RU2205652C1 - Method for isolating arboviruses, tick- borne encephalitis, in paticular - Google Patents

Abstract

FIELD: medicine, virology. SUBSTANCE: the resent innovation deals with virological diagnostics of arboviral infections, tick-borne encepalitis, in particular. One should collect infection-suspicious material, its preparation for trial and isolation of arboviruses due to combined infectioning in 1-3-d-aged experimental animals both into brain and subcutaneously with suspension of this material. In animals with manifestation form of infection one should sample brain to prepare suspension and conduct the next 3-4-fold passages into brain of experimental animals with this suspension to accumulate neurotropic viruses being in sufficient quantity for identification. In animals with inapparatus form of infection one should additionally study neuro- and immunocompetent organs by well- known immunoenzymatic method on 7th, 14th, 21st d. Samples for immunocompetent organs at the presence of positive test for antigen of arboviruses should be selected to prepare suspension, then one should carry out 3-5-fold passages due to infecting experimental animals with this suspension to store immunotropic viruses at sufficient quantity for identification. EFFECT: increased number of diagnostic methods in case of arboviral infection. 2 tbl

Description

 The invention relates to medicine, namely to virology, and can be used for virological diagnosis of arbovirus infections, in particular tick-borne encephalitis.

 A known method of isolation of arboviruses (RF patent 1836422 A3, MKI 5 C 12 N 7/00, 04/23/91), including blood sampling in patients and in persons with a tick bite, isolation of leukocyte blood mass, release from inhibitors and the subsequent 2-fold passage to the monolayer cell culture SPEV.

 The disadvantage of this method is the low percentage of isolation of the virus due to the use of cell culture SPEV, which does not secrete non-virulent virus strains.

 The closest in technical essence is the method of isolation of arboviruses (Results of science and technology. Virology series, v.25, M., 1991, pp. 22-27), including the collection of suspicious material for infection, preparing it for research and isolation of highly virulent strains of arboviruses by intracerebral and subcutaneous infection of 1-3-day-old experimental animals with a 10% suspension of the test material, followed by selection of diseased animals, taking the brain from them and 3-4-fold passages to accumulate neurotropic viruses in the brain in sufficient quantity.

 The disadvantage of this method is the low percentage of virus isolation due to the isolation of only highly virulent neurotropic strains.

 The objective of the invention is to increase the qualitative and quantitative indicators of virological diagnostics by isolating not only highly virulent, but also weakly virulent strains of arboviruses, in particular tick-borne encephalitis.

The problem is solved as follows:
- collection of material suspicious of contamination,
- preparing him for research,
- virus isolation due to combined infection in the brain and under the skin of 1-3-day-old experimental animals,
- selection of animals with a manifest form of infection,
- the fence of their brain and the subsequent 3-5-fold passage for the accumulation of neurotropic viruses in the brain in sufficient quantity to identify it,
- a study of the brain and spleen, animals with an inapparent form of infection in a known manner in the enzyme immunoassay (ELISA) on the 7th, 14th, 21st day,
- sampling with the presence of a positive test for arbovirus antigen,
- Conducting subsequent 3-5-fold passages for the accumulation of immunotropic viruses in sufficient quantities for their identification.

The essence of the method is as follows. As the test material can be used:
- direct carriers of arboviruses - ticks, mosquitoes, etc .;
- blood of persons with a “bite” of insects and ticks;
- leukocyte mass of blood of people and animals;
- blood and organs of sick people and animals with suspected infection with arboviruses.

 The test material is prepared in accordance with generally accepted methods. To isolate arboviruses, 1-3 days old white mice or other experimental animals are infected, in particular, newborn hamsters, rat suckers or guinea pigs, combined by intracerebral and subcutaneous methods. Sample material that can pass through the brains of white mice and cause a clinic of the disease in them has neurovirulent properties. The brain of these mice is mistaken for a typical neurotropic strain.

 In animals infected with samples that do not cause disease, the brain and spleen are examined in a known manner in an enzyme-linked immunosorbent assay (ELISA). 2-day-old white mice are infected with samples that give a positive result in ELISA, combined with intracerebral and subcutaneous methods. Infected animals are monitored for 2–3 weeks. In these mice, the brain and spleen in ELISA are examined on the 7th, 14th and 21st day of observation. The brain of clinically healthy mice with negative results of ELISA does not expose further passages. 10% suspension is prepared from positive samples of the spleen, passages are performed on 2-day-old white mice. The possibility of multiple passaging of the isolate only through immunocompetent organs indicates the isolation of an immunotropic strain of TBE virus. Identification of virus strains is carried out in MFA and rtga.

