CULTIVATION OF POTATO LEAFROLL VIRUS (PLRV) IN MAMMALIAN CONTINUOUS CELL LINES

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 (BHK21), 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 TCD50/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 o in the absence of bivalent ions of magnesium, IP was in the range of 60–65 o under our experimental conditions. The optimal temperature for the reproduction of PLRV in the cell culture was c. 24 ° . 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, -21, PTV, MD 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.


INTRODUCTION
Potato leafroll virus (PLRV), a representative of the Polerovirus genus, Luteoviridae family, is a single stranded RNA-virus, of which the sequence of nucleotides is completely determined [1]. The virions of PLRV are isometric, 23 to 25 nm in size and its pure RNA has ratio of A 260 /A 280 of 1.78. The virus remains infectious when diluted up to 10 -4 in sap from infected plants, and in sap j after 5-10 days at 2 ºC, the virus temperature inactivation point (TIP) in crude sap when heated for 10 minutes is 70 to 80 °C [2].
PLRV often occurs in potato plants along with other viruses, and to our knowledge there are no data in the scienti¿ c literature regarding the method of obtaining its isolates from plants with a mixed infection into pure culture. The above mentioned characteristics of PLRV complicate its detection and identi¿ cation and hinders the development of fully speci¿ c diagnostic methods. The routine diagnosis of PLRV involves the application of immunological methods. These include the now outdated double immunodiffusion in gel, enzyme-linked immunosorbent assay (ELISA), the immunochromatographic assay detection of the virus (lateral À ow immunochromatographic assay), Luminex × MAP® Technology -a novel method of analyzing different antigens, which combines immunological, À uorescent methods and laser technologies and allows the simultaneous detection of several viruses in plant material [4,5]. Detecting virus by the method of reverse transcription polymerase chain reaction (RT-PCR) is ranked the ¿ rst among the genetic molecular methods for the detection and identi¿ cation of RNA viruses [6,7].
Multiplication of PLRV is done using potato plants or indicator plants such as Datura stramonium L. and Physalis angulata L., onto which the virus is transmitted from a stock of known infected plants using grafting or aphids. After the multiplication period, which takes about 30 days, the leaves of infected plants are used to obtain PLRV preparations.
Cultivation and multiplication of PLRV can also be performed in protoplasts of tobacco or potato mesophyll. It envisages obtaining a culture of protoplasts, infecting it with the puri¿ ed and concentrated preparation of PLRV using poly-L-ornithine and incubating protoplasts at permanent illumination and temperature [8].
In 2009 we detected a phenomenon of productive potato leafroll virus infection in mammalian continuous cell lines. The procedure to obtain an infected mammalian cell is, in short, as follows: To isolate PLRV vegetative parts of potato plants with the of PLRV (reference strain 879 from the Institute's phytopathogenic viruse collection) are used, in which the presence of the virus was con¿ rmed by electron microscopy (EM) and ELISA. Plants samples are clari¿ ed with chloroform and placed in culture vials with cultures cells of pigs embryonic kidney (PEKV). The inoculated culture vials with the continuous cell line are incubated in a thermostat at 37 °C for appearance of virus cytopathic effect (CPE). Degenerative changes of PEKV showed appearance and gradual increase in number of single rounded cells with enhanced refractivity. The infected cells are moved away from glass. Areas without cells increase in size up to complete destruction of cell monolayer. On average the detection of CPE due to PLRV infection takes 5 to 15 days.
After adaptation of PLRV to the continuous cell line via four passages, the new system «virus-cell» is used for the accumulation of viral biomass. The EM of virus preparations after ultracentrifugation demonstrated aggregates of whole isometric particles and absence of empty capsids. After the manifestation of a CPE, the virus was identi¿ ed using the reverse transcription polymerase chain reaction (RT-PCR) with the primer pair sense-5 ' -CgCgCTAACAgAgTTCAgCC and antisense-5 ' -gCAATgggggTCCAACTCAT, that should yield a 336 bp product, corresponding to the RNA of PLRV [6].
