CULTIVATION OF VIRUSES — cultivation of viruses in simulated conditions by infection of animals, cultures of cells and fabrics. To. make in the diagnostic purposes century (allocation from patients and carriers), during the experimental work (studying of viruses), for production of virus vaccines and diagnosticums.
V. Galtier for the first time carried out cultivation of a rhabdovirus in 1879, having infected a rabbit with a brain of a sick dog. Levenstein (A. Lowenstein, 1919) the first published data on successful transfer of a virus of herpes from the person to a rabbit. Gryuter (W. Gruter, 1920) proved a possibility of cultivation of a virus of herpes on rabbits. Ability of a virus of a vaccine (vaccinia) to be reproduced in fabric culture was proved by Parker and Nai (F. Parker, V. to N. Nye) in 1925. In 1931 Woodruff (A. M. of Woodruff) and E. Gudpascher showed an opportunity To. century on chorion-allantoisnoy to a cover of embryos of hens (a virus of smallpox of birds).
Viruses are reproduced only in living cells therefore for their accumulation infect with viruses of animals or cultures of cells and fabrics. At the same time there is an adaptation of the virus got from an organism of the patient or the carrier to new conditions. The less the artificial system from natural differs, the adaptation of a virus is carried out easier.
For an optimum reproduction of a virus it is necessary to use the most sensitive system and to carry out infection with strongly divorced fresh material as the inactivated virus particles can slow down reproduction of infectious virions. The system, in a cut a virus passes a complete cycle of a reproduction, carries the name of permissive (allowing). In not permissive (not allowing) system there is an incomplete cycle of a reproduction of a virus or it is not reproduced at all. The system, permissive for this virus, can become for it not permissive at change of culture conditions, napr, at change of temperature.
On animals cultivate those viruses which cause in them a clear clinical or pathoanatomical picture (e.g., development in mice of paralyzes at infection with a rhabdovirus or pneumonia at an influenzal infection). Many viruses grow in the small differentiated fabrics of embryos of birds and newborn mammals better, than in an organism of adult individuals.
For To. use mice, rats, Guinea pigs, rabbits, the Syrian hamsters, the African polecats, monkeys, hens century, etc. On adult mice cultivate influenza viruses, rage, many togavirusa; mice suckers are irreplaceable at cultivation of a number of viruses of Koksaki and togavirus of a number of arenovirus — causative agents of viral hemorrhagic fevers.
Suckers of white rats and the Syrian hamsters are often used for cultivation of oncogenous viruses. Guinea pigs serve for cultivation of viruses of a foot-and-mouth disease, the Marburg disease, etc. From monkeys most often use green African monkeys and different types of a macaque. So, studying of viruses of poliomyelitis and yellow fever became possible after their adaptation to an organism of macaques. Cultivation of causative agents of some slow infections (a chicken, Kreyttsfeldt's diseases — Jacoba), and also viruses of hepatitises A and B for the first time worked well at infection of a chimpanzee. Sensitive to a virus of hepatitis And also were South American monkeys of a marmozeta.
For obtaining standard results the animals used for work with viruses shall be genetically homogeneous. To this purpose serves inbredny crossing a lab. animals — brothers and sisters or parents and children, than the increasing degree of homozygosity is reached.
For successful To. century, in addition to a look and age of animals, the way of administration of material matters that is caused by a tropnost of a virus. Therefore in most cases his inoculation in sensitive fabric is necessary for reproduction of a virus in an organism of an animal. Only some viruses pathogens for animals at any ways of inoculation (e.g., a virus of the Venezuelan encephalitis of horses for mice).
The majority of neurotropic viruses cultivate by introduction them in cerebral hemispheres of animals. This way infect mice with various togavirusa, bunyavirusa and other arbovirus. The rhabdovirus is entered in the same way to mice, rabbits, sheep and dogs, a virus of a lymphocytic choriomeningitis — to mice and Guinea pigs. At cultivation of a virus of poliomyelitis on monkeys it is inoculated in a spinal cord or a thalamus of a brain. Often at cultivation of neurotropic viruses they are entered an animal into an abdominal cavity, however this way of inoculation concedes on sensitivity to intracerebral. Respiratory viruses cultivate usually by intranasal infection — dig in them in Nov a narkotizirovanny animal or enter in the form of an aerosol in the special camera.
