RU2431148C1 - Method for producing cultural, polyvalent epidemic haemorrhagic fever diagnosticum for indirect immunofluorescence - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: method for producing EHF Diagnosticum by reproduction of four hantaviruses - Puumala strain PUU-TDK/VERO, Dobrava strain DOB-EAT-VERO, Hantaan strain Ussuri-4590 and Seoul strain SK-515/Georgia-88, in a monolayer passaged cell culture of VERO-line grass monkey kidneys, followed by virus-specific antigen concentration test, polyvalent antigen production, antigen fixation on a slide and UV processing. ^ EFFECT: invention provides a more sensitive diagnostic test, presenting a low-price production process and more effective use of the EHF Diagnosticum. ^ 4 cl, 2 ex, 1 tbl

Description

The present invention relates to the field of virology, in particular to a method for producing test systems intended for specific diagnosis of viral infections, namely, hemorrhagic fever infection with renal syndrome.

Hemorrhagic fever with renal syndrome (HFRS) is an acute, viral disease of zoonotic nature, characterized by systemic lesions of small blood vessels, hemorrhagic diathesis and a peculiar kidney damage, high morbidity, severe course (often fatal) and persistent disability. The absence of specific treatment and prophylaxis agents determines the high social and medical significance of HFRS in Russia and in many countries of the world.

Cases of HFRS disease are recorded in 57 administrative regions of the Russian Federation and make up 8-10 thousand patients annually, as a whole throughout the country.

The causative agents of HFRS - the viruses Puumala, Hantaan, Seoul and Dobrava - belong to the genus Hantavirus (Bunyaviride family), which currently includes more than 30 different hantavirus serotypes or genotypes. These include not only pathogenic hantaviruses for humans, but also viruses with currently undetermined epidemiological significance. The carriers of hantaviruses and the source of human infection are wild mouse-like rodents. To date, the etiological role of hantaviruses in the structure of the incidence of HFRS has been proven: Puumala and Dobrava viruses cause disease in people in the European part of Russia and in Europe, and Hantaan and Seoul viruses in the Russian regions of the Far East and Asian countries. That is, under one name “HFRS”, at least four etiologically independent hantavirus infections are recorded. The dependence of the clinical and epidemiological manifestations of these infections on their etiological condition, expressed by the pathogen belonging to a particular hantavirus genotype or serotype, is becoming increasingly apparent. In addition, it is possible that other known and not yet known hantaviruses can exhibit properties that are virulent for humans under certain conditions and cause new clinical forms of hantavirus infection. In connection with the foregoing, the problem of the incidence of hantavirus infections caused by different viruses is of particular importance for public health in terms of the effective diagnosis, treatment and prevention of these infections. The pronounced immunological differences between the viruses that cause hantavirus infections create difficulties associated with their specific diagnosis. To solve this problem, it is necessary to develop and use universal test systems capable of detecting and identifying not only currently known, but also new, previously unknown viruses that cause hantavirus infections in sick people and animals.

Currently, for the serological diagnosis of HFRS, the company PROGEN, Heidelberg, Germany produces antigenic preparations, which, in contrast to the proposed Diagnosticum HFRS, are monovalent antigens of the 3 hantaviruses Puumala, Hantaan and Seoul. A significant drawback of the use of such antigens in the combined foci of circulation of several hantaviruses at the same time is the need to use three monovalent antigens in parallel for the determination of antibodies in a single serum sample of a patient with HFRS. The 3-stage research procedure leads to a significant increase in both the number of manipulations and the duration of the experiments and, accordingly, increases the cost of a diagnostic examination of patients. In addition, PROGEN does not produce a diagnosticum for the detection of HFRS caused by the Dobrava hantavirus, which is a common causative agent of HFRS in European foci of infection.

The principle of action of the invention is based on the specific immunochemical interaction of hantavirus antigens with antibodies to these viruses. Visualization of antichantavirus antibodies bound to the antigen is carried out using antispecies antibodies labeled with fluorescein isothiocyanate (FITC). The result is recorded using a fluorescence microscope.

The process of obtaining a diagnosticum includes: reproducing hantaviruses of different serotypes in an inoculated culture of VERO cells, collecting cells containing antigen, applying such cells to a glass slide and fixing them with acetone. To obtain a diagnosticum, hantavirus strains are used: Puumala (PUU-TKD / VERO strain), Dobrava (DOB-EAT-VERO strain), Hantaan (Ussuri-4590 strain) and Seoul (SK-515 / Georgia-88 strain), causing HFRS in countries of Europe and Asia.