The aims of our present work were: 1) to isolate PLRV isolates, circulating in Ukraine's territory (Chernihiv's region); 2) to investigate physical-chemical, biological and antigen properties of PLRV isolates; 3) to study the pathogenicity of PLRV for plants, after it was multiplied and cultured in mammalian cells; 4) to investigate an alternative method of PLRV identi¿ cation, namely the neutralization test in the continuous mammalian cell line.

MATERIALS AND METHODS
Potato material (18 plants with symptoms of leafroll and three aphids samples (per sample 5-10 Myzus persicae aphids, directly collected from plants) selected in the ¿ elds of Chernihiv's region, and the reference strain of PLRV, kept in the virology laboratory of Institute in potato clone G 879, were used to conduct the virological research.
Plants and aphids samples were clari¿ ed with chloroform (1 : 4) and placed in culture vials with cultures cells of pigs embryonic kidney (PEKV). The multiplication of virus was done in these cell cultures which were grown in growth medium 199 («BioTestLab», Kyiv, Ukraine) at the culture vials. Prior to introducing the virus, the nutrient medium was drained, and the cell monolayer was rinsed twice with a 0.9 % NaCl solution and Hank's solution. An amount of 0.5 ml of the virus suspension was introduced per vial and placed in an incubator at 37 qɋ to contact for one hour. After the contact the cell monolayer was washed with Hank's solution, the solution was removed and the monolayer of cells further incubated at 37 °C up to a stage of 75 % CPE, subsequently the cultures were stored deep frozen at -18 qɋ in triplicate. Pure populations of PLRV were obtained via three times cloning of viruses by the method of limiting dilutions with subsequent threetimes cloning by Melnick's method of plaque technique [9]. Virus accumulated in the previous dilution, was used for each subsequent passage.
The isolation, concentration and puri¿ cation of PLRV from the infected mammalian cell lines was performed as follows: Two parts of virus suspension were added to one part of chloroform with subsequent hand shaken homogenization for 30 min. Then the mixture was kept for 12-18 h at 4 qɋ, and subsequently centrifuged for 20 min at 1500 g. To the supernatant ammonium sulfate was added until saturation of 50 % and the suspension kept at 4 qɋ for 1 h. The precipitate was removed by centrifugation at 5-10 qɋ for 20 min at 1500 g, resuspended in 0.9 % NaCl, and dialyzed against the same solution for 16-18 h. Control of purity of virus preparations was done spectrophotometrically using a spectrophotometer (SF-46, Leningrad, USSR) by measuring the ratio of Ⱥ 260 /Ⱥ 280 , and by electron-microscopy using a transmission electron microscope (UEMB-100V, Sumy, USSR).
For the hyperimmunization of rabbits puri¿ ed and concentrated virus-containing suspensions of PLRV were used. To the prepared antigen, used for subcutaneous administration, adjuvant Montanide ISA 25 (SEPPIC, France) was added according to the manufacturer's instructions. The immunization of rabbits was done according to the scheme, developed by us. Immunization of rabbits is carried out through ¿ vetime administration of concentrated virus antigen in turns subcutaneously with adjuvant Montanide ISA 25 in an amount of 1 mg of protein/2 ml intracutaneously without adjuvant along spinal column to 8-10 points in amount of 1 mg of protein/1 ml with an interval between introductions of 7, 3, 4, 3 days respectively.
We did not determine genetic markers of PLRV when cultured in mammalian cells, but we analysed resistance of virus isolates to lipid-dissolving solvents [11], the sensitivity to certain media at different pH values, thermo-resistance [12] and thermal stability in order to properly identify the isolates as PLRV.
The stability of isolates of PLRV at various pH values of the solution (0,1 M Na 2 CO 3 , 0.1 M Na 3 C 6 H 5 O 7 and their combinations) was studied in a BHK-21 cell line. For this purpose, the virus isolates LT, LB, LS at a dose of 1 lgTCID 50 were kept in the solution with a pH value of 2.0, 3.0, 4.0, 7.2, 10.0 and 11.0 respectively at room temperature for 10 minutes. After that, in all samples, the pH was adjusted to 7.2. The sensitivity of the virus to the acid and alkaline pH values was determined by the difference between the titre of the virus isolates as compared to that of the PLRV control strain (pH 7.2). The experiment was performed in three replications.