Adenoviruses inoculate to the Syrian hamsters subcutaneously or in a mucous membrane of zashchechny bags, a virus of herpes of monkeys — to rabbits vnutrikozhno, and smallpox viruses — on the scarified skin (rabbits, calfs, hens). Viruses of smallpox and herpes can be cultivated on the scarified cornea of a rabbit. Introduction of viruses to a muscle, intravenously, through a mouth and per rectum is applied seldom. Intravenous infection of Guinea pigs, hamsters and polecats is technically difficult, instead material is entered more often into a cardial cavity.
To. century on animals very much is at a loss existence in their organism of various bacteria, mycoplasmas and viruses which can contaminate the cultivated virus. Sometimes the virus which is in an organism of an animal creates immunity to the cultivated virus, napr, the activator of an ektromeliya at mice to a virus of a vaccine.
For reduction of risk of pollution of the cultivated viruses foreign activators even more often use the animals who are grown up in the conditions of isolation. For this purpose receive pathogenic agents, animal, free from specific to this look — SPF (specific pathogen free). Their mothers should have no infections which are transmitted through a placenta. Cubs are taken by means of Cesarean section, enter them into intestines apatogenous bacteria, napr, lactic then they are raised by SPF females. Further these animals breed in the regular way. Support them in the enclosed space where sterile air, food, water and so forth moves.
Animal, free from any activators, contain in special boxes in the conditions of even more strict isolation (see. Germ-free animal ).
Embryos of birds with their low-differentiated fabrics are suitable for cultivation of very many viruses. For obtaining optimum results the look and age of embryos, a way of infection, the entered dose and temperature of an incubation matters. Most often use embryos of hens. They are most sensitive till 13th day of an incubation. Inoculate viruses usually on chorion-allantoisnuyu a cover, in a vitellicle, an allantoisny and amniotic cavity; and intravenously (in vessels of covers) viruses enter into a brain of embryos seldom. In a vitellicle cultivate many togavirusa; influenza viruses and inf. parotitis are well cultivated in an amniotic cavity 10 — 11-day embryos, at the same time the influenza virus breeds not only in cells of amnion, but also in a trachea and lungs of an embryo. Viruses of smallpox group, etc. cultivate on chorion-allantoisnoy to a cover, infecting 10 — 13-day embryos through a natural trapped air or through an opening on a side surface of egg after creation of an artificial trapped air. At infection with any way embryos can be injured therefore their death in the first 24 hours is regarded as nonspecific. Optimum quantity of a virus at infection — 1000 — 10 000 infectious doses. To. century in embryos usually occurs at t ° 36 — 37 °. Some viruses, napr, a virus of a vaccine, can breed at a temperature over 40 ° while the causative agent of natural smallpox needs to be cultivated at a temperature not over 38,5 °. The temperature and sensitive mutants of viruses having, as a rule, reduced pathogenicity cultivate at t ° 25 — 28 °.
At reproduction in embryos viruses can cause their death (many arbovirus, a virus of an entsefalomiokardit, etc.), emergence of changes on chorion-allantoisnoy to a cover (smallpox viruses) or in a body of an embryo, accumulation in embryonal liquids of hemagglutinins (influenza viruses, parotitis) and a complement-linked viral antigen.
The majority of the known viruses can be grown up in cultures of cells and fabrics (see). Most often use the single-layer primary or intertwined cellular cultures on glass, apply suspension cultures less often. Viruses adapt to primary cultures of cells easier, than by intertwined. Viruses of the person best of all breed in cultures of human cells and renal cells of monkeys.
An optimum dose of a virus at infection — 0,1 — 0,001 50% of a fabric cytopathic dose of a virus for a cell. The volume of an inoculum shall be small. Adsorption of a virus is carried out during 1 — 2 hour at t ° 37 ° then the inoculum is deleted if it is toxic for cells. A medium shall have pH 6,9 — 7,2. If to it serum is added, the last shall not contain antibodies or nonspecific inhibitors in relation to the cultivated virus. The most intensive reproduction of the majority of viruses occurs at t 36 — 37 °; at lower temperature (33 °) cultivate rhinoviruses.
