Delputte, Peter

Lefebvre, David

Misinzo, Gerald

Nauwynck, Hans

English

Ghent University Academic Bibliography

Arch Virol (2008) 153: 337–342DOI 10.1007/s00705-007-1092-0Printed in The NetherlandsBrief ReportIncreased yield of porcine circovirus-2 by a combined treatmentof PK-15 cells with interferon-gamma and inhibitors of endosomal-lysosomal system acidificationG. Misinzo, P. L. Delputte, D. J. Lefebvre, H. J. NauwynckLaboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, BelgiumReceived 14 June 2007; Accepted 29 September 2007; Published online 17 December 2007# Springer-Verlag 2007SummaryTreatment of porcine kidney (PK-15) cells witheither interferon-gamma (IFN-g) or endosomal-lysosomal system acidification inhibitors increasesreplication of porcine circovirus type 2 (PCV2). Inthe present study, the effect of a combination ofthese treatments on the number of infected cellsand virus yield was tested. The number of PCV2(Stoon-1010)-infected PK-15 cells increased incells treated with ammonium chloride (445 39%increase), IFN-g (446 8%), ammonium chlorideþIFN-g (1721 283%), chloroquine diphosphate(1037 121%), chloroquine diphosphateþ IFN-g(2199 255%), monensin (950 178%) andmonensinþ IFN-g (1948 60%). Combined IFN-g and endosomal-lysosomal system acidificationinhibitors increased PCV2 yield by up to 50 timescompared to untreated PK-15. This augmented vi-rus replication in PK-15 cells may be helpful in theproduction of PCV2 vaccines.Porcine circovirus type 2 (PCV2) is a memberof the genus Circovirus, family Circoviridae [30].PCV2 infections are mainly associated with a mul-tifactorial disease in pigs, postweaning multisyste-mic wasting syndrome (PMWS) [1, 7, 9, 12, 17,27]. Apart from PMWS, PCV2 has also been as-sociated with various disease syndromes in pigsincluding porcine dermatitis and nephropathy syn-drome [3, 5, 19, 21, 31, 35, 37], congenital tremors[6, 14], reproductive failure [16, 18, 21, 28, 29,33], exudative epidermitis [38], porcine respiratorydisease complex [13], proliferative and necrotizingpneumonia [11] and enteritis [4, 15]. In vitro, PCV2replicates in porcine kidney epithelial cells [2, 20,25, 32], and PCV2 isolation and production isroutinely performed using porcine kidney (PK-15)and swine kidney (SK) epithelial cell lines [2, 32].When PCV2 replication kinetics were studied in aPK-15 epithelial cell line that was inoculated withthe prototype PCV2 strain Stoon-1010 [9], 1.8% ofcells were infected with a maximum yield of 3.9log10 TCID50=ml in culture supernatant [22]. Thislow yield slows down the production of PCV2vaccines. Therefore, increasing the replication ofPCV2 in PK-15 cells will allow vaccine productionto be scaled up, making the production more effi-cient and profitable.Correspondence: Hans J. Nauwynck, Laboratory of Virol-ogy, Faculty of Veterinary Medicine, Ghent University,Salisburylaan 133, 9820 Merelbeke, Belgiume-mail: hans.nauwynck@UGent.beEarlier studies have shown that the number ofPCV2-infected cells can be enhanced by treatmentof PK-15 cells either with interferon-gamma (IFN-g)[23] or inhibitors of endosomal-lysosomal systemacidification such as ammonium chloride (NH4Cl),chloroquine diphosphate (CQ) and monensin [25].In the study by Meerts et al. [23], the effect of IFN-g treatment of PK-15 cells on the PCV2 yield wasalso investigated, and it was shown that IFN-gtreatment increased the yield of progeny PCV2 by20 times. Up till now, neither the effect of treatmentof PK-15 cells with the endosomal-lysosomal sys-tem acidification inhibitors alone nor the effect oftreatment of PK-15 cells with the combination ofIFN-g and inhibitors of the endosomal-lysosomalsystem acidification on PCV2 yield have been in-vestigated. It was the aim of the present study toinvestigate the combined effect of treating PK-15cells with IFN-g and inhibitors of endosomal-lyso-somal system acidification on PCV2 infection andPCV2 yield.To investigate the effect of IFN-g and inhibitorsof the endosomal-lysosomal system acidificationon the number of PCV2-infected cells, 2104PK-15 cells that were free of porcine circoviruseswere seeded per well of a 96-well cell culture