A special laboratory named this virus. Methods for cultivating viruses in the laboratory, a method for indicating virus reproduction

Areas of activity

Direction scientific activity– infectious virology and epidemiology, problems of viral infections of humans and laboratory primates, viral hepatitis of various etiologies; development of models of the most significant human viral infections in laboratory primates to study unresolved issues of pathogenesis and immunogenesis, testing of developed means of prevention and treatment.

Tasks and functions

The work of the laboratory consists of research and scientific-practical sections.
The main objectives of the laboratory’s research work are:
1) Study of spontaneous viral infections of monkeys of the gastrointestinal and respiratory tract in a comparative aspect with similar human infections.
2) Study of specific markers of viral hepatitis A, E, C, G in monkeys.
3) Study of the characteristics of strains of viral agents isolated from monkeys.
4) Study of the role of rota-, adeno-, entero-, noro- and other intestinal viruses in the pathology of the gastrointestinal tract of laboratory primates. Molecular genetic study of strains.
5) Study of epizootic and epidemiological aspects of spontaneous viral infections of monkeys.
6) Study of the features of the formation of humoral immunity in spontaneous hepatitis and other viral infections of monkeys.
7) Study of the prevalence of hepatitis E among monkeys and the population in the non-endemic territory of the South of Russia (Greater Sochi region).
8) Study of anti-measles immunity in monkeys of the Adler Primatological Center and the population of the surrounding region.
9) Study of the diagnostic value of various methods and diagnostic test systems for viral infections of monkeys.
10) Simulation of human viral diseases on monkeys with the aim of further using the developed models to study issues of pathogenesis, immunity, methods of treatment and prevention of these diseases.

The scientific and production function is as follows:
1) specific diagnosis of viral infections in the event of outbreaks of diseases of unknown etiology.
2) implementation of serological and genetic monitoring of viral infections among laboratory primates and the population of the surrounding region for the purpose of epidemiological surveillance.

Research topics

1. Intestinal viruses of laboratory primates (2010-2012). Head and responsible executor of the work: manager. lab., doctor of medical sciences Korzaya L.I. Performers: researcher Keburia V.V., junior researcher Goncharenko A.M.
2. Study of anti-measles immunity in monkeys of the Adler Primatological Center and the population of the surrounding region (2013-2014) Scientific supervisor and executive officer: head. lab., doctor of medical sciences, Korzaya L.I.
3. Characteristics of rotavirus strains in laboratory primates (2013 – 2015) Head: L.I. Korzaya. Responsible executor: D.I. Dogadov.
4. Molecular genetic characteristics of intestinal viruses circulating among monkeys of the Adler Primatological Center (2015-2017). Work manager and responsible executor: manager. lab., doctor of medical sciences Korzaya L.I. L.I. Performers: junior researcher Dogadov D.I., researcher Keburia V.V., junior researcher Goncharenko A.M.
5. Study of the spread of human respiratory viruses among laboratory primates (2016-2018). Head and responsible executor of the work: manager. lab., doctor of medical sciences Korzaya L.I. Performers: researcher Keburia V.V., junior researcher Goncharenko A.M.

Results

Spontaneous and experimental hepatitis A has been comprehensively studied in several species of lower monkeys of the Old World (Z.V. Shevtsova, L.I. Korzaya). It has been established that in terms of serological, virological, biochemical and morphological manifestations, hepatitis A does not differ from the human disease of the same name. A new fact of chronicity and long-term persistence of the virus has been established. Spontaneous and experimental hepatitis A have been proposed as models for studying similar human infections.
A new phenomenon has been discovered in the infectious pathology of the lower monkeys of the Old World - natural infection with an HCV-like virus. A new HCV-like infection in rhesus macaques has been characterized. Not only serological, but also virological markers of infection (HCV RNA) were detected. Features of the indicators of the humoral response to the virus in a comparative aspect with humans were revealed, which were expressed in a wide variety of variants of the spectrum of antibodies in relation to the main diagnostic proteins of HCV, the predominance of antibodies to non-structural proteins of the virus, as well as their low reactivity. Markers of HCV-like infection in monkeys have been detected for several years. The infection is practically asymptomatic, accompanied by only a slight increase in the level of alanine aminotransferase without morphological liver damage. The data obtained are not only theoretical, but also of great practical importance and are the basis for the further development of an experimental model of hepatitis C in lower apes.
For the first time, data have been obtained on the widespread circulation of the hepatitis E virus not only among the macaques of the Adler nursery, but also among the population of the surrounding territory - the Greater Sochi region. The detection of anti-HEV IgM in human sera indicates the presence of cases of acute HEV in an area that is not endemic for HEV. The percentage of detection of anti-HEV among nursery staff was significantly lower than among the population of the surrounding region, which suggests the presence of a different source of HEV infection for humans than monkeys.
Infection of laboratory primates with intestinal viruses (rota-, entero-, adeno-, HAV, HEV) was revealed. Most viral infections were asymptomatic, accompanied only by laboratory signs. A certain role of rotavirus, adenovirus and enterovirus in diseases of the gastrointestinal tract of monkeys, as well as virus carriage in “clinically” healthy animals, is shown. In dead monkeys with diarrhea (rhesus macaques), a mixed infection associated with intestinal viruses was observed in more than half of the cases (58.3%). In clinically healthy animals, mixed infection was detected almost 5 times less often (12.5%). The different sensitivity of commercial test systems for detecting rotavirus and adenovirus in materials from monkeys has been shown. Methods for detecting intestinal viruses (PCR, ELISA, RNGA, RTNGA) have been introduced into the practice of the laboratory of infectious virology to clarify the etiology of diarrheal diseases in monkeys, which will avoid the unjustified use of antibiotics and other drugs.
An extremely low degree of anti-measles immunity was revealed among monkeys of the Adler Primatological Center (14.9±1.6% due to individuals over 22 years old). There has been no circulation of the measles virus among monkeys since 1993. The emergence of a source of infection among non-immune monkeys (85.1%) can lead to an outbreak of measles. When the measles epidemic situation worsens, vaccination of seronegative monkeys is proposed.
Important data were obtained when studying the immunostructure of the young population of Sochi to the measles virus (indicator group aged 18 to 35 years). Lack of immunity to measles in 42.2±7.4% of RUDN University students (18-25 years old) and low level antibodies to the virus in the vast majority of other individuals in this group indicates an unfavorable measles situation among the susceptible population. Among the employees of the Research Institute of Medical Sciences, 14.5% of persons seronegative for the measles virus were also registered, and 22.2% were under the age of 35 years. The presence of seronegative individuals requires measles vaccination.

