online ISSN:2312-3389 print ISSN:2312-3370 DOI:10.15407/agrisp

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https://doi.org/10.15407/agrisp5.03.019
CULTIVATION OF POTATO LEAFROLL VIRUS (PLRV) IN MAMMALIAN CONTINUOUS CELL LINES
I. V. Volkova, L. M. Reshotko, T. O. Bova, O. O. Dmytruk, S. V. Derevianko

Institute of Agricultural Microbiology and Agroindustrial Manufacture, NAAS 97, Shevchenko Str, Chernihiv, Ukraine, 14027

E-mail: volkova1212@ukr.net, reshotko_lm@ukr.net, tetyanabova@gmail.com, oks.dmytruk@gmail.com, biopreparat@i.ua

Received August 22, 2018 / Received September 05, 2018 / Accepted November 21, 2018
Abstract
Aim. To use the ability of potato leafroll virus (PLRV) to infect and multiply in mammalian continuous cell lines to purify PLRV isolates from the vegetative plant material, and to study the pathogenicity of those isolates for plants (after culturing in mammalian continuous cell line), to investigate morphological, physical-chemical, biological and antigen properties of PLRV isolates from mammalian cells and to study an alternative diagnostic method – the neutralization test in the mammalian continuous cell lines. Methods. The methods of cultivating animal viruses in the mammalian continuous cell line, microscopical biochemical, and serological methods, the method of artifi cial nutrition of aphids are detailed under Material and Methods. Results. It was demonstrated that successful cultivation of PLRV in mammalian continuous cell line allowed obtaining pure virus isolates from potato plants and aphids and preserving them for a long time (over a period of 7 years). The cultivation of PLRV in the mammalian continuous cell line did not impact its pathogenic properties and allowed transmitting the virus to plants. Continuous cells lines of pig embryonic kidney (PEKV), of kidney Syrian hamster (BHK- 21), of testicles of piglets (PTP), of kidneys of the bull (MDBC), and of carcinoma rabbit kidney (RK-13) were found to be sensitive to PLRV, Con tinuous cell lines of human (HeLa, Hep-2 and of African green monkey kidney (Vero) were not infected by the virus. The infectious activity of PLRV in the sensitive continuous cell lines was 20–8.5 lg TCD 50 /ml depending on the cell line. The isolates of PLRV were resistant to lipid- dissolving solvents, multiplied in a pH range from 4.0 till 10.0 and were thermoresistant at 50 oС in the absence of bivalent ions of magnesium, ТIP was in the range of 60–65 oС under our experimental conditions. The optimal temperature for the reproduction of PLRV in the cell culture was c. 24 °С. The use of neutralization test in the mammalian continuous cell line allowed isolation in pure culture and identifi cation of PLRV reliably in a time span of c. 14 days. Conclusions. It was proven that PLRV can be cultivated in the mammalian continuous cell lines of PEKV, ВНК-21, PTV, MDВК and RK-13. It was established that the cultivation of PLRV in these continuous cell lines did not impact its biological, pathogenic, antigenic and physical-chemical properties. The identifi cation of pure cultures of PLRV obtained in mammalian cells can be reliably performed by the use of neutralization reaction.
Key words: phytopathogenic viruse, mammalian cell culturing, neutralization test.
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