Thermo-resistance was studied via determining the infectious activity of the three PLRV isolates in cell culture after heating them at 50 qɋ for 1 h in the presence of 1 Ɇ of a MgCl 2 solution and without it. The experiment was performed in three replications.
The impact of temperature on the functioning of the PLRV-animal cell system was studied in the BHK-21 cell line, infected with isolate LT at a dose of 1 lgT-CID 50 . The incubation was conducted at 2, 10, 24 and 37 °ɋ. The degeneration changes in the monolayer, the time of their occurrence and infectious activity of the virus were noted. The experiment was performed in three replications.
The antigenic af¿ nity between virus isolates was established in the cross reaction of virus neutralization using a stable dose of the virus (100 TCID 50 ) and antiserum to PLRV isolates (20 neutralizing doses) and 10-times dilutions of the virus with a stable dose of antiserum (20 neutralizing doses). Normal rabbit serum in 1 : 5 dilution and blood serum, obtained from the culture of BHK-21 cells, were used for the control. The antigenic af¿ nity was calculated by the formula [9]:  To study the pathogenicity of PLRV for plants, after it was multiplied and cultured in mammalian cells, the method of Rochow [13] was used to transmit the virus to plants using aphids (Myzus persicae Sulz.), which were fed through an arti¿ cial membrane (Fig. 1). VOLKOVA et al. To study an alternative method of PLRV identi¿ cation, namely the neutralization test, two samples of potato leaves (on 1 sheet with 10 plants) of variety Suvenir Chernihivsky with mild mosaic symptoms and variety Tyras without any disease symptoms. Samples were selected in the hydroponic greenhouse of Scien-ti¿ c Production Association "Chernihivelitkartopli a".
A pure culture of PLRV reference strain obtained via the PEKV cell line, was used to obtain the reference antiserum.
The preparations, obtained from both samples of potato leaves, were used to infect the PEKV cell line and incubated in the thermostat at 37 °ɋ till the manifestation of the features of cytopathic effect of the virus, which appeared on the third day.
The samples were typed in the serological neutralization test in the PEKV cell line. Prior to that, the titer of the obtained reference antiserum was determined to be 1:256 in the neutralization test.
To identify the isolated viruses in the neutralization test, 0.5 ml, containing 100 TCID 50 of virus antigen in 0.1 ml, was mixed with 0.5 ml of reference serum to PLRV, containing 20 neutralizing doses per milliliter. After incubating the mixture at 37 °ɋ for 60-90 min, 0.2 ml was introduced into a test tube to which 0.8 ml of the supporting medium was added before. Control and experiment test tubes were further incubated at 37 °ɋ and the results registered on the 4 th and 7 th day.

RESULTS AND DISCUSSION
Three isolates (LB, LS and LT) of PLRV were obtained from 18 symptomatic potato and 3 aphid samples after being cultured in the PEKV cell line (Table 1). The LB, LS, LT isolates of PLRV were kept at -18 °ɋ in a domestic freezer and did not lose their infectivity over a period of 7 years and maintained in a pure culture by passaging them in PEKV and BHK-21 cell cultures.
Virus isolates, extracted from plant samples and aphids, were found to be resistant to lipid-dissolving solvents (ether, chloroform) which demonstrated the absence of a lipid-containing envelope in them.
As seen from the results, presented in Table 2, at different pH values the infectious titer almost did not change in the range from 4 to 10.0, decreased by 4 lg TCD 50 /ml at ɪɇ 3.0, and at ɪɇ 2.0 there was complete inactivation.
The study of thermal stability of extracted PLRV isolates established that the thermal TIP in the culture of PEKV after heating for 10 min was in the range from 60 ºɋ to 65 ºɋ. It may be that the difference in TIP values was conditioned by different chemical composition of the media, where the viruses were placed while heated, although we did not test this supposition.