Since 2002, scientific research by employees of the Laboratory of Infectious Viruses has been supported by grants from the Russian Foundation for Basic Research (RFBR) and RFBR-YUG.
The laboratory trains virologist specialists through graduate school.
Laboratory staff carry out scientific and pedagogical work at the Sochi branch of the Peoples' Friendship University of Russia at the departments of physiology, medical veterinary medicine and veterinary and sanitary examination. A course of lectures and practical classes is conducted in the disciplines “Virology”, “Microbiology”, “Biological safety in laboratories”. Thesis works are carried out in the laboratory.
The laboratory conducts contract research institutes for preclinical testing of vaccines.

Staff

• Korzaya Lidiya Ivanovna – head. lab., doctor of medical sciences
• Keburia Victoria Velodievna – researcher
• Dogadov Dmitry Igorevich – researcher
• Goncharenko Alexandra Mikhailovna – junior researcher

Main publications

1. Korzaya L.I., Shevtsova Z.V., Lapin B.A., Dyachenko A.G., Krylova R.I. Obtaining and characterizing cultural strains of hepatitis A virus from humans and monkeys. Question Virol., 1997, No. 2, p. 60-63.
2. Korzaya L.I., Lapin B.A., Shevtsova Z.V., Krylova R.I., Esvandzhia N.Ch. Characteristics of experimental models of hepatitis A in hamadryas baboons. Question Virol., 1998, No. 2, pp. 67-70.
3. Korzaya L.I., Shevtsova Z.V., Lapin B.A., Krylova R.I., Dzhelieva Z.N. Repeated infection with hepatitis A in rhesus monkeys in the experiment. Question Virol., 1998, No. 4, p. 158-163.
4. Lapin B.A., Dzhikidze E.K., Krylova R.I., Korzaya L.I., Gvozdik T.E., Dzhelieva Z.N., Simavanyan K.V., Kukava G.G. Spontaneous disease of monkeys of unknown etiology. Baltic. J.Lab. Anim. Sci., 1999, P.19-28.
5. Korzaya L.I., Lapin B.A., Shevtsova Z.V., Krilova R.I. Spontaneous and experimental hepatitis A in Old World monkeys are the models of human hepatitis A. // J. Lab. Anim. Sci., 2001, 11(2), P. 135-141.
6. Korzaya L.I., Lapin B.A., Keburia V.V., Chikobava M.G. Natural infection of primates with hepatitis C virus // Bulletin. Expert Biol. – 2002. – .T. 133, No. 2. - P. 211-214.
7. Lapin B.A., Shevtsova Z.V., Korzaya L.I., Krylova R.I. Spontaneous and experimental hepatitis A in Old World monkeys and their use in studying this infection. // World of viral hepatitis. - 2006. - No. 6. - P. 3-9.
8. Korzaya L.I., Keburia V.V., Lazareva I.Ya., Lapin B.A. Viral hepatitis (A, E) in laboratory primates. Epidemiological aspects of the problem. // In the book: Fundamental and applied problems of medicine and biology in experiments on monkeys. Proceedings of the international scientific conference. September 19-22, 2007, Sochi-Adler. – 2007. - P. 94-103.
9. Keburia V.V., Korzaya L.I., Krylova R.I., Lapin B.A. Long-term dynamic observation of HCV-positive macaques at the Adler Primatological Center. // In the book: Fundamental and applied problems of medicine and biology in experiments on monkeys. Proceedings of the international scientific conference. September 19-22, 2007, Sochi-Adler. – 2007. - P. 132-141.
10. Korzaya L.I., Lapin B.A., Keburia V.V., Lazareva I.Ya. Frequency of detection of antibodies to the hepatitis E virus in service personnel and in macaques of the Adler nursery. // Question virusol. – 2007. - P. 36-40.
11. Korzaya L.I., Keburia V.V., Lazareva I.Ya., Lapin B.A. Frequency of spread of antibodies to the hepatitis E virus among the population of the Adler region and monkeys of the primatological center. // In the book: Viral hepatitis - epidemiology, diagnosis, treatment and prevention. Materials of the YII Russian scientific and practical conference with international participation. May 29-31, 2007, Moscow. – pp. 113-114.
12. Korzaya L.I., Keburia V.V., Lazareva I.Ya. Analysis of the detection of hepatitis E markers in monkeys of the Adler nursery. // In the book: The contribution of fundamental research to the development of the modern innovative economy of the Krasnodar region. Conference of grant holders of the regional competition of the Russian Foundation for Basic Research and the administration of the Krasnodar Territory “SOUTH OF RUSSIA”. Collection of abstracts. – Krasnodar 2007. – P. 146-148.
13. Keburia V.V., Korzaya L.I. Spontaneous HCV-like infection of laboratory primates. // Materials of the Y scientific-practical conference of young scientists and students of the south of Russia. "Medical Science and Healthcare". Krasnodar 2007. – P. 37-40.
14. Korzaya L.I., Keburia V.V., Goncharenko A.M. Markers of enteral hepatitis E and A in the population of the Adler region and monkeys of the primatological center. // Abstracts of the Fourth International Conference dedicated to the 85th anniversary of the St. Petersburg NIIEM Pasteur and the 120th anniversary of the Paris Pasteur Institute. St. Petersburg, June 2-4, 2008. P. 32.
15. Korzaya L.I., Keburia V.V., Lazareva I.Ya., Goncharenko A.M. Analysis of the detection of hepatitis E markers in monkeys of the Adler nursery. // In the book: The contribution of fundamental research to the development of the modern innovative economy of the Krasnodar region. Conference of grant holders of the regional competition of the Russian Foundation for Basic Research and the administration of the Krasnodar Territory “SOUTH OF RUSSIA”. Collection of abstracts. – Krasnodar 2008. – P.56.
16. Korzaya L.I., Keburia V.V., Goncharenko A.M., Lapin B.A. Epidemiological aspects of the study of viral hepatitis E in humans and laboratory primates. // Materials of the II Annual All-Russian Congress on Infectious Diseases. – Moscow, March 29-31, 2011, p. 154.
17. Korzaya L.I., Keburia V.V., Goncharenko A.M., Lapin B.A. Molecular diagnostics of enterovirus infections in monkeys. // On Sat. proceedings of the VII All-Russian Scientific and Practical Conference with international participation “Molecular Diagnostics - 2010”. – Moscow, November 24-26, 2010,

