Comparison of sensitivity of transplantable cell cultures to caninae coronaviridae

Abstract

With the advent of the cell culture method, the diagnosis of viral infections has risen to a qualitatively higher level. Cultures of cells are widely used in cytogenetic, biochemical, molecular and various laboratory studies. The article presents data on the indication of the field isolate of coronavirus of dogs on the territory of Ukraine, allocated in 2016 from the pathological material of a dead animal with characteristic clinical signs. The work was carried out at the Faculty of Veterinary Medicine of the Zhytomyr National Agroecological University in the virology laboratory. All work on cell cultures was carried out in a specialized mini-box under ultraviolet radiation. To isolate the viral isolate, a fragment of the intestine of the dog was used, after euthanasia, which was ill with coronavirus enteritis. In the experiment, cell cultures were used: kidney hamster (VNK-21), rabbit kidney (RK-13) and kidney pig (SPEV) It was found that for incubation of virus-isolating isolates, the most sensitive are freshly transplanted, after 24 hours of incubation, with at least 70 % and no more than 80 % of the cell monolayer. Control over the number of cells introduced during transplanting was carried out using a chamber with a grill Goryaev. The most optimum was the sown concentration from 1,0×105 to 2,0×105 cl. / ml Cultivation of the crossover lines of cell cultures was carried out using a nutrient medium consisting of medium 199 and DMEM – 90 %, fetal bovine serum – 10 %, and antibiotics. The support medium for the infected cell culture was as nutritious as it was, without serum alone. Cultivations were performed according to generally accepted techniques, evaluating the cytopathogenic effect on the system of four pluses under an inverted microscope, compared with control. Before conducting cultural studies, the monoinfection was confirmed by the ELISA method. It was determined that the cytopathogenic effect of the virus was observed 72 hours after infection, the more intensive process of cell destruction was recorded in the line of cultures of VNK-21 cells, where at 5–6 days almost always stabilized 90–100% cytopathogenic action of the virus, and the titre of infectious activity increased with each new passage of viral material 1.2 lg TCD50/cm, after the third passage rose to 3,7±0,03 lg TCD50/cm, and after the fifth – increased to 4,8±0,04 lg TCD50/cm.