Reactive oxygen species regulate the replication of porcine circovirus type 2via NF-κB pathway
Xingxiang Chen a ,Fei Ren a ,John Hesketh b ,Xiuli Shi a ,Junxian Li a ,Fang Gan a ,Kehe Huang a ,⁎
a College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,PR China
b
Institute for Cell and Molecular Biosciences,University of Newcastle,The Medical School,Framlington Place,Newcastle Upon Tyne NE24HH,UK
a b s t r a c t
a r t i c l e i n f o Article history:
Received 22October 2011
Returned to author for revision 29November 2011
Accepted 10January 2012
Available online 11February 2012Keywords:
Porcine circovirus type 2PCV2replication
Reactive oxygen species NF-κB
Intracellular redox state has been suggested to have various effects on the replication of different viruses within host cells.The aim of the present study was to investigate the in fluence of reactive oxygen species (ROS)on replication of porcine circovirus type 2(PCV2),in PK15cells.Following PCV2infection there was a time-dependent increase in ROS.Antioxidant N-acetyl-L -cysteine treatment of cells resulted in lower ROS levels and lower PCV2replication.In contrast,treatment by buthionine sulfoximine (BSO),a GSH synthesis inhibitor,resulted in elevation of ROS levels and increased PCV2replication.Furthermore,inhi-biting the activity of NF-κB,a redox-responsive transcription factor,suppressed BSO-mediated increase of PCV2replication,indicating that increased PCV2replication likely occurs via ROS activation of NF-κB.Taken together,our results indicate that the generation of ROS during PCV2infection is involved in its replication and this progression is associated with the alteration in NF-κB activity induced by ROS.
©2012Elsevier Inc.All rights reserved.
Introduction
Porcine circovirus (PCV)is classi fied in the genus Circovirus of the family Circoviridae (Todd et al.,2005).Two genotypes of PCV have been identi fied.PCV type 1(PCV1),which was first recognized in 1974as a contaminant of a continuous porcine kidney cell line (PK15)(Tischer et al.,1982),is known to be non-pathogenic to pigs (Allan et al.,1995).Infection with PCV type 2(PCV2)has been associ-ated with postweaning multisystemic wasting syndrome (PMWS)in young weaned pigs,a disease first recognized in Canada in 1991(Clark,1997).Usually PMWS appears in pigs at the age of 5to 18weeks and affected pigs show fever,wasting or unthriftiness,respi-ratory distress,enlarged lymph nodes and,occasionally,jaundice and diarrhea (Darwich et al.,2004;Segales and Domingo,2002).Mortality rates may vary from 1%to 2%up to 30%in complicated cases when co-infections with porcine reproductive and respiratory syndrome virus,porcine parvovirus,or Mycoplasma hyopneumoniae .Several studies have suggested that severely PMWS affected pigs may develop immu-nosuppression (Segales et al.,2004).Nowadays,PMWS and other re-lated PCV2-associated diseases are occurring in all swine-producing areas of the world and have become increasingly serious threats to global pig production (Allan and Ellis,2000;Allan et al.,1998;Choi et al.,2000;Fenaux et al.,2000;Grau-Roma et al.,2011;Mankertz et al.,2000;Patterson and Opriessnig,2010).However,not all pigs infected with PCV2will develop PMWS.Even if PMWS occur,severity of the disease is different in different pig farms.Obviously,infection of
pigs with PCV2and other infectious/noninfectious triggers are re-quired for PMWS to occur (Grau-Roma et al.,2011;Patterson and Opriessnig,2010).Several studies have linked PMWS expression to management measures,presence of concurrent viral infections,stim-ulation of the immune system,nutrition,male castration and lower piglet weight at weaning (Grau-Roma et al.,2011),but the pathogenic mechanism of PCV2remains poorly understood.
Oxidative stress results from an imbalance between ROS produc-tion and anti-oxidant activity conferred by enzymes such as the thior-edoxin,reductases,glutathione peroxidases and glutathione reductase (Schafer and Buettner,2001).Viral infection is often ac-companied by alteration of intracellular redox state (Baruchel and Wainberg,1992;Casola et al.,2001;Jamaluddin et al.,2009;Korenaga et al.,2005;Seet et al.,2009;Wang et al.,2001;Waris et al.,2005).Infection by the HIV is associated with decreased levels of GSH and increased production of ROS (Ciriolo et al.,1997;Garaci et al.,1997;Palamara et al.,1995).Additionally,the viral TAT protein in-creases intracellular ROS levels by inhibiting the antioxidant enzyme manganese superoxide dismutase (Flores et al.,1993).Direct interac-tion of core protein with mitochondria is an important cause of the oxidative stress seen in chronic hepatitis C (Korenaga et al.,2005).ROS have also been shown to affect viral replication,promoting HIV replication (Baruchel and Wainberg,1992;Staal et al.,1990)but being associa
reactive toted with lower bovine leukemia virus proviral loads (Bouzar et al.,2009).However,there is no information available con-cerning the production of ROS induced by PCV2infection and the ef-fects of ROS on PCV2replication,which may contribute to interpret why there are different incidences of PMWS in different pig farms with PCV2infection.
Virology 426(2012)66–72
⁎Corresponding author.Fax:+862584398669.E-mail address:khhuang@njau.edu (K.
Huang).