Scientific works in 5 years

1. Korzaya L.I., Keburia V.V., Goncharenko A.M., Dogadov D.I., Lapin B.A. Markers of viral infections in laboratory primates. //In the book: Fundamental and applied aspects of medical primatology. Proceedings of the second international scientific conference. Volume 1. (August 8-10, 2011, Sochi-Adler). – 2011. - P. 79-88.
2. Keburia V.V., Korzaya L.I., Goncharenko A.M., Gvozdik T.E. Studying the role of viral agents in the pathology of the gastrointestinal tract of monkeys. //In the book: Fundamental and applied aspects of medical primatology. Proceedings of the second international scientific conference. Volume 1. (August 8-10, 2011, Sochi-Adler). – 2011. - pp. 216-221.
3. Korzaya L.I., Keburia V.V., Dogadov D.I., Lapin B.A. Enteric viral infections of laboratory primates. // Materials of the IY Annual All-Russian Congress on Infectious Diseases. – Moscow, March 26-28, 2012, p.186.
4. Korzaya L.I., Dogadov D.I., Keburia V.V., Lapin B.A. Rotaviruses of laboratory primates. Materials of the V Annual All-Russian Congress on Infectious Diseases, March 25-27, 2013 Moscow, p. 208-209.
5. Dogadov D.I., Korzaya L.I., Keburia V.V. Determination of adenoviruses in laboratory primates by polymerase chain reaction. Materials of the V Annual All-Russian Congress on Infectious Diseases, March 25-27, 2013 Moscow, p. 129.
6. Korzaya L.I., Keburia V.V., Dogadov D.I., Lapin B.A. Comparative study of serological markers of enteric viral hepatitis in rhesus monkeys (Macaca mulatta). // Materials of the VI Annual All-Russian Congress on Infectious Diseases, March 24-26, 2014 Moscow, p. 146.
7. Korzaya L.I., Dogadov D.I., Keburia V.V., Lapin B.A. Study of the epidemiological aspects of measles using a model of natural infection in laboratory primates. // Materials of the VI Annual All-Russian Congress on Infectious Diseases, March 24-26, 2014 Moscow, p. 146-147.
8. Dogadov D.I., Korzaya L.I., Keburia V.V. Molecular genetic markers of intestinal viral infections of laboratory primates. //On Sat. proceedings of the YIII All-Russian scientific and practical conference with international participation. – Moscow, 2014. - Volume I. – P.385.
9. Lapin B.A., Dzhikidze E.K., Yakovleva L.A., Eorova T.P., Korzaya L.I. Diseases of monkeys that are dangerous to humans. Rules for keeping and working with monkeys in quarantine when animals arrive from external sources, as well as during experimental infection. // Methodological recommendations - M. - 2014. P. 52.
10. Dogadov D.I., Korzaya L.I., Keburia V.V. Molecular genetic markers of intestinal viral infections of laboratory primates. //On Sat. proceedings of the YIII All-Russian scientific and practical conference with international participation. – Moscow, 2014. - Volume I. – P.385.
11. Dogadov D.I., Korzaya L.I., Kyuregyan K.K., Carlsen A.A. Detection of hepatitis A virus in green monkeys (CHLOROCEBUS PYGERYTHRUS) arriving from their natural habitat (Tanzania). // Medical virology, volume XXIX (2). Application. Proceedings of the conference dedicated to the 60th anniversary of the Institute of Poliomyelitis and Viral Encephalitis named after M.P. Chumakova. (December 8-9, 2015). Moscow, 2015. P. 84.
12. Korzaya L.I., Keburia V.V., Dogadov D.I., Lapi B.A., Kyuregyan K.K., Mikhailov M.I. Markers of hepatitis E in the population of Greater Sochi and monkeys of the Adler Primatology Center. // Question Virol, 2015 (accepted for publication!)

