Varianytsia V. V., Vysekantsev I. P.

MICROBIOLOGY

Scentific article

We studied various compositions of preserving media for long-term storage at the temperatures of –20 and –80о С of the rabies virus standard strain CVS, which is used for control in manufacture of rabies biologicals. The virus was accumulated in cell culture of BHK-21 (clone 13) in the growth medium based on DMEM culture medium supplemented with 0.1% (v / v) BSA. Sucrose, glycerol, DMSO, gelatin, sodium alginate and peptone in various concentrations by volume were added to the viral suspension. The resulting samples were placed in freezers for storage at –20 and –80о С. The cooling rates to these temperatures were not controlled. Assessment of the samples safety was carried out by determining the infectious activity before storage, after a week and after 1, 3, 6 and 12 months of storage (observation period). To determine the infectious activity of the virus, the titration method in cell culture of BHK-21 was used. The cell monolayer was stained with specific rabies monoclonal antibodies labeled with fluorescein isothiocyanate and counted using a laboratory microscope with a fluorescence module. The virus infectious activity value was calculated using Spearman-Karber’ method and expressed in decimal logarithm of 50% cell culture infectious dose (lg CCID50). As the initial control, the infectious activity of the virus in the growth medium before manipulations (hereinafter, the initial infectious activity of the virus) was used. It was amounted to (6,01 ± 0,16) lg CCID50. It was established that after storage at the temperature of –80ºС for a year (observation period), the indicators of the infectious activity of the rabies virus CVS strain exceed the virus safety values at the temperature of –20о С. During storage at these temperatures for a year (observation period), the highest safety indices of the rabies virus CVS strain were provided by preserving media based on growth medium (DMEM with 0.1% BSA) without additives and with the addition of sucrose, glycerol, DMSO and gelatin. In preserving media with sucrose, glycerol and DMSO, the dynamics of the virus preservation during storage was the same. The decrease in infectious activity occurred until the first 3 months and between 6 and 12 months of storage. After 12 months at –20о С, 66% of the initial infectious activity of the virus remained in the growth medium without additives. In samples with 5–10% sucrose, 3% gelatin, and 5–7.5% glycerol, the infectious activity of the virus was significantly higher than in the growth medium without additives. It was 73–74, 71, and 69% of the initial control, respectively. Along with this, at –80ºС after 12 months, the growth medium without additives ensured safety of 80% of the initial activity of the virus. Significantly higher safety indices were provided only by samples with 5–10% sucrose. It was 82–86% of the initial virus activity. The minimum indicators of the virus infectious activity at both temperatures were in media with sodium alginate and peptone. Taking into account that only sucrose-supplemented medium showed significant efficacy compared to the medium without additives at –80о С, the protective medium based on the virus growth medium with the addition of sucrose was recommended for long-term storage of the rabies virus standard strain CVS in manufacture of rabies biologicals (5–7.5%) and storage temperature of –80о С.

rabies virus, cell culture, long-term storage, protective media, virus preservation, virus infectious activity

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«Bulletin of problems biology and medicine» Issue 4 Part 1 (153), 2019 year, 205-211 pages, index UDK 578.824.11:57.043:578.22:578.72:615.371

10.29254/2077-4214-2019-4-1-153-205-211