The way to select arboviruses, particularly tick-borne encephalitis

 

The invention relates to medicine, namely to Virology, and can be used for virological diagnosis of arboviral infections, particularly tick-borne encephalitis. The invention consists in collecting suspected to be infected material, its preparation research and selection arboviruses by combined infection of 1-3-day-old experimental animals in the brain and under the skin of a suspension of this material. In animals with the first form of infection produce fence brain, made from his suspension and spend the next 3-4-multiple passages in the brain of experimental animals of this suspension for accumulation of neurotropic viruses in sufficient number for identification. In animals with inapparent form of infection additionally investigate neuro - and immunocompetent organs known by ELISA on days 7, 14, 21 days. Samples of immunocompetent organs with the presence of a positive test for antigen arboviruses select and prepare the suspension, spend 3-5-fold passages by infecting experimental animals this suspension for accumulation of immunotropic viruses sufficient for identification. The technical result - expanding Arsenal of diagnostically is to Virology, and can be used for virological diagnosis of arboviral infections, particularly tick-borne encephalitis.

There is a method of allocating arboviruses (RF patent 1836422 A3, MKI 5 12 N 7/00, from 23.04.91), including blood sampling in patients and in persons with a tick bite, the allocation of leukocyte mass of blood, the release of inhibitors and subsequent 2-fold passage in monolayer cell culture spew.

The disadvantage of this method is the low percentage of isolation of the virus through the use of cell culture spew, which are not allocated avirulent strains of the virus.

The closest in technical essence is a way of separating arboviruses (Results of science and technology. A series of Virology, T. 25, M, 1991, S. 22-27), including the collection is suspected to be infected material, prepare it for the research and selection of highly virulent strains of arboviruses by intracerebral and subcutaneous infection 1-3-day-old experimental animals 10% suspension of investigated material with subsequent selection of diseased animals, fence their brain and 3-4-fold passages for accumulation in the brain of neurotropic viruses in sufficient quantity.

The disadvantage of this method is the low percentage of isolation of the virus C is improving qualitative and quantitative indicators of the virological diagnosis by highlighting not only highly virulent, but slaboperemennykh strains arboviruses, particularly tick-borne encephalitis.

The problem is solved as follows: - collection of suspected contamination of the material, its preparation for the survey - virus isolation due to combined infection in the brain and under the skin of 1-3-day-old experimental animals, the selection of animals with the first form of infection - fence their brain and subsequent 3-5-fold passage for accumulation in the brain of neurotropic viruses sufficient for its identification, the study of the brain and spleen of animals with inapparent form of infection known manner in the enzyme-linked immunosorbent assay (ELISA) for 7, 14, 21 days, - sampling with the presence of a positive test for antigen arboviruses, - follow-3-5-fold passages for the accumulation of immunotropic viruses in sufficient number to identify them.

The method consists in the following. As the study material can be used: - direct media arboviruses - ticks, mosquitoes, and others;
- the blood of persons with "bite" insects and mites;
- leukocytic mass blood of people and animals;
- the blood and the bodies of sick people and animals with suspected infection with arboviruses.

Studies the expression of 1-3-day-old white mice or other experimental animals, in particular newborn hamsters, suckling rats or Guinea pigs, combined intracerebral and subcutaneous ways. Material samples can passivates through the brain of mice, and cause the clinic disease, has neurovirulence properties. The brain of these mice mistaken for typical neurotropic strain.

In animals infected samples, which do not cause disease, brain and spleen examine the known way in the enzyme-linked immunosorbent assay (ELISA). Samples that gave a positive result in ELISA, combined intracerebral and subcutaneous ways infect 2-day white mice. For infected animals are observed within 2-3 weeks. In these mice, examined the brain and spleen in IFA on days 7, 14 and 21 days of observation. The marrow of healthy mice with negative ELISA results further passages do not expose. Of the positive samples of the spleen prepare a 10% suspension, conduct passages on 2-day-old white mice. The possibility of repeated passage isolate only through immunocompetent organs indicates the allocation of immunotropic strains of TBE virus. Identification of virus strains is carried out in the IAF, rtha.

Sample leukocyte mass of blood from persons with bite cuppa isolated 53 neurotropic strains of TBE virus, that amounted to 8.0%. In the second group isolated 48 strains, which amounted to 52.2 per cent. One is marked 14 neurotropic strains (15.2%) and 34 immunotropic strains (36,9%). The differences between the first and second groups was 6.5 times. This suggests that using a previously used methods were not allocated immunotropic strains of viruses that constitute the bulk of viral populations, the caller typically erased and inapparent form of the disease.

An example of the accumulation of specific antigen in the spleen of mice infected with 10 viscerotropic isolates, was the experience of the titration of a 10% suspension of immune organs (101-1010with subsequent contamination of the cell culture spew. From these cells after 5 days were prepared slides, according to the usual method IFA. For each strain the indicators of the accumulation of the virus for 7 and 14 days using the indirect method of fluorescent antibodies using immune serum of the patient TBE in a dilution of 1: 20 and conjugated serum against globulin person. In table. 1 presents data confirming the presence of immunocompetent organs, particularly the spleen, specific antigen (ELISA) and TBE virus (in the abilities to TBE virus these 10 isolates, obtained by the proposed method are the results of their identification. For DSA, rtga prepared prodominantly antigen from vaccinated immunocompetent organ. When setting rtha used mouse immune serum to regional strains Pixelskin (isolated from brain dead patient) and 828 (isolated from human blood with inapparent form EC), as well as commercial immune serum to the standard TBE virus.

From the data table. 2 shows that all strains had hemagglutination properties in the credits 64-128 AE. In rtga revealed a higher specific due to the strain 828. Immune serum to the strain Pixelskin reacted weaker. This indicates antigenic vicinity of the studied isolates for strain 828, which is also isolated from the blood of a patient with inapparent form EC.

An example of a specific application. The blood of persons with a tick bite (92 people) taken from a vein in an amount of 5 ml heparin solution at the rate of 25 UNITS per 1 ml, defend in thermostat at a temperature of 37oC for 30 minutes, taken leukocyte suspension and get leukocyte mass. It infects the brain and subcutaneous 92 family of 6 two-day white mice. Watching them within 14 days. From the total the RIT about that 15.2% of the surveyed persons with tick bite are carriers of neurotropic viruses.

Of the remaining families with mouse inapparent form of current infection at 14 days taken marrow and spleen of mice-suckers, prepared samples for research in ELISA and analyzed according to the instructions for use of test kits ELISA for antigen detection of TBE virus (instructions attached). However, none of these samples the TBE virus antigen in the brain is not detected. Sample spleens with negative results, further studies are not exposed.

The remaining samples from spleens with the presence of antigen EC was passively on the suckling of white mice. While every 7, 14, 21 days conducted research spleens in ELISA. From all investigated samples selected 34 immunotropic strains that was 36.9%. The results obtained indicate that these individuals infected tick bite immunotropic strains of TBE virus. Such cases had not been previously diagnosed, people remained without specific treatment.

Thus, the proposed method can improve the qualitative and quantitative indicators of the virological diagnosis through the use of immunocompetent organs.

 

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