Microbiological laboratories are part of state sanitary and epidemiological surveillance centers (TSGSEN), infectious diseases and large hospitals, dermatovenerological and tuberculosis dispensaries. Depending on the departmental affiliation, diagnostic work is carried out in bacteriological, virological, mycological, etc. laboratories, which is regulated by relevant instructions and legislation. In accordance with these legislative acts, each of the laboratories can only work with certain groups of microorganisms.

The bacteriological laboratories of the Central State Medical University work with microbes of groups III and 1U, conducting etiological diagnostics of infectious diseases of an airborne, intestinal, purulent-inflammatory, etc. nature.

Virology laboratories carry out virological diagnostics of diseases caused by viruses (influenza, polio, herpes, etc.), as well as chlamydia (ornithosis, genitourinary diseases, etc.) and rickettsia (Q fever, typhus, etc.).

Laboratories for especially dangerous infections diagnose especially dangerous microbial infections (plague, cholera, anthrax, brucellosis, tularemia, etc.), and some of them of viral etiology (Marburg, Ebola, smallpox, etc.). The work is carried out according to particularly strict regulations.

The skin and venereological group of diseases and tuberculosis are diagnosed in the appropriate dispensaries.

Bacteriological laboratories TsGSEN should be located in standard buildings or premises designed for the volume of work carried out, in accordance with the purpose. Each bacteriological laboratory must have a full set of units necessary for them : a registry for receiving tests and issuing results, boxes for working with bacteria of different groups, a room for immunodiagnostic and molecular genetic tests, rooms for preparing culture media, sterilization, washing, a vivarium with boxes for healthy and experimental animals is possible.

Each room is equipped with appropriate equipment and equipment.

The sterilization room must contain a steam sterilizer of a certain model - vertical or horizontal, etc.

In terms of equipment for growing, storing microorganisms and other work, you must have:

Refrigerators for separate storage of cultures and other biological preparations, according to the instructions.

Centrifuges for sedimentation of corpuscular substances, including microorganisms. Centrifuges must be refrigerated.

Thermostats for growing bacterial cultures at a given temperature.

Micronaerostats, for growing bacteria called anaerobes in oxygen-free conditions.

Distiller for producing distilled water.

Apparatus for making cotton-gauze plugs of different sizes.

In the box rooms it is necessary to have: biological immersion microscopes (including a phase-contrast device illuminator, a dark-field condenser), water baths, refrigerators, a set of instruments (bacteriological loops, needles, scalpels, tweezers, spatulas, etc.), sets of dyes, alcohol , reagents, filter paper, pencils for writing on glass, acids, alkalis, alcohol lamps and gas burners. Jars with disinfectant solution (signed, with the date of preparation of this disinfectant solution). Laboratory glassware : pipettes, test tubes, flasks, vials, Petri dishes, mattresses, pasters, etc.

The laboratory must have a certain supply of commercial culture media, test systems and diagnostic sera, and various sets of diagnostic drugs.

Some rules for working in microbiological laboratories:

1. All employees work in medical gowns, caps, and slippers. If necessary, wear a mask.

2. Smoking, eating, and drinking water are prohibited in the laboratory, except in specially designated areas.

3. The workplace is kept in exemplary order.

4. If infected material gets on a table, floor or other surface, it is necessary to treat the area with a disinfectant solution.

5. Storage, shipment, issuance of microorganism cultures is carried out as prescribed.

6. At the end of work, wash your hands thoroughly with soap. If necessary, hands can be treated with one of the disinfectant solutions, etc.

Virology laboratories. They must be completely isolated from other units. There are certain differences in the structure of virology laboratories, depending on specialization, but there are main divisions : freezer, washing, disinfection, pure and infected cell cultures, boxes for working with viruses, a room for immunodiagnostic studies, a vivarium, a room for chicken embryos - clean and infected.

The virology laboratory must have : locker room - for changing clothes, shoes, tiled floors covered with linoleum. Windows covered with mesh. Ultraviolet lamps. Refrigerators are required - at 4 o C and an ultra-fridge at -20-40 o C or more, porcelain mortars, homogenizers, pestles, tools (scissors, needles, syringes, scalpels, tweezers, etc.). Centrifuges with cooling up to 5-6 thousand vol. and ultracentrifuges up to 30 thousand vol. per minute or more. Luminescence microscope. Incubators at 37 o C, for the purpose of containing chicken embryos and tools for working with them. Thermostats with automatic parameters for incubating infected chicken embryos. Glassware and test tube racks, burners, etc.

