RU1799598C - Marker for estimating attenuation of poliomyelitis viruses - Google Patents

Abstract

The invention relates to the field of virology. The goal is to use propolis extract as a marker. SUMMARY OF THE INVENTION: The dynamics of suppressing the reproduction of vaccine variants of poliomyelitis virus in cell culture when propolis extract is present in the medium is ahead of a similar process for wild variants. Positive effect: expanding the range of markers.

Description

The invention relates to virology, and in particular to a means for assessing the degree of attenuation of viruses. The use of propolis and propolis preparations in medicine for therapeutic purposes is known.

The purpose of the invention is to expand the range of markers used to assess the degree of attenuation of viruses by identifying new additional propolis properties previously unknown.

Propolis extract is used as a marker to achieve the goal.

The marker is based on the fact that the dynamics of suppressing the reproduction of vaccine variants of poliomyelitis virus in cell culture, in the presence of propolis extract in the supporting medium, is significantly ahead of the same process for poliomyelitis variants. If the difference in the intensities of suppressing the reproduction of poliomyelitis virus isolates in cell culture in the presence and absence of propolis (0.8%) in the support medium is 2.0 Ig CPDbO / ml, then they are wild. At 3.0 Ig, CPDbO / ml - to vaccine, and if the difference between these indicators is in the range of 2.0-3.0 Ig CPD50 / M / 1, then they relate to

in between. The technology for determining the marker we offer is simple to implement, not associated with significant material costs, as it eliminates the use of expensive reagent materials.

The use of the proposed marker for assessing the degree of attenuation of viruses has shown that the information obtained with this trait fully correlates with the antigenic trait. In all experiments, a complete coincidence of the antigenic (known) marker with the new marker proposed by us was noted.

Example. We assessed the degree of attenuation of 60 poliomyelitis virus isolates isolated from poliomyelitis patients from samples of water bodies (wastewater, open reservoir water), as well as standard polio virus strains, wild and vaccine, according to a number of known, as well as according to our proposed marker.

Production of propolis extract (marker).

Use an 8.0% aqueous propolis extract collected in apiaries by beekeepers of the Pobeda collective farm of the Rybinsk district of the USSR, Moldova, which is obtained as follows

VI

Oh Oh

ate about

00

Methodology: 20.0 g of propolis is weighed, triturated in a mortar, then this ground mass is poured into a glass flask, to which up to 100.0 ml of distilled water is added. The extraction is carried out at 42.0 + 0.1 ° C for 4 days, periodically shaking (for 10 minutes the contents of the flask are 5-6 times a day. The resulting solution is sterilized through an asbestos filter and stored in a volume of 20-25 ml in dark bottles, well corked at a temperature of + 40 ° C. After stabilization, a yellow-light (lemon) color is established. When using, the aqueous extract of propolis is diluted so that the final concentration in the supporting medium of the cells is 0.8 ± 0.1% .

The preparation of the transplantable HEP-2 culture is carried out as follows.

Before sowing the culture, a monolayer is reviewed and evaluated. A culture with a continuous monolayer of cells having a morphology typical of a given line, with distinct boundaries between cells, is selected. Cells are removed from the glass of the mattresses with trypsin-versene. For this, a mixture of equal volumes of a 0.25% trypsin solution and versene solution is prepared, and it is heated in a water bath to 30-35 ° C. At the beginning, the nutrient medium is poured from the mattress, the cell layer is washed 3 times with a phosphate-buffered solution (pH 7.5) that does not contain calcium and magnesium ions. Then, a monolayer of cell culture is poured with a thermal mixture (trypsinversen) so that all areas are covered with liquid. In mattresses of 100, 250 and 1000 ml, 10, 15 and 30 ml, respectively, are added. The mattresses are shaken and placed in a thermostat (37 ° C) for 15-20 minutes until the cells are completely separated from the glass surface. Large conglomerates of cells disperse, pipetiry several times the cell suspension. The cell suspension was centrifuged for 10 minutes at 2000 rpm, and the trypsin-versene mixture was removed. Cells precipitated by centrifugation are resuspended in a small volume of growth medium, the number of cells is counted in a counting chamber Goreva. Cell suspension is monitored for bacterial sterility, diluted with nutrient medium so that 1.0 ml contains 60 thousand cells. The medium used for growing cells was Eagle Medium, Medium 199. Eagle-MEM supplemented with calf bovine serum 10% and antibiotics. Transplantable

