Reproduction and apoptosis of EBV- latent infected cells under influence a TRIZ-created antiviral drugs

Abstract

Introduction. We had worked persistently and in 1997, the first model of such a structure based on oligopeptides of horse albumin was revealed. We named this project and called Albuvir (combined from the word Albumin and the word virus) and applied in veterinary medicine, where viral infections represent more than 95% of all pathologies. In 2000 an application was filed for the first modification of this drug. Subsequently, for the production and sale of this product, registration certificates of the State Committees in Veterinary Medicine of Eastern European countries were obtained.  In 2010, we signed a license agreement to transfer of limited rights in the field of veterinary medicine for the production and sale of Albuvir in Eastern Europe. The drug was produced commercially and was successfully used to cure hundreds of thousands of chickens, rabbits, sheeps, cows, pigs, pigeons, fish (industrial - sturgeon), cats, dogs, geese, ducks, and other types of farm birds and animals. Thus, the task of this work was to study the ability of Albuvir to inhibit EBV reproduction and induce apoptosis of latently infected cells in the culture. Materials and methods. Cell culture. The inhibitory effect of Albuvir on EBV was studied in the following cell cultures: Raji - cell culture of the B-phenotype of Burkitt's lymphoma. Namalwa is a B-phenotype cell culture from Burkitt's lymphoma that lacks the EBV genome. B95-8 is a marmoset monkey B-lymphocyte cell line containing the complete EBV genome, producing complete viral particles. To control cell viability, a 0.4% trypan blue dye (Sigma, United States) was used. EBV was isolated from a lymphoblastoid culture of B95-8 cells (B-lymphocytes of monkeys-marmazetka), which produces this virus, according to the method of Walls, Crawford. Investigated substances. Albuvir (N. 1) - dynamic acylated acidic peptides - antagonists of signal peptides of nuclear (nucleolar) localization and polyribosomes. Lysine tris-succinamide (N. 2). Acyclovir was used as a reference drug (references data)  MTT test. The cytotoxic concentration was determined in the Raji lymphoblastoid cell system using the colorimetric MTT method. This method is widely used to determine the CC50 of potential drugs in the study of the cytopathic action of viruses in vitro. Raji cells were plated into 96 well culture plates, 100 μl per well, 25 μl MTT (final concentration 5 μg / ml) was added and incubated for 3 h at 37 ° C in an atmosphere of 5% CO₂. After incubation, cells were washed with PBS and resuspended in 96% ethanol to dissolve formazan. The results were analyzed spectrophotometrically on a Dynatech reader (Sweden) at a wavelength of 540 nm. Polymerase chain reaction. The degree of influence of drugs on EBV reproduction was determined using PCR test systems "AmpliSens-100-R". A fragment encoding the VCA protein of the virus with a size of 290 nucleotide sequences was the chosen genome region of the Epstein-Barr virus. The control was cells that, after infection with the virus, were incubated in the growth medium without the addition of the substances that were studied. We determined the percentage of inhibition of the level of accumulation of viral DNA in the samples treated with the test substances in relation to the control sample, the value of which was taken as 100%. Results and discussion. Effect of the dynamic antiviral drug Albuvir on reproduction and apoptosis latently infected with the Epstein-Barr virus cells in vitro. Modern approaches to the treatment of herpes infection, in particular the Epstein-Barr virus (EBV), include the use of ethyotropic drugs, as well as sensitizing therapy. The range of drugs active against EBV remains very limited to ganciclovir and acyclovir. The search for new compounds active against EBV remains relevant. The aim of this study was to find out the anti-EBV activity of the drug Albuvir in Raji, B95-8, Namalwa lymphoblastoid cells. The cytotoxicity index (CC50) was determined, which was 3000 ug/mL, and the concentration of the drug that inhibits the reproduction of the virus (EC₅₀) was 0.1 ug/mL. The ability of the drug Albuvir to inhibit the reproduction of the Epstein-Barr virus in all studied cell cultures was revealed. When the economic efficiency of creating static drugs in accordance with the S-shaped curve decreases, the need arises for a transition to a supersystem, namely, the creation of dynamic drugs systems. It has been proven that the drug Albuvir is able to inhibit the reproduction of the Epstein-Barr virus in Raji, B95-8 and Namalwa cell cultures. It is determined that the drug has a high activity in the culture of Raji cells (SI 8400), respectively, the drug is promising enough to develop as a treatment for EBV-associated diseases.

DOI: 10.5281/zenodo.4038923