Some rules for working in virology laboratories:

1. Only employees are allowed to enter the virology laboratory. It is mandatory to change into special clothing in accordance with the work being performed (robe, cap, shoes).

2. Before entering, be sure to have a mat soaked in a disinfectant solution.

3. All types of work with contaminated or suspected contaminated material should be carried out strictly in accordance with the instructions.

4. Working in boxes is mandatory with additional clothing attributes: mask, double robe, gloves, towel, safety glasses, etc.

6. In the event of an “accident” (spraying of virus-containing material), you should call the manager or another doctor (without leaving the box by pressing the bell button) and carry out disinfection together according to the instructions.

7. If necessary, get vaccinated according to the instructions.

8. Spent virus-containing material is destroyed by autoclaving at 1.5 atm for 30 minutes.

9. After completing the work, you should change into a regular robe and shoes, a cap or scarf in the box room, wash your hands with soap, and disinfect if necessary.

10. Before work, boxes are treated with formalin vapor and ultraviolet light. After work, treat with a 2% solution of chloramine, etc.

Rules for student work at the Department of Microbiology and Virology :

1. Duties of duty officers:

Together with the laboratory assistant, check the presence and quantity of instruments (bacterial loops, tweezers, etc.), materials intended for classes (test tubes and cups with cultures, pencils for writing on glass, papers with gentian violet, dyes, etc.), the condition of microscopes, object and cover glasses, etc. Make entries in the appropriate journal for those on duty. Receive educational material from the laboratory assistant and distribute to students. Take cups and test tubes with microorganism inoculations individually.

At the end of classes, the person on duty checks the condition of the work tables, eliminates all defects in their cleaning, checks the availability of instruments, microscopes, etc. Test tubes and cups with cultures are handed over individually to the laboratory assistant, fills out the notebook of the person on duty, hands over the room to the laboratory assistant, and turns off the lights.

2. Student responsibilities :

Before you start. Put on a medical gown, fasten it, put on a cap or scarf. Place briefcases and books in a desk drawer or in bags. Check the condition of the work table and microscope. Place a notebook, pen, pencils, including colored ones, on the desktop.

During work. Handle the microscope, glassware, instruments, etc. with care. Be attentive to all stages of working with bacterial cultures. Label dishes with germs. Sterilize the loop after finishing working with cultures. In the event of an accident (contamination of the table surface, floor, clothing, skin, etc. with infectious material), report the incident to the teacher and jointly eliminate the contamination.

After work. Tidy up your workspace. Hand over inoculated cups and test tubes to the person on duty. Hand over the waste material to the duty officer. All used instruments should be burned over the fire of an alcohol lamp, glass should be placed in jars with disinfectant solution. Tidy up the microscope and workbench. Wash your hands thoroughly with soap.

Enter and work in training rooms without a gown or cap. Eat food in the premises of the department. Smoking, littering. Place briefcases, hats, and textbooks on the table and window sills. Damage equipment. Dirty tables. Talk loudly and laugh, run and interfere with the work of the department.

VIROLOGICAL LABORATORY- an institution engaged in the study of viruses and viral diseases or the production of viral preparations (vaccines, diagnostics, antiviral immune serums, etc.).

V. l. separated from the bacteriological ones and began to exist as independent units in the 20th century. In the USSR, the first V. l. were created in the 30s. Now there are several medical institutes in the country. virology, combining V. l. various profiles where viral diseases are studied, the nature of viruses is investigated, viral drugs are developed and produced (Institute of Virology named after D.I. Ivanovsky, Institute of Poliomyelitis and Viral Encephalitis, All-Union Research Institute of Influenza, Moscow Research Institute of Viral Preparations, etc.) . V. l. There are also in most Union and Republican institutes of microbiology and epidemiology, usually profiled in relation to the study of one or several diseases. In addition there is approx. 150 V. l. at republican, regional and city sanitary-epidemiological stations, as well as laboratories at large to lay down. institutions; They mainly do diagnostic work. In addition to honey V. l., there are laboratories for studying viral infections of animals and plants.

Sizes and states of V. l. depend on the purpose and scope of work. In all cases, it is imperative to ensure the safety of personnel and the ability to work in sterile conditions.

V. l. consists of the laboratory itself and utility rooms - for processing and sterilizing utensils, preparing nutrient media (for growing cell cultures, identifying bacteria and mycoplasmas), lyophilizing viruses, an incubator, vivarium, etc. If V. l. is part of a larger institution (institute, sanitary-epidemiological center, station, etc.), utility rooms can be common to a number of laboratories or the entire institution.

Actually V. l. is built as a bacteriological laboratory (see), taking into account the specifics of the work - growing cell and tissue cultures, ultracentrifugation, storing viruses at low temperatures, etc. It is equipped for diagnostic work (isolation of viruses and serological reactions), for studying the properties of viruses and their structure , conducting genetic research, etc.

Room V. l. should be easy to wash and treat with disinfectant solutions. For this purpose, the walls are painted with oil paint or tiled, the floor is covered with linoleum or tiles. The room is equipped with supply and exhaust ventilation with approximately 10-fold air exchange; it must be provided with cold and hot water, as well as lighting gas. It is advisable to have centralized system compressed air (pressure up to 1.0-1.2 atm) and vacuum (up to 700-760 mm Hg residual pressure). Shower rooms for employees must be provided. When working with particularly dangerous viruses, it is necessary to neutralize (by boiling) wastewater.