 Samples of leukocyte masses of blood from individuals with a tick bite were examined using a culture of SPEV cells (655 samples) and the proposed method (92 samples). In the first group, 53 neurotropic strains of TBE virus were isolated, which amounted to 8.0%. In the second group, 48 strains were isolated, which amounted to 52.2%. Of these, 14 neurotropic strains (15.2%) and 34 immunotropic strains (36.9%) were isolated. The difference between the indicators between the first and second groups was 6.5 times. This indicates that, using previously used methods, immunotropic strains of viruses that make up the bulk of the viral population, which usually causes erased and inapparent forms of the disease, were not isolated.

An example of the accumulation of a specific antigen in the spleen of mice infected with 10 viscerotropic isolates was the experience of titration of a 10% suspension of immunocompetent organs (10 ¹ -10 ¹⁰ ) followed by infection of the cell culture SPEV. After 5 days, slides were prepared from these cells using the standard MFA technique. For each strain, virus accumulation indices were obtained on the 7th and 14th days using the indirect method of fluorescent antibodies using immune serum from a patient with CE in a dilution of 1: 20 and conjugated serum against human globulins. In the table. 1 presents data confirming the presence in the immunocompetent organs, in particular in the spleen, of specific antigen (in ELISA) and virus CE (in MPA). Its titers reached 10 ^-7 .

 Another example, testifying in favor of the antigenicity of the TBE virus of the same 10 isolates obtained by the proposed method, are the results of their identification. To conduct RGA and RTGA, a protamine sulfate antigen was prepared from a virus-containing immunocompetent organ. When rtga was used, mouse immune sera were used against regional Pischelkin strains (isolated from the brain of a deceased patient) and 828 (isolated from human blood with an inapparent form of TBE), as well as commercial immune serum to the standard TBE virus.

 From the data table. 2 shows that all strains had hemagglutinating properties in titers 64-128 AE. In rtga, higher specific bonds to strain 828 were detected. Immune sera to Pischelkin strain responded weaker. This indicates the antigenic proximity of the studied isolates to strain 828, which is also isolated from the blood of a patient with an inapparent form of EC.

An example of a specific application. The blood of individuals with a tick bite (92 people) is taken from a vein in an amount of 5 ml to a heparin solution at a rate of 25 PIECES per 1 ml, set in a thermostat at a temperature of 37 ^o C for 30 minutes, a leukocyte suspension is taken and a leukocyte mass is obtained. It infects 92 families of 6 two-day-old white mice in the brain and subcutaneously. Observe them for 14 days. Of the total number of infected families, 14 showed symptoms of the disease (immobility, paralysis, death). This suggests that 15.2% of the examined individuals with a tick bite are carriers of neurotropic viruses.

 From the remaining murine families with an inapparent form of the course of infection, the brain and spleen of sucker mice were taken on day 14, samples were prepared for ELISA testing and analysis was performed according to the instructions for using the enzyme-linked immunosorbent assay for detecting CE virus antigen (instructions attached). At the same time, in none of these samples was the CE virus antigen found in the brain. Samples of spleens with negative results are not further investigated.

 The remaining samples from spleens with the presence of CE antigen were passaged on suckers of white mice. Moreover, every 7, 14, 21st day, spleen studies were performed in ELISA. Of all the studied samples, 34 immunotropic strains were isolated, which amounted to 36.9%. The results obtained indicate that these individuals with a tick bite are infected with immunotropic strains of TBE virus. Such cases were not previously diagnosed, people were left without specific treatment.

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

Claims (1)

 A method for isolating arboviruses, in particular tick-borne encephalitis, including collecting suspicious material for infection, preparing it for research and isolating arbovirus by combined infection of 1-3-day-old experimental animals into the brain and under the skin with a suspension of this material, selection of animals with a manifest form of infection, sampling they have a brain, preparation of a suspension from it, and subsequent 3-4-fold passages to the brain of experimental animals with this suspension for the accumulation of neurotropic viruses in sufficient quantities for identification, exc characterized in that in animals with an inapparent form of infection, neuro- and immunocompetent organs are additionally examined by the known enzyme immunoassay on days 7, 14, 21, and brain samples with a negative result are destroyed, and samples of immunocompetent organs with a positive test for arbovirus antigen are taken , a suspension is prepared from them and 3-5-fold passages are carried out by infection of experimental animals with this suspension to accumulate immunotropic viruses in sufficient quantity for identification.

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