SUMMARY OF THE INVENTION

The invention relates to a method for producing a diagnosticum for the specific diagnosis of hemorrhagic fever with renal syndrome, which is a multivalent antigenic preparation, by reproducing four hantaviruses: Puumala (PUU-TKD / VERO strain), Dobrava (DOB-EAT-VERO strain), Huntaan (Ussuri strain 4590) and Seoul (strain SK-515 / Georgia-88) in a monolayer transplantable culture of green monkey kidney cells, the VERO line, checking for virus-specific antigen, obtaining a multivalent antigen, fixing the antigen on the subject with tekle and ultraviolet treatment. When reaching 90% or more cells containing the antigen, a mixture of suspensions of cells infected with the viruses of Puumala, Dobrava, Hantaan and Seoul and uninfected VERO cells combined in a ratio of 2: 1: 1: 1: 1 is used.

The invention also relates to a kit for the diagnosis of hemorrhagic fever with renal syndrome, including: a) a polyvalent antigenic preparation obtained by reproducing four Puumala hantaviruses (PUU-TKD / VERO strain), Dobrava (DOB-EAT-VERO strain), Hantaan (Ussuri-strain 4590) and Seoul (strain SK-515 / Georgia-88) in a monolayer transplantable culture of green monkey kidney cells, VERO line, checking for virus-specific antigen, obtaining polyvalent antigen, fixing antigen on a glass slide and processing ultraviolet vym exposure; b) three control samples, which are 2 positive samples - one ampoule with blood serum containing antibodies that interact with the antigens of the hantavirus Hantaan, Dobrava and Seoul, and antibodies that interact with the antigen of the hantavirus Puumala, and 1 negative sample - an ampoule with normal serum that does not contain antibodies to hantaviruses; c) luminescent serum against human globulins (for example, produced by the NF Gamalei Institute), processed to contrast the background with Evans blue solution.

The antigen can be applied to slides of 6 spots in 2 rows for rapid diagnosis of sporadic HFRS incidence, when it is necessary to examine only one large or 7 spots in 3 rows for rapid diagnosis of large outbreaks of HFRS, when it is necessary to examine at the same time a large group of patients.

DETAILED DESCRIPTION OF THE INVENTION

In more detail, obtaining a diagnosticum is as follows. A VERO cell culture is grown in one-liter roller bottles in a MEM Eagle medium with a double set of vitamins and amino acids and 5% of the blood serum of cow fruits. Upon reaching the monolayer (3-4 days), a pool of the virus, previously diluted with Igla 2MEM medium, is added to the bottles to a concentration that ensures a multiplicity of infection of 0.1-0.5 IU / cell with an inoculum volume of 15 ml per 1 liter roller bottle. To adsorb the virus onto the cells, the infected bottles are placed in a roller unit and incubated for 1 hour at a temperature of (37 ± 1) ° C. After the specified time, 100 ml of the medium supporting cell growth is introduced into each bottle and again placed in a roller unit in a thermostatic room with a temperature of (37 ± 1) ° C. The collection of cells containing viral antigens is carried out on 10-11 days. The cells are removed from the glass with a mixture of 0.02% versene and 0.5% trypsin, washed with a 0.85% NaCl solution 5 times, a suspension of infected cells (450-550 thousand cells / ml) is prepared and 0.005 ml applied per slides for immunofluorescence. After drying, the glass is placed for 20 minutes in chilled acetone to fix the cells and inactivate the virus and then dried at room temperature. The resulting antigenic (monovalent, that is, prepared for each individual virus) preparations are examined by indirect immunofluorescence for the content of the viral antigen using control sera of people who have had HFRS, containing antibodies to the viruses of Puumala, Dobrava, Khantaan and Seoul, as well as human serum that does not contain antibodies to huntviruses. In the presence of 90% or more cells containing antigen, suspensions of cells infected with the viruses of Puumala, Dobrava, Hantaan and Seoul, and uninfected cells are combined in a ratio of 2: 1: 1: 1: 1. The resulting cell suspension (polyvalent antigen) is applied at 0.005 ml onto a slide for immunofluorescence in two rows of 6 drops in a row or in 3 rows of 7 drops. Slides are kept at room temperature until the suspensions are completely dry, after which the preparations are fixed in chilled (at a temperature of minus 15 ° C) acetone for 15-20 minutes. After extraction from acetone, the preparations are dried and treated with ultraviolet radiation for 3 hours to completely inactivate the infectious activity of viruses.