In V. l. It is mandatory to have a separate room for sterile work, consisting of two compartments separated by a glass partition. The interior space - the box - should be small (6-8 m2), with a low ceiling (see Boxes, microbiological). The door should open into a anteroom used for putting on additional clothing, separated by a second partition from the rest of the room. To sterilize the box and pre-box, bactericidal lamps made of uviol glass with a predominant wavelength of 254 nm are used (see Germicidal irradiators). For this purpose, you can use BUV lamps, which are installed at the rate of 2-2.5 W per 1 m 3 of room; average lamp life 1500 hours. It is mandatory to provide the boxes with sterile air through forced ventilation with 4-5 times exchange; To sterilize the air, LAI K filters made of Petryanov fabric - FPP type - can be used.

The box should contain only the tools and utensils necessary for work, a sterilizer for tools, wide-mouth jars with a disinfectant solution, and a tank with a lid for contaminated material. When working with pathogens of particularly dangerous infections (smallpox, encephalitis, etc.), an additional table box is installed in the box, in which both incoming and outgoing air are sterilized by filtration. The basic rules for working with particularly dangerous pathogens are regulated by special instructions.

For disinfection in V. l. Lysol 1-5%, chloramine 1-5%, formaldehyde 2.5-5% are most often used.

In addition to the usual bacteriological glassware, V. l. must have a homogenizer for grinding tissue, magnetic stirrers, microscopes (light for research in ordinary and ultraviolet light, as well as electronic), centrifuges of various powers (at 3-5 thousand rpm, and also with cooling at 12-15 thousand). and 60 thousand rpm), having a set of rotors. Thermostats are required that simultaneously operate at different temperatures (from 25 to 40°), including with the supply of carbon dioxide, or thermostatic rooms. V. l. equipped with refrigerators or cold rooms with t° +4°, -20° and -40°. To store cell cultures, Dewar flasks with liquid nitrogen or refrigerators with temperatures below -90° are required.

Laboratories studying the biochemistry of viruses are equipped like chemical laboratories. Work with radioactive isotopes is carried out in a specially equipped room.

In the media preparation departments, in addition to the usual chemical utensils, it is necessary to have installations for water treatment, which is double distilled in glass apparatus or deionized on columns with ion exchange resins. To sterilize solutions that cannot be autoclaved, use Seitz asbestos paper sterilizing plates mounted in Salnikov-type filters (F-140, FS-3, FS-7, etc.); filtration is carried out at a pressure of 0.5 atm. For the same purpose, you can use glass plates (candles) and Millipore filters with pore sizes from 0.22 to 1.2 microns (see Bacterial filters).

The vivarium (see) must be separated from the premises of the V. l. It must have rooms for quarantine of incoming animals, for their infection and separately for autopsy. Work with small laboratory animals is carried out for protective glass. It is better to disinfect animal cages using steam. It is also necessary to have a crematorium for burning animal corpses and garbage.

Bibliography: Kravchenko A. T. Principles of organization and operating mode of virological and rickettsial laboratories, Guide to Laboratories, diagnosis of viral and rickettsial diseases, ed. P. F. Zdrodovsky and M. I. Sokolov, p. 219, M., 1965; Laboratory diagnosis of viral and rickettsial diseases, ed. E. Lenneth and N. Schmidt, trans. from English, p. 9, 123, etc., M., 1974; Guide to the laboratory diagnosis of smallpox for smallpox eradication programs, Geneva, WHO, 1969; Virologische Praxis, hrsg. v. G. Starke, Jena, 1968, Bibliogr.

In the virology laboratory, work is carried out to isolate virus strains, their identification and cultivation, and various scientific studies are carried out. When working with viruses, you must first of all:

1. Prevent contamination of virus strains with foreign microflora;

2. Ensure the safety of working personnel from possible infection by viruses;

3. Ensure the safety of the surrounding population from infection with viral infections through wastewater, corpses of experimental animals, etc.

When studying materials obtained from patients with viral infections, for the purpose of laboratory diagnosis of these diseases, they use various methods:

· Methods of electron and, to a lesser extent, light microscopy;

· Methods for isolating and cultivating viruses in cell cultures;

· Methods for isolating and cultivating viruses in developing chicken embryos and in the body of sensitive experimental animals;

· Identification of viruses by their hemagglutinating ability;

· Various serological research methods: traditional and express methods;

· Molecular genetic research methods - molecular hybridization and polymerase chain reaction.

1.1.2. Materials studied for viral infections

When taking infectious material from people and animals, it is necessary to take into account the tropism of viruses for certain tissues and organs, the routes of virus release into the external environment and the characteristics of the pathogenesis of a particular viral infection.

There are pneumotropic, enterotropic, hepatotropic, lymphotropic, neurotropic and dermotropic viruses. Depending on the tropism, various materials are subjected to research. For example, they examine mucus from the throat, sputum, etc., if the virus is pneumotropic; bowel movements - with enterotropic viruses; liquid from vesicles or pustules, crusts - if the virus is dermotropic, etc.

1.1.3. Processing of virus-containing material

Infectious materials, taken taking into account the tropism of viruses and in compliance with asepsis, are placed in sterile containers, carefully sealed and sent to the laboratory, placed in a thermos with ice.

