Cultivation of potato leafroll virus (PLRV) in mammalian continuous cell lines

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 arti¿ 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 lipiddissolving solvents, multiplied in a pH range from 4.0 till 10.0 and were thermoresistant at 50 ºɋ in the absence of bivalent ions of magnesium, ɌIP was in the range of 60-65 ºɋ under our experimental conditions. The optimal temperature for the reproduction of PLRV in the cell culture was c. 24 °ɋ. The use of neutralization test in the mammalian continuous cell line allowed isolation in pure culture and identi¿ 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, ȼɇɄ-21, PTV, MDȼɄ 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 identi¿ cation of pure cultures of PLRV obtained in mammalian cells can be reliably performed by the use of neutralization reaction.

Improvement of system diagnostic and specific prophylax of teschovirus encephalomyelitis (Teschen disease)

“…Виділення та культивування вірусів проводили в перещеплюваних культурах клітин нирки ембріону свині (СНЕВ) та нирки новонародженого сірійського хом'яка (ВНК-21). Гіперімунні сироватки крові одержували на кролях за схемою розробленою нами [7].…”

Improvement of diagnostic tools and specific prophylaxis of Teshen disease

“…As a result of studying the antigenic properties of the strain Dniprovsky-34 in RN with reference strains, it was found that the strain indicated antigenically related to PTV-1 Talfan, Teschen 199, Tirol, C 2372 at 100% and does not enter into serological responses with reference strains of other serotypes of PTV, PSV and EV-G. High antigenic affinity to the epizootic and reference strains of PTV-1 and high immunogenicity led to the selection of the strain Dniprovsky-34 as a production. According to the results of commission tests, the Dniprovsky-34 strain was deposited with the Depository of the State Scientific-Control Institute for Biotechnology and strains of microorganisms under the registration number 486 and the patent of Ukraine was obtained for a utility model [8].…”