At To. century for the purpose of their allocation from the infected bodies cultivation of cells of the most studied fabric after its tripsinization is very effective (e.g., fabrics of almonds for allocation of adenoviruses).
Reproduction of the majority of viruses is followed by cytopathic changes. The maximum quantity of a virus in culture is usually observed at a degeneration of 75% of cells. Reproduction of the viruses which do not have cytopathic activity can be established by means of a hemadsorption virus test (many mikso-and togavirusa), method of an immunofluorescence, way of a research of cultural liquid on availability of hemagglutinins (e.g., myxoviruses) or a complement-linked viral antigen, and also in animal experiments (rhabdovirus). Some viruses can be revealed on their ability to suppress reproduction of a cytopathic virus, i.e. on an interference phenomenon (e.g., in cultures of cells of embryos of the hens infected with viruses of a leukosis of birds the virus of sarcoma of Raus does not breed). Some viruses form in cells of inclusion.
The majority of viruses after reproduction in cells leaves in culture medium, some other remains connected with cells (viruses of smallpox, adenoviruses), some herpetic viruses need to be oversown together with the unimpaired cells as at destruction of cells they are inactivated. Sometimes at interaction of a virus and a cell the infection develops hron. E.g., the intertwined human cells infected with a virus of a lymphocytic choriomeningitis can produce inf. a virus throughout many generations.
For cultivation of coronaviruses of the person and some other use fabric cultures, i.e. fabric fragments cultivated out of an organism. Most often use tissue of a trachea of a rabbit. About reproduction of a virus in this case judge by the termination of the movement of cilia of culture of fabric.
It is necessary to consider a possibility of presence at cultures of cells and fabrics of various viruses and mycoplasmas. They can be brought together with cells if the last are taken from the infected organism, to get from trypsin or the medium of serum used as ingredient.
Adaptation of a virus to simulated conditions of reproduction demands carrying out several passages, quickly following one after another at infection with a small dose of a virus. Usually intensity of a reproduction of a virus at the same time considerably increases. Sometimes the virus after adaptation to one system gains ability to breed also in other systems. Svezhevydelenny viruses are more plastic, than long cultivated in any one conditions. To. century in simulated conditions quite often leads to decrease in their pathogenicity for feral hosts, than use for receiving vaccinal strains. In unfavorable conditions of cultivation (insensitive systems or at infection with too high dose) the defective virus particles which are containing only a part of a genome or not having nucleinic to - you can form. Some viruses do not manage to be cultivated in simulated conditions at all or their reproduction stops after several passages.
It is possible to keep viruses within several days at t ° 4 ° in the environment with pH apprx. 7,0. Their stability increases during removal of cellular fragments and addition of serum (10%), glycerin (50%) or skim milk (50%). All viruses well remain at t ° — 70 ° below in bottletight vessels; many remain viable months and even years. Smallpox viruses and enteroviruses well remain at t ° — 20 °. Freezing of a virus shall happen quickly. For increase in stability of a virus add serum protein, a chicken yolk, peptone, sucrose or glucose by Wednesday. Influence of stabilizers on different viruses is unequal. Viruses can remain viable a long time and after lyophilizing. As stabilizers at the same time use peptone (10%), milk (50%), sucrose with gelatinous or a chicken yolk (on 10%). The lyophilized virus shall remain in vacuum or neutral gas (e.g., nitrogen) at t ° 4 ° or — 15 °.
Bibliography: Laboratory diagnosis of viral and rickettsial diseases, under the editorship of E. Lennet and N. Schmidt, the lane with English, M., 1974; With about to about l about in M. I., With and - N and c to and y A. A. and Remezov P. I. Virologic and serological researches at viral infections, L., 1972; Stark G., etc. Practical virology, the lane with it., M., 1970, bibliogr.; Comparative diagnosis of viral diseases, ed. by E. Kurstak a. C. Kurstak, v. 1—2, N. Y. a. o., 1977.
AA. P. To a saw.