English, M., 1970, bibliogr.; Methods of virology and molecular biology, the lane with English, M., 1972; P sh e of N and h N about in V. A., Semenov B. F. izeze-r about in E. G. Standardization of methods of virologic researches, M., 1974, bibliogr.; The guide to laboratory diagnosis of viral and rickettsial diseases, under the editorship of P.F. Zdrodovsky and M. I. Sokolov, M., 1965; M. I. Falcons, With and N and c to and y A. A. and Remezov P. I. Virologic and serological researches at viral infections, L., 1972; Virologische Praxis, hrsg, v. G. Starke, Jena, 1968, Bibliogr.
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