Study of reproduction cycles and yield dynamics of the classical swine fever virus in PK-15 cultured cells using a fluorescent probes technique

The authors have elaborated some methodological approaches necessary to solve certain biotechnological problems. This approaches permit to study the classical swine fever virus reproduction and accumulation in a swine established cell line, PK-15, the virus propagation being accompanied with no cytopathic effect The reliable and promising fluorescent probe technique needing no previous virus adaptation and multiplication in permissive cells is successfully confirmed to be useful for such purposes.

Introduction.The classical swine fever (CSF) caused by the homonymous RNA-containing virus (CSFV), a member of Pestivirus genus (Flaviviridae family), is still among the most dangerous swine infections.The widest investigations concerning recombinant vaccine construction are now carrying out.However, such vaccines are not yet completely studied.In some countries belonging to the former Soviet Union several anti-CSFV vaccines have been elaborated and are now produced containing vaccine virus strains.To cons truct any up-to-date antiviral vaccines aimed for the CSF prevention, a biotechnological problem of the large scale CSFV cultivation followed by a high virus yield is to be successfully solved.The solving of this problem, however, is almost impossible without full information concerning reproductive cycles of both vaccine and virulent CSFV strains; such information is to be obtained using virus-sensitive cell culture infection.
The in vitro CSFV reproduction has been pre viously investigated with a fluorescent antibody ap proach [1 ], the virus reproduction having been regis tered according to fluorescent foci appearance on the cell monolayer surface; a reproduction curve presen ted in the paper [1 ] gives, however, no possibility to understand clearly the dynamics of the CSFV accu mulation in infected cells.Some biological properties of the CSFV including also its in vitro multiplication accompanied by no cytopathic effect (CPE), its inti mate association with cell membranes, and usual low virus yields make difficult any investigation con cerning both CSFV reproduction cycles and its accu mulation dynamics.The CSFV as well as the bovine viral diarrhea virus (BVDV) and the border disease virus (BDV) of sheep belong to Pestiviruses, the CSFV possessing a one-sided antigenic relation with the BDV (anti-BVDV antibodies are able to neutralize CSFV) [2].It is already known that almost all the batches of untreated bovine sera do contain the CSFV neutralizing anti-BVDV antibodies, the CSFV accu mulation level during its in vitro cultivation being markedly decreased [3 J.
The goal of this work was to study the CSFV reproductive cycles and its accumulation dynamics in an established monolayer cell culture, PK-15 (of swine kidney origin).To realize such an investigation we were to take into consideration all the data mentioned above.
That is why a fluorescent probe technique (FPT) has been used permitting to study in vitro virus reproduction even without any detectable CPE 14-8 ].Besides, in our investigation a bovine serum batch has been taken for virus-producing cell cultivation freed previously from antibody fraction by the polyethylenglycol (PEG) treatment [91; the absence of anti-BDV antibodies permits to increase the CSFV titers following this pathogen in vitro cultivation [101.
Materials and Methods.Virus.This study was made using two virulent CSFV strains -Alfort (re ceived from the National Veterinary Research In stitute in Pulawy, Poland) and Shi-min' (a kindly gift of the Epizootology Laboratory of our Institute) as well as a vaccine strain LK-K (received from the Vaccine Preparations Laboratory of our Institute) adapted previously to the PK-15 cell line.
Cell culture.The cells of the PK-15 line were grown forming a monolayer in the MEM-Eagle me dium supplemented by hemohydrolysate and 10 % RPMI as well as by PEG-treated bovine serum (5-7,5 %) (the untreated serum, batch 4, had been purchased from the Konotop slaughter-house, Uk raine) .
Cell infection and cultivation.In our experiments the PK-15 monolayer cultures grown in penicillin vials (PVs) were washed three times by a supporting serum-free medium and then infected by the CSFV strains listed above; the infecting virus doses are given in the Table .The virus adsorption was made during 1 h at 37 °С.Free virus particles present in the medium were washed out (three times), and the supporting medium containing the PEG-treated serum (2 %) was added into each PV.Three first virusinfected PVs (for each strain) were frozen at once, the rest of them were incubated then in a thermostat (37 °С) and three PVs for each strain were randomly taken from the thermostat in 3, 5, 7, 19, 24, 48, 72, 96, and 120 h following the beginning of incubation.All the PVs were thrice frozen-thawed and then three PVs culture media (for each strain and each time of cultivation) were united and clarified by centrifu gat і on, the supernatant fluids were then used for titration.Control PV cultures were incubated without any strain infection.
Virus titration.The summary virus strain yields (intra-and extracellular) were determined by titra tion in PK-15 suspensions pretreated by a fluorescent probe to realize the FPT as described early [4][5][6][7][8].The cell photometry was made using a «LUMAM 1-2» fluorescent microscope supplemented by a special equipment unit, FMEL-1 (LOMO, Sankt-Petersburg).An average value of the fluorescence intensity obser ved in probe-treated intact cells (25 or more) is taken as a unit of fluorescence.The fluorescence К value is used to simplify the calculation of results.К is a ratio of the fluorescence intensity of tested (infected) cells (F { ) to the fluorescence intensity of control ones (F c ): The К values above or equal to 1.5 indicate the presence of the virus tested in a sample investigated.The values below 1.5 prove the absence of the virus tested or its neutralization by specific (antiviral) antibodies in homologous systems containing both virus particles and immune serum.The diagnostical values of the К criterion have been originally deter mined to correspond to the P < 0.01, i. e. the statistical significance of results obtained is 99.9 %.
The experiments described above were realized three times with each CSFV strain.
Results and Discussion, Our results confirm no CPE takes place during any CSFV strain reproduction in vitro.The data concerning three virus strains (Alfort, Shi-min\ LK-K) titrations during 120 h of their cultivation are given in the Table and in the Figure .Our investigations concerning extracellular and intracellular virus titers following virus absorption show the infectious virus of any strain studied to be bound by infected cells, no infectious virus particles