HEP-2 culture is poured into 1.0 ml tubes. As the support medium, the same nutrient media as mentioned above without the addition of serum are used.

The renewal of a continuous monolayer of new generation cells is observed after 5-6 days. Titration of polio virus isolates of one or another immunological type in order to study the degree of their attenuation,

as well as standard vaccine and wild strains of the polio virus, they are carried out in the Hep-2 cell culture by the final dilution method according to the cytopathogenic effect and are expressed in Ig (CPDzo / ml). For titration

Five strains of poliomyelitis virus are initially prepared for breeding, usually tenfold from 10 to. 9.0 ml of sterile saline or Hanks solution is added to all tubes. Then, 1.0 ml of virus-containing material is added to the first tube and a dilution (1:10) is obtained. Using a new pipette, the contents of the first tube are thoroughly mixed and 1.0 ml is transferred into the second

5 test tube, receiving a dilution (1: 100), etc. up to 108.

Then, an aqueous extract of propolis (8.0%) in a volume of 0.5 ml is combined with an equal volume of virus-containing material

0 of each dilution. A mixture of propolis extract and virus was incubated for 1 h at 37 ° C. Before infection, the monolayer of the HEp-2 culture mixture (virus + propolis extract) is produced 3 times

5 washing the cell culture from serum with Hanks solution, pH 7.4 or Earle's solution - pH 7.6. For each ten-fold dilution of virus-containing material, 4 tubes with cell culture are used. A mixture of propolis extract and virus in a volume of 0.2 ml is added to test tubes with cell culture, in which a maintained medium in a volume of 0.8 ml is pre-filled. The tubes are placed in a thermostat in special trays in a horizontal position. The condition of the infected cultures is periodically checked under a light microscope for 7 days. The result of the cytopathic effect of the CPD virus

0 is considered positive if at least half of the cells in the culture undergo specific degeneration.

The experience is accompanied by the following control:

5 a). Reproduction control of standard poliomyelitis virus strains: poliomyelitis virus is titrated in the HEp-2 cell culture in the absence and presence of propolis extract in the cell support medium at a concentration (0.8%):

b) Cell culture control: use an intact HEp-2 cell culture in the absence and presence of propolis extract in a support medium (0.8%). The titer (50.0% of the effective dose) is calculated according to the Reed-Meng method and the punched method using special tables (Guidelines for the sanitary-virological control of environmental objects, 1982, M., 75, p. Drozdova S.G. and doctor of medical sciences Kazantseva V.A.).

The results of assessing the dynamics of suppressing the intensity of reproduction of poliomyelitis virus strains in HEp-2 cell culture in the presence and absence of an aqueous extract of propolis in a support medium are taken into account only if the following controls are observed:

1. A monolayer of non-infected HEP-2 culture (in the presence and absence of propolis extract in a supporting medium at a final concentration of 0.8%) should be maintained until the end of the experiment, i.e. up to the seventh day, inclusive after infection (control of cell culture).

2. The difference in the intensity of reproduction in the culture of HEP-2 cells in the absence

and the presence of propolis extract in a support medium for attenuated standard polio virus strains is more than 103 ° CPDbo / ml, whereas for wild standard virus strains

polio this indicator is

 10 ° CPD50 / ml.

Claims (1)

The formula is also invented. The use of a 0.8% propolis solution as a marker for assessing the attenuation of poliomyelitis viruses.