plate(Nunc, Roskilde, Denmark). Cells were maintainedat 37 C in a humidified 5% CO2 incubator in cul-ture medium containing 10% fetal bovine serum(FBS; Invitrogen, Grand Island, U.S.A.), 0.3 mg=mlL-glutamine (BDH Chemicals Ltd., Poole, England),1% nonessential amino acids (100 ; Invitrogen),1 mM sodium pyruvate (Invitrogen), 100 U=mlpenicillin, 0.1 mg=ml streptomycin and 0.1 mg=mlkanamycin in RPMI-1640 (Invitrogen). Recombi-nant porcine IFN-g (R & D systems, Abingdon,U.K.) was dissolved and stored as previously de-scribed [23]. Cell culture medium with or without500 U=ml IFN-g was added to PK-15 cells at6 h post-seeding. At 24 h post-seeding, cells werewashed and inoculated with prototype PCV2 strainStoon-1010 (5.3 log10 TCID50, 20th passage) at aFig. 1. Effect of IFN-g and in-hibitors of endosomal-lysosomalsystem acidification on PCV2 in-fection of PK-15 cells. Representa-tive light microscopic images ofPK-15 cells treated with or withoutIFN-g and=or CQ after performingan IPMA staining to identifyPCV2-infected cells. Bar: 200mm338 G. Misinzo et al.multiplicity of infection (moi) of 0.3 at 37 C. After1 h, the viral inoculum was washed off and cellswere incubated in cell culture medium with orwithout 25 mM, 125 mM or 6mM of NH4Cl, CQor monensin, respectively. At 24 h post-inoculation(hpi), cell culture medium with or without the en-dosomal-lysosomal system inhibitors was replacedwith fresh cell culture medium without inhibitorsof endosomal-lysosomal system acidification. Cellswere fixed with methanol at 20 C for 10 min af-ter the first cycle of PCV2 replication at 36 hpi [22].PCV2-infected cells were identified by an immuno-peroxidase monolayer assay (IPMA) using porcinepolyclonal anti-PCV2 antibodies [34] and horserad-ish peroxidase-conjugated polyclonal rabbit anti-swine immunoglobulins (DakoCytomation, Glostrup,Denmark). Substrate was added to stain-infectedcells, which were then counted by examinationunder a light microscope (Olympus Optical Co.,Hamburg, Germany). The number of infected cellsper well in untreated PK-15 cells was used as areference, and all results were expressed as relativepercentages to this reference. All experiments wereperformed three times with each condition in a sin-gle experiment in quadruplicate.Treatment of PK-15 cells either with IFN-g orinhibitors of endosomal-lysosomal system acidifi-cation increased the number of PCV2-infected cells(Figs. 1 and 2), in agreement with previous studies[23, 25]. Combined treatment of PK-15 cells withIFN-g and inhibitors of endosomal-lysosomal sys-tem acidification also increased the number ofPCV2-infected cells. The percentages of PCV2-infected PK-15 cells at 36 hpi in untreated and treat-ed PK-15 cells are shown in Table 1. From theseresults, it can be concluded that inhibiting endo-somal-lysosomal system acidification gives an ad-ditional enhancement of PCV2 infection in PK-15cells treated with IFN-g. This additional effect waspossible because IFN-g and inhibitors of endoso-Fig. 2. The effect of IFN-g and inhibitors of endosomal-lysosomal system acidification on the number of PCV2-infected PK-15 cells. PK-15 cells were pre-treated with(filled bars) or without (empty bars) IFN-g before they wereinoculated with PCV2. After PCV2 inoculation, cells wereincubated with or without inhibitors of endosomal-lysosom-al system acidification for 24 h. Cells were fixed at 36 hpiand PCV2-infected cells were counted following IPMAstaining. The number of PCV2-infected cells in untreatedcell cultures was taken as reference, and all values are re-lative percentages of this referenceTable 1. Total PCV2 yield in untreated PK-15 cells and PK-15 cells treated with IFN-g, inhibitors of endosomal-lysosomalsystem acidification, or a combination of IFN-g and inhibitors of endosomal-lysosomal system acidificationLysosomotropicagentIFN-g (U=ml) % of PCV2-infectedcells at 36 hpiPCV2 titre log10 (TCID50 per 105 cells)1 hpi 24 hpi 48 hpi 72 hpiControl 0 0.38 0.03 1.9 1.5 2.9 3.1Control 500 1.71 0.12 2.0 2.1 3.4 3.9NH4Cl 0 1.70 0.06 2.1 1.9 3.0 3.8NH4Cl 500 6.67 1.57 1.6 3.0 4.0 4.8CQ 0 3.97 0.31 1.4 1.8 3.3 4.3CQ 500 8.47 1.23 1.6 2.3 4.0 4.6Monensin 