Journal of microbiology, epidemiology and immunobiologyJournal of microbiology, epidemiology and immunobiology0372-93112686-7613Central Research Institute for Epidemiology40910.36233/0372-9311-2018-3-18-25DETERMINATION OF THE RABIES VIRUS-INFECTED VERO LINE CELL PORTION BY FLOW CYTOMETRYKravtsovA. L.Saratovfake@neicon.ruGeneralovS. V.Saratovfake@neicon.ruKozhevnikovV. A.Saratovfake@neicon.ruGavrilovaYu. K.Saratovfake@neicon.ruAbramovaE. G.Saratovfake@neicon.ruKochkinA. V.Saratovfake@neicon.ruNikiphorovA. K.Saratovfake@neicon.ruRussian Research Institute for Plague Control «Microb»2507201895318252408201924082019Copyright © 2018, Kravtsov A.L., Generalov S.V., Kozhevnikov V.A., Gavrilova Y.K., Abramova E.G., Kochkin A.V., Nikiphorov A.K.2018<p><strong>Aim.</strong> Experimental substantiation of possibility to determine the rabies virus-infected Vero cell line portion in culture by flow cytometry (FC) and FITC labeled diagnostic anti-rabies immunoglobulin (DAI), manufactured in Russia. <strong>Materials and methods</strong>. Fixation and permeabilization of Vero cells, infected by rabies virus strain «Moscow 3253», was carried out by means of Суtofix/Cytoperm reagent (BD Biosciences, USA) according the Vengatesan D. et al. method (2006) and then intracellular rabies antigen was stained by DAI. Percentage of infected cells was determined by FC in 24, 48 and 72 h and as well in 48 h when the cell cultures were infected with tenfold dilutions of virus-containing fluid from 10-1 to 10-8. <strong>Results</strong>. There was a significant increase in the percentage of infected cells on average from 30 to 70% in time interval from 24 to 48 h. With 1000-fold dilution of viral-containing fluid the FC method detected the 6,9±0,21% of infected cells in Vero cultures (P0,001, n=3). <strong>Conclusion</strong>. FC has proved to be a fast, sensitive and reliable method for determining the relative number of virus- infected Vero cells in cultures. The drug DAI had a sufficient activity for its effective use in the automated version of MFA based on the FC method. The use of FC is possible at various stages of anti-rabies drug production and control, and is also promising in terms of further improving of the rabies diagnosis.</p>rabies virusflow cytometryVero cell cultureвирус бешенствапроточная цитометриякультура клеток Vero[1. Ашмарин И.П., Воробьёв А.А. Статистические методы в микробиологических исследованиях. Л., 1962.][2. ГОСТ 26075-2013 Животные. Методы лабораторной диагностики бешенства. М., Стандартинформ, 2014.][3. Генералов С.В., Абрамова Е.Г., Матвеева Ж.В., Жулидов И.М., Лобовикова О.А., Свинцов Р.А., Комиссаров А.В., Киреев М.Н., Никифоров А.К. Культуральный антиген в технологии получения антирабического иммуноглобулина из сыворотки крови лошади. Проблемы особо опасных инфекций. 2012, 4 (114): 65-68.][4. Кравцов А.Л. Совершенствование диагностики инфекционных болезней, связанное с использованием технологии импульсной проточной цитометрии. Биотехнология. 2013, 3: 8-23.][5. Сибиряк С.В., Хайдуков С.В., Зурочка А.В. Оценка апоптоза в иммунологических исследованиях. В: Вопросы современной проточной цитометрии. Клиническое применение. Челябинск, 2008.][6. Alvarez-Barrientos A., Arroyo J., Canon A.R. et al. Applications of flow cytometry to clinical microbiology. Clin. Microbiol. Rev. 2000, 13 (2): 167-195.][7. Bordignon J., Ferreira S., Caporale G. et al. Flow cytometry assay for intracellular rabies virus detection. J. Virol. Methods. 2002, 105 (1): 181-186.][8. Halfmann P., Kim J. H., Eblhara H. et al. Generation of biologically contained Ebola viruses. PNAS. 2008, 105 (4): 1129-1133.][9. Hanners N.W., Eitson J.L., Usui N. Western Zika virus in human fetal neural progenitors persists long term with partial cytopathic and limited immunogenic effects. Cell Reports. 2016, 15 (11): 2315-2374.][10. McSharry J.J. Detection and quantitation of human immunodeficiency virus-infected peripheral blood mononuclear cells by flow cytometry. J. Clin. Microbiol. 1990, 28: 724-733.][11. Rudd R. J., Trimarchi C.V. Comparison of sensitivity of BHK21 cells and murine neuroblastoma cells in the isolation of a street strain rabies virus. J. Clin. Microdiol. 1987, 25: 1456-1458.][12. Sydow F., Santiago M.A., Neves-Souza P.C. et al. Comparison of Dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen. Memento Instituo Oswaldo Cruz. 2000, 5:483-489.][13. Steen H.B., Boye E., Starsted K. et al. Applications of flow cytometry on bacteria: Cell cycle kinetics, drug effects and antibody binding. Cytometry, 1982, 2 (4): 249-256.][14. Vengatesan D., Raj G.D., Raja A. et al. Detection of rabies virus antigen or antibody using flow cytometry. Cytometry. 2006, 70B (5): 335-343.][15. Woldeheiwet Z. Clinical laboratory advances in the detection of rabies virus. Clin. Chem. Acta. 2005, 351: 49-63.]