To identify specific antibodies to HFRS pathogens, the studied blood serum is applied to preparations with a polyvalent culture antigen in dilutions starting from 1:16 and above. Three controls are used in the experiments: a positive control sample — blood serum containing antibodies that interact with the antigens of the hantaviruses Hantaan, Dobrava, and Seoul; the positive control sample is serum containing antibodies to the Puumala hantavirus, and the negative control sample is normal serum containing no antibodies to the Hantaviruses. The drugs are placed in a humid chamber and kept at 37 ° C for 30 minutes or at 4 ° C for 18-20 hours. After the exposure, the glass is washed 3 times with 3 minutes each time with a 0.85% NaCl solution, rinsed with water and dried at room temperature. After that, luminescent serum is applied to the antigen wells against human globulins (or against the globulins of the animal species whose blood serum is examined) in a working dilution, which is determined in a preliminary experiment. To contrast the background, Evans blue is used in a final concentration of 1: 10000. The preparations are again incubated in a humid chamber at a temperature of 37 ° C for 30 minutes, washed with a 0.85% NaCl solution 3 times for 3 minutes, rinsed with water, dried at room temperature and examined using a luminescent microscope under a water-immersion lens (x60 or x65).

Hantaviruses are characterized by the localization of a fluorescent antigen with a clear granular structure in the cell cytoplasm. Depending on the stage of antigen accumulation, it is possible to observe both small or large granules in the perinuclear zone, as well as clumps of fluorescent material filling the entire cytoplasm of the cell. The bright emerald green granular glow of the hantavirus antigen is clearly visible against a background of gray-brown to dark brown or brick-red staining of the structure of uninfected cells. The same background is noted on antigen with normal serum, used as a control. Serum titer is considered its highest dilution, capable of detecting a specific HFRS virus antigen.

The proposed method allows to obtain diagnostic of HFRS, which has the following advantages:

1) universality, manifested in the fact that a diagnosticum containing antigens of all currently known hantaviruses that cause HFRS on the Eurasian continent can be used with equal efficiency in various endemic regions, regardless of the type of hantaviruses circulating in the territories of these regions.

2) high sensitivity of detecting antibodies from the first days of the clinical manifestations of the disease, which provides an early specific diagnosis of this disease.

The fundamental difference between the proposed “Diagnosticum of hemorrhagic fever with renal syndrome of culture, multivalent for the indirect method of immunofluorescence” from the previously developed Diagnosis of HFRS (Tkachenko EA, Dzagurova TK, etc. A method for obtaining a diagnosticum of hemorrhagic fever with renal syndrome. . 1365918, 1985) and subsequently produced according to FSP 42-0070592404 is that the strains of the Puumala and Dobrava viruses are replaced by more active strains adapted for propagation in the culture of cells VERO label with a high level of intracellular antigen production, which in turn increases the sensitivity of the HFRS Diagnostic by 2-4 times. Active replication of the virus in culture helps to reduce the period of antigen accumulation in cells from 14 to 10-11 days and, accordingly, reduces the material costs of its production (the amount of nutrient medium used to maintain cell activity is reduced, as well as the electricity consumption for the operation of the roller unit and temperature control). The strain of the Puumala virus CG-1820-Ufa, isolated from the bank vole, was replaced by the strain PUU-TKD-VERO, isolated from a patient with HFRS. The strain of the Dobrava virus AR-47 / Sochi-01, isolated from the Caucasian forest mouse, was replaced by the strain DOB-EAT-VERO isolated from a patient with HFRS. In addition, the proposed HFRS Diagnosticum is completed with antigenic preparations with the design of a slide of two types: in addition to the option of applying a hantavirus antigen of 7 spots in three rows, the kit will include glasses on which the antigen is applied in 6 spots in 2 rows. In this way, the number of antigenic preparations in the Diagnosticum kit will increase from 10 to 15. The inclusion of the antigen in the form of 12 spots on the glass in the Diagnosticum kit creates savings and convenience in express diagnostics of the sporadic HFRS incidence when it is necessary to examine only one patient. The introduced changes can increase the sensitivity of the diagnostic test, reduce the cost of production and the effectiveness of the use of Diagnostic drugs HFRS compared to the Diagnostic, which was previously produced under the conditions of FSP 42-0070592404. The strain PUU-TKD / VERO (PUU-TKD / VERO) of the virus "PUMALA" (Puumala, genus Hantavirus, family Bunyaviridae) was deposited on November 24, 2009 under No. 1026 in the State Virus Collection (DI Ivanovsky RAMS Research Institute of Virology ) The strain DOB-EAT / VERO (DOB-TEA / VERO) of the virus "DOBRAVA" (Dobrava, genus Hantavirus, family Bunyaviridae) was deposited on November 24, 2009 under No. 1027 in the State Virus Collection (D.I. Ivanovsky Research Institute of RAMS )

Example 1. Examination of paired blood serum of a patient with suspected HFRS taken on the 2nd and 5th day of illness.