It is recommended to examine the material as soon as possible, since viruses are quickly inactivated. The preservation of the virus is facilitated by placing the test material (in a 50% glycerin solution) in a refrigerator at a temperature not exceeding 5 o C. But the most reliable method is frozen storage at a temperature of -45 o C and below; under such conditions the virus can remain viable for a long time.

Processing of dense material containing viruses begins with grinding it in a mortar or grinding it in special devices - homogenizers. Then a 10% suspension is prepared in saline solution, which is centrifuged at 2000-3000 rpm for 15-30 minutes to sediment large particles. The viruses remain in the supernatant, which is subjected to further study.

The liquid virus-containing material is directly centrifuged and the supernatant is also obtained.

If there is doubt about the bacteriological sterility of the test virus-containing supernatant, antibiotics are added to it to destroy foreign microorganisms. Antibiotics do not affect viruses, and they remain viable.

1.1.4.Microscopic research methods in virology

- Electron microscopy

Electronscopic preparations are prepared from purified and concentrated virus-containing suspensions or ultrathin sections of tissue infected with viruses. Viral objects are applied to special substrate films placed on support meshes. Substrate films must be very thin (no more than 30 nm thick), transparent and sufficiently strong, for example, colloidal carbon. The films are applied to supporting copper meshes (2-3 mm in diameter) with numerous holes. The drugs are then processed in various ways.

Metal spraying methods used to obtain contrast agents. Vapors of heavy metals (gold, platinum, uranium, etc.), formed in a special device under vacuum and high temperature conditions, are directed at an acute angle onto the virus-containing drug. Viruses are coated with a thin layer of metal.

Negative contrast method is based on the fact that when the drug is treated with certain salts of heavy metals, for example, a 1-2% solution of phosphotungstic acid, a denser layer is created that does not allow electrons to pass through, and in which more electron-transparent objects under study are clearly visible.

Ultrathin sectioning combined with negative contrast is the best for studying the fine structure of virions and studying the stages of interaction of viruses with the cell, but at the same time it is the most complex. The examined pieces of infected tissue or other virus-containing material are fixed in a special fixative (for example, osmium). Dehydrate by sequential placement in alcohols of increasing strength. The samples are filled with special plastic, after polymerization of which solid transparent blocks are formed. Ultrathin sections with a thickness of 10-20 nm are prepared from the blocks on a special microtome. The resulting sections are contrasted by placing them in a solution of phosphotungstic acid.

The preparations prepared by the methods described above are studied in a transmission electron microscope, the resolution of which reaches 0.2-0.3 nm. The image of the preparation is observed on the fluorescent screen of an electron microscope and special photographic plates are photographed from which prints are obtained. Received magnifications: ×100000-×400000.

Scanning electron microscopy is carried out using a scanning electron microscope, in which a thin beam of electrons quickly moves across the object under study, that is, scans its surface. As a result, radiation of secondary electrons arises, which, passing through a cathode ray tube, is converted into a three-dimensional image of an object on a fluorescent screen.

Scanning microscopy makes it possible to obtain a three-dimensional image of virions (the preparation is first sprayed with metals), to distinguish the details of the structure of their surface, but does not reveal their internal structure. The resolution of a scanning microscope is 7-20 nm.

- Light microscopy

In a light microscope you can see large viruses, the sizes of which are within the resolution of the microscope - at least 0.2 microns. As well as intracellular inclusions in tissues affected by the virus.

Large viruses, for example, poxviruses, and inclusions are detected using special staining methods, in phase contrast, in a dark field of view; Fluorescent microscopy is also used.

Large viruses are identified by Morozov staining (silvering). To identify intracellular inclusions, histological sections from affected tissues, smears or prints are prepared. Usually the preparations are stained according to Romanovsky-Giemsa, sometimes by other methods. The detection of Babes-Negri inclusions in nerve cells of the brain during rabies is of greatest practical importance. For this purpose, the preparations are stained according to Mann.

Luminescence microscopy. Preparations prepared from materials containing large viruses, intracellular inclusions, and accumulations of viral antigens are stained with solutions of fluorochrome dyes. With fluorescent microscopy in UV light, acridine orange-stained accumulations of RNA genomic viruses and the inclusions they form are visible as luminous red granules against the background of pale green cell cytoplasm; DNA genomic viruses give off an emerald green glow.

Immunofluorescent method is based on the combination of viruses, intracellular inclusions, and accumulations of viral antigens with specific antiviral antibodies labeled with fluorochrome dyes. The resulting complexes glow under fluorescence microscopy.

A group of Canadian scientists from the University of Alberta in Canada conducted a rather risky experiment, as a result of which they were able to resurrect an extinct species of horsepox that is not dangerous to humans. It turned out to be relatively simple and cheap and caused a predictable panic attack among ordinary people.

Horsepox viruses are relatives of smallpox, which has claimed about half a million lives in the last century, although it has been known to humanity for thousands of years. It took scientists centuries and a lot of money and effort to defeat the smallpox virus. Canadian scientists led by virologist David Evans managed to restore the virus using genetic engineering methods for only $100,000. To recreate the virus, scientists used source code that was delivered by regular mail. The team purchased the overlapping DNA fragments, each about 30,000 base pairs long, from Geneart (Regensburg, Germany), which synthesizes DNA commercially. This allowed them to stitch together the virus's 212,000 base pair genome. The virus was introduced into cells already infected with another poxvirus, and they began to produce pieces of the horsepox virus. The virus was then grown, sequenced and characterized, and had a predicted genome sequence. This technology was described back in 2002 in an article in the Proceedings of the National Academy of Sciences, but was used for the first time.