0 3.61 0.39 1.9 2.3 3.9 3.6Monensin 500 7.52 1.17 2.0 2.8 4.6 4.8Enhancement of PCV2 production in PK-15 cells 339mal-lysosomal system acidification are known toaffect different stages of PCV2 infection. Previousstudies have shown that treatment of cells withIFN-g increases PCV2 infection by increasing in-ternalization of PCV2 particles into the cell [23],while treatment of cells with inhibitors of the en-dosomal-lysosomal system acidification increasesPCV2 infection by increasing disassembly of inter-nalized PCV2 particles within the cell [25].For virus yield assays, 2105 cells were seededper well of a 24-well cell culture plate (Nunc).Cells were pre-treated with or without 500 U=mlIFN-g at 6 h post-seeding. At 24 h post-seeding,cells were washed and inoculated with the proto-type PCV2 strain Stoon-1010 (5.3 log10 TCID50,20th passage) at an moi of 0.3 for 1 h at 37 C.The viral inoculum was washed off, and cells werefurther incubated in culture medium with or with-out 25 mM NH4Cl, 125 mM CQ or 6mM monensinfor 24 h. Then, the culture medium with or with-out endosomal-lysosomal system acidification in-hibitors was replaced with fresh culture mediumwithout inhibitors of endosomal-lysosomal systemacidification. All inhibitors of endosomal-lysosom-al system acidification were removed at 24 hpi be-cause one of the inhibitors, monensin, is a classicalexocytosis blocker [8, 10, 26, 36]. At 1, 24, 48 and72 hpi, the cell culture supernatant was collected.Cells were subjected to three freeze-thaw cycles.Total virus yield (intra- and extracellular virus titres)was determined on PCV2-negative PK-15 cellsby inoculation of a tenfold dilution series on PK-15 cells in quadruplicate, as previously described[24]. After 36 h of cultivation at 37 C in the pres-ence of 5% CO2, cells were fixed in methanol at20 C for 10 min. Viral antigens were detectedusing an IPMA as described above, and PCV2 titreswere expressed as log10 TCID50 per 105 cells.Treatment of PK-15 cells either with IFN-g orinhibitors of endosomal-lysosomal system acidifi-cation increased PCV2 yield (Table 1). Combinedtreatment of PK-15 cells with IFN-g and inhibitorsof endosomal-lysosomal system acidification fur-ther increased PCV2 yield. PCV2 yield increasedgradually under all conditions, with the highest yieldobserved at 72 hpi. The highest recorded PCV2yield was 4.8 log10 TCID50 per 105 treated cellscompared to 3.1 log10 TCID50 per 105 untreatedcells at 72 hpi. This is a 50-fold increase in PCV2yield. The highest increase in PCV2 infection wasobserved when PK-15 cells were treated with acombination of IFN-g and CQ or NH4Cl.The results of the present study show that a com-bined inhibition of endosomal-lysosomal systemacidification and IFN-g treatment of PK-15 cellsincreases the number of PCV2-infected cells andPCV2 yield. Treatment of cells with a combinationof inhibitors of endosomal-lysosomal system acidi-fication and IFN-g may be of use in increasing theyield of PCV2 for PCV2 vaccine production.AcknowledgmentsThe authors are grateful to Carine Boone and Chris Brackefor their skillful technical assistance. This work was fundedby the European Union (Sixth Framework Programme,Project No. 513928, coordinated by G. Allan). G.M. wassupported with a scholarship for candidates from developingcountries from the Special Research Fund (BOF) of GhentUniversity. P.L.D. was supported with a fellowship (B=06524) from BOF of Ghent University.References1. Allan GM, McNeilly F, Kennedy S, Daft B, Clarke EG,Ellis JA, Haines DM, Meehan BM, Adair BM (1998)Isolation of porcine circovirus-like viruses from pigswith a wasting disease in the USA and Europe. J VetDiagn Invest 10: 3–102. 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Increased yield of porcine circovirus-2 by a combined treatment of PK-15 cells with interferon-gamma and inhibitors of endosomal-lysosomal system acidification

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Increased yield of porcine circovirus-2 by a combined treatment of PK-15 cells with interferon-gamma and inhibitors of endosomal-lysosomal system acidification

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