Before use, a glass slide with an antigenic preparation, stored at a temperature not exceeding minus 20 ° C, is dried at room temperature (16-24 ° C).

Twice dilutions of the studied blood serum in a 0.85% NaCl solution are prepared in the wells of the plastic panels and, starting with a 1:16 dilution, one drop (5 μl) is applied to a separate “spot”. 3 control sera in working dilutions are also applied to a separate “spot”. Preparations with applied test and control samples are placed in a humid chamber and incubated at 37 ° C for 30 minutes or at 2-8 ° C for 18 hours. After incubation, the preparations are 3 times 3 minutes in physiological saline, washed in for 1 min with water and dried at room temperature. On each "spot" is applied luminescent serum against human globulins in a working dilution of 1 drop (5 μl). The preparations are incubated in a humid chamber at a temperature of 37 ° C for 30 minutes, after which they are kept in physiological saline for 3 times for 3 minutes, rinsed with water and dried at room temperature.

The experience is taken into account using a LUMAM-2 luminescent microscope or another brand under a water-immersion lens (x60 or x65). First, the wells with control sera are microscopic. Upon detection of a fluorescent antigen with a clear granular structure in the cytoplasm of cells in wells with control positive sera and the absence of luminescence in the control with normal serum, they begin to look at the studied blood sera. Serum No. 1 detects hantavirus antigen in dilutions from 1:16 to 1: 256, which is determined by the characteristic granular emerald green glow. A similar glow is recorded in dilutions from 1:16 to 1: 1024 in wells coated with serum No. 2. Thus, the titer of serum No. 1 - 256; serum No. 2 - 1: 1024.

Example 2. According to the method described above, the blood serum of patients with suspected HFRS was studied using Diagnosticum (1), produced under the terms of FSP 42-0070592404, and Diagnosticum (2), proposed for patenting. The results presented in the table show the benefits of the proposed Diagnosticum for the early diagnosis of HFRS.

Illness day The number of examined sera Detection of antibodies against hantaviruses Diagnosticum-2 Diagnosticum-1 abs. (%) abs. (%) one 8 6 (75%) 2 (25%) 2 13 12 (92%) 9 (69%) 3 25 24 (96%) 20 (80%) four 32 32 (100%) 30 (94%) 5 58 58 (100%) 58 (100%)

Claims (4)

1. A method for obtaining a Diagnosticum for the specific diagnosis of hemorrhagic fever with renal syndrome, which is a multivalent antigenic preparation, by reproducing four hantaviruses: Puumala strain PUU-TKD / VERO, Dobrava strain DOB-EAT-VERO, Hantaan strain Ussuri-4590 and Ce -515 / Georgia-88 in a monolayer transplantable culture of green monkey kidney cells, VERO line, checking for the content of virus-specific antigen and when 90% or more cells containing the antigen are reached, to obtain a multivalent antigen, see Suspensions of cells infected with the indicated viruses and non-infected VERO cells are diluted in a ratio of 2: 1: 1: 1: 1, antigen fixation and ultraviolet irradiation. 2. A kit for the diagnosis of hemorrhagic fever with renal syndrome, including: a) a polyvalent antigenic preparation obtained by reproducing the four Puumala hantaviruses, the PUU-TKD / VERO strain, the Dobrava strain DOB-EAT-VERO, the Hantaan strain Ussuri-4590 and the Seoul strain SK- 515 / Georgia-88, in a monolayer transplantable culture of green monkey kidney cells, VERO line, checking for virus-specific antigen, obtaining polyvalent antigen, fixing antigen on a glass slide and processing with ultraviolet radiation;
b) three control samples, which are 2 positive samples — blood serum, containing antibodies that interact with the Hantavirus antigens Hantaan, Dobrava and Seoul, and antibodies that interact with the Puumala hantavirus antigen, and 1 negative sample — normal serum that does not contain antibodies to hantaviruses control samples packaged in ampoules,
c) luminescent serum against human globulins, processed to contrast the background with Evans blue solution. 3. The kit according to claim 2, characterized in that the antigen is applied to slides of 6 spots in 2 rows. 4. The kit according to claim 2, characterized in that the antigen is applied on a glass slide of 7 spots in 3 rows.