Genie out of the bottle

Horsepox is harmless to humans, is not a serious threat to agriculture, and is considered extinct. However, the success of Evans and his group excited his colleagues. “If this can be done with horsepox, then there is no doubt it can be done with human smallpox,” said virologist Gerd Sutter of the Ludwig-Maximilians-Universität München (LMU) in Germany. WHO has already called the experiment of Canadian scientists “a genie out of the bottle.” According to experts, terrorists or rogue states could take advantage of the potential of biotechnology. The work has sparked renewed debate over whether the last two known living specimens of Variola vera, or smallpox, should be destroyed. In 1979, the World Health Organization officially declared victory over this terrible disease through global vaccination. At the same time, an agreement was reached to destroy all remaining samples of the virus, with the exception of two, which were transferred to secret and highly guarded laboratories in the United States and Russia (to the Centers for Disease Control and Prevention (CDC) in Atlanta and to the Russian Research Institute of Viral Drugs in Moscow). The Russian samples were later transferred to the State Scientific Center of Virology and Biotechnology in Novosibirsk (in the village of Koltsovo). Some scientists believe that studying the virus will help the world prepare for future outbreaks.

Genie or good fairy?

However, Evans’ research is not only the genie out of the bottle: as the scientist himself believes, his experiment also has scientific value. By developing technologies for creating viruses, scientists expect to create effective vaccines against various diseases, learn how to control viruses and, possibly, develop an effective cure for cancer. Tonix, a New York-based pharmaceutical company with which Evans has collaborated, hopes to use the equine virus to improve a vaccine against human smallpox. The current vaccine causes serious side effects in some people. Synthesizing poxviruses, for example, could help develop viruses that can kill tumors, Evans said. The scientist believes that, of course, it is always necessary to remember the “dual purpose” of such research, but at the same time it is necessary to “use the incredible power of this approach” in the fight against dangerous diseases.

Laboratory contaminations: stories of genies released from bottles

Research into infectious diseases has always been dangerous and sometimes ends tragically

• First cases and intra-laboratory infection with typhoid fever were noted in 1885, a report about them was published by K. Kisskalt (1915). In subsequent years, the number of reported cases of infection in laboratories increased, which is explained by the expansion of research with pathogenic microorganisms in many countries around the world. By 1950, the number of documented laboratory infections reached 6,000.
• Plague fort "Emperor Alexander"I". Two outbreaks of plague are widely known at Fort Emperor Alexander I in Kronstadt, which was used as a laboratory for the disease. Despite strict control and all the safety measures taken, in the “plague fort” in 1904, Dr. V.I. Turchinovich-Vyzhnikevich died and fell ill, but paramedic S. Poplavsky was cured, and in 1907, Dr. Manuil Fedorovich Schreiber died and fell ill, but Dr. L.V. Padlevsky was cured
• First disaster in Marburg. An officially registered outbreak of the disease caused by this virus occurred in 1967 in the city of Marburg in Germany at one of the pharmaceutical plants. The animal caretaker died two weeks after he noticed symptoms of a mysterious disease in green monkeys brought to Behring's laboratory from central Africa. The vaccine was grown in the laboratory using kidney cells from these monkeys. Very soon the rest of the laboratory workers also fell ill. Similar cases were noted in Behring's laboratories in Frankfurt and Belgrade, into which green monkeys from the same batch were imported. Twenty-four people working in the laboratory became victims of some unknown disease, and later six nurses who cared for them fell ill. Seven of all those infected died.
• In 1976, an intra-laboratory contamination occurred. Ebola virus in the UK, as a result of an injection with a contaminated needle.
• Laboratory "Vector". In 1988, at the State Research Center for Virology and Biotechnology (“Vector”), Koltsovo, an employee was infected with Marburg fever when punctured by a needle. The sick employee, Nikolai Ustinov, died.
• The second case of intra-laboratory infection at Vector. In 1990, laboratory employee Sergei Vyazunov became infected with the Marburg virus. Presumably, the infection occurred through the eyes. Vyazunov
• Laboratory contamination with Ebola virus in Côte d'Ivoire. A scientist fell ill after performing an autopsy on a wild chimpanzee. The patient was treated in Switzerland and recovered.
• 48 Central Research Institute of the Ministry of Defense of the Russian Federation: In 1996, a laboratory assistant at the Virology Center of the Research Institute of Microbiology of the Ministry of Defense of the Russian Federation in Sergiev Posad died, who became infected with the Ebola virus through negligence by pricking her finger while giving injections to experimental guinea pigs.
• Fort Detrick, Maryland (USA): In 2004, a laboratory employee became infected with the Marburg virus, also due to a needle puncture. In this case, the sick person recovered
• "Vector" again. On May 19, 2004, Antonina Presnyakova, a 46-year-old senior laboratory assistant at the Department of Particularly Dangerous Viral Infections of the Research Institute of Molecular Biology of the State Scientific Center of Virology and Biotechnology, died of Ebola.