While studying the thermal resistance, the infectivity of the three PLRV isolates, heated without 1 Ɇ of the solution of MgCl 2 , did not change compared to the unheated control, and in the presence of 1 Ɇ of MgCl 2 , it decreased by 2-3.5 lg TCID 50 /ml, which demonstrated the absence of stabilization of virions with bivalent cations of magnesium (Table 3).
The results of the determination of temperature impact are presented in Table 4.
There was a noted considerable slowing down of PLRV replication at temperatures below 24 qɋ. For in-stance, after 7 days of incubation at 2 and 10 °ɋ, there were no degenerative changes observed in the monolayer, and the infectious titer of PLRV decreased to 2 lg TCID 50 /ml. At 24 qɋ on the 3 rd and 4 th day after the inoculation, single round cells were observed. The destruction of 75 % of the monolayer was observed after 7 days. On the 3 rd and 4 th day, the infectious activity was 4.5-5.5 lg TCID 50 /ml respectively, on the 7 th day it was 6.5 lg TCID 50 /ml. At the optimal temperature of incubation, i.e. 37 qɋ, the cytopathic action of the virus developed already after 24 h, and the infectious titer of the virus was 7.5 lg TCID 50 /ml.
The antigenic af¿ nity of the three PRLV isolates from three different sources, namely aphids, leaves and tubers of potato, was 100 % i.e. they were serologically identical.
PLRV had a cytopathic effect on the PTP cell line 12-24 h after inoculation, for the PEKV and the BHK-21 cell lines it was 24-48 h after inoculation. The cytopathic effect was visible as symptoms of degeneration in the cell culture in the form of single rounded cells with increased refractivity, in increasing numbers. The affected cells came loose from glass, and clear empty spots appeared in the monolayer, increasing in size up to complete and visible destruction of cell groups. After 48-96 h, destructive changes were also observed in the cultures of the RK-13 and MDBK cell lines, here the cells also formed symplasts.
No degenerate changes were detected in the cultures of HeLa, Hep-2 and Vero cells after consecutive passaging, PLRV did not multiply in these cell lines.
Twenty-four days after the 10 M. persicae aphids per PLRV isolate had been feeding for 24 h and were killed by insecticides there was a noted manifestation of interveinal chlorotic zones on old and young leaves in P. angulata plants and a delay in growth of D. stramonium plants, which indicated successful transmission of PLRV with the preservation of virus pathogenicity after long-term cultivation in mammalian cell culture.
We also studied the possibility of using the mammalian cell culture for PLRV diagnostics in plant samples.
The neutralization of viruses with the reference serum of rabbit blood demonstrated PLRV infection in potato leaves of varieties Suvenir chernihivsky and Tyras. The virological analysis with the mammalian cell culture takes c. 10 days. Thus, it was established that the application of neutralization test -a method, previously not applicable for identi¿ cation of phytoviruses -in the continuous mammalian cell culture allows isolation and identi¿ cation of PLRV in potato plants reliably.

CONCLUSIONS
It was demonstrated that the cultivation of PLRV in some mammalian continuous cell lines allowed isolation of pure virus isolates from potato plants and aphids and preserving them for a long time (up to 7 years).
The investigated three isolates of PLRV were resistant to lipid-dissolving solvents, were multiplying in media with pH values from 4.0 till 10.0 and were thermo-resistant at 50 ºɋ in the absence of bivalent ions of magnesium; TIP was in the range of 60-65 ºɋ under our experimental cond itions. The optimal temperature for the replication of PLRV was c.24 °ɋ.
It was established that continuous cell line lines of PEKV, BHK-21, PTP, MDBK and RK-13 were sensitive to PLRV. The human cell lines HeLa and Hep-2, and a primate cell line (Vero) were not infected by the virus. The infectivity of PLRV in the sensitive cell cultures was 2.0-8.5 lg TCID 50 /ml depending on the cell culture.
The cultivation of PLRV in continuous mammalian cell lines did not impact its pathogenic and other physic-chemical properties and allowed transmitting the virus to plants.