0042-6822/$–see front matter ©2012Elsevier Inc.All rights reserved.doi:
10.1016/j.virol.2012.01.023
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Intracellular redox state has been suggested to affect the replica-tion of viruses through different mechanism within host cells accord-ing to previous studies.Inhibition of influenza infection by GSH appears to occur via GSH-dependent inhibition of apoptosis which may slow virus release(Cai et al.,2003).Increased HIV replication likely occurs via ROS activation of NF-κB(Korenaga et al.,2005). However,a mechanism of action on Sendai virus replication is not known(Ciriolo et al.,1997).It has been reported that NF-κB activa-tion occurs principally in virus infected cells through a direct mecha-nism because a co-localization of ORF1protein with nuclear translocation of p65was detected in the P
CV2-infected cells but not in UV-irradiated PCV2-infected cells(Wei et al.,2008).However,it is still unclear whether activation of NF-κB as an oxidant sensitive pathway contributes to PCV2replication in the host cells.
The present study was performed to investigate whether PCV2 infection is accompanied by alteration of ROS generation,whether PCV2replication in PK15cells is affected by intracellular ROS and whether PCV2replication is affected by the activation of oxidant sensitive NF-κB pathways.
Results
PCV2infection of PK15cells induces ROS production
To determine whether PCV2infection induced ROS production, PK15cells were infected with PCV2at MOI of1at different time points.At the same end time point DCFH-DA was added to cultures and incubated for30min at37°C.Fluorescence was measured within 60min according to the manufacturer's instruction.When compared with controls,a significant production of ROS was observed at differ-ent time points after infection,reaching a plateau at around72h (Fig.1B).At96–120h post infection,ROS levels had declined from the maximum level but were still elevated above those in untreated cells.As a positive control for DCF-DA oxidation,PK15cells exposed to100μM ROSUP(Beyotime,China)for20min
also exhibited a sig-nificant increase over un-stimulated cells(data not shown).These re-sults demonstrate that ROS are produced following PCV2infection.
Reduction of ROS levels induced by NAC can decrease PCV2replication
Thefinding that ROS are generated during PCV2infection prompted us to examine whether decreasing the production of ROS would alter PCV2replication.Firstly,cells were treated with the anti-oxidant NAC,a precursor in glutathione synthesis which can promote the generation of cysteine and indirectly increase glutathione levels. PK15cells were seeded at a density of1×104/well in96-plate and in-cubated with NAC(5mM)at different time points.At the same end time point PK15cells were collected for measuring DCFH-DA oxida-tion.ROS levels were significantly decreased at24–120h after 5mM NAC treatment compared with control cells(Fig.1).After incu-bation with1,3and5mM NAC for72h,ROS levels were reduced sig-nificantly when compared with control cells(Fig.2A).After PK15cells were incubated with various concentration of NAC for24h before PCV2infection and for a further72h,ROS levels were increased when compared with uninfected cells but were lower when com-pared to infected control cells(Fig.2A).These results indicate that ROS levels were lowered by NAC.
To determine whether reduction of ROS levels play any role in the replication of PCV2,the effects of N
AC on PCV2replication were eval-uated.PK15cells were seeded in24-well cell culture plates and cul-tured overnight with various concentration of NAC before PCV2 infection at MOI of1and incubation with NAC for72h.
Results
Fig.1.Time course of ROS production after PCV2infection,BSO and NAC treatments.
(A,B)PK15cells were infected with PCV2at MOI of1at different time points.At the
same end time point DCFH-DA was added to cultures and incubated for30min at
37°C.Fluorescence was measured within60min at485nm for excitation and
530nm for emission.(C,D)PK15cells were treated with50μM BSO or5mM NAC at
different time points.At the same end time point PK15cells were loaded with DCFH-
DA andfluorescence was measured within60min.Values shown are means±SD
from four independent experiments.Groups were compared by a1-way ANOVA fol-
lowed by least-significant difference test(*P b0.05,**P b
0.01).
Fig.2.Reduction of ROS levels induced by NAC decreases PCV2replication.(A)ROS
levels in NAC-treated PK15cells at72h were determined with DCFH-DA.For the
PCV2-infected groups,PK15cells were incubated with various concentration of NAC
for24h before PCV2infection and then incubated with NAC for a further72h.Fluores-
cence was measured within60min at485nm for excitation and530nm for emission.
Values shown are means±SD mean from four independent experiments.(B/C)PK15
cells were cultured overnight with various concentration of NAC before PCV2infection.
PCV2-infected PK15cells at72h in the presence of various concentration of NAC were
assayed for the viral DNA copies by real-time PCR and the amount of infected cells by
IFA.Values shown are means±SD mean from three independent experiments.Aster-
isks indicate groups statistically significantly different from control by a1-way
ANOVA followed by least-significant difference test(*P b0.05,**P b0.01).
67
X.Chen et al./Virology426(2012)66–72
showed that NAC treatment decreased the progression of the infec-tion as measured by PCV2DNA copies and the number of infected cells(Figs.2and3).The PCV2log10DNA copies significantly de-creased(Fig.2B)and the percentage of PCV2infected cells dropped by74%,61%and55%after72h incubation with1,3and5mM NAC respectively when compared with the control cells(Figs.2C,3).A high titer viral stock solution was treated with NAC at5mM for1h at37°C to determine whether NAC affected infectivity and replica-tion of virus.No effect of NAC treatment on virus infectivity was ob-served when these cells were compared with a mock treatment control(data not shown),indicating that NAC is not directly inacti-vating the virus.Overall,the results suggest reduction of ROS levels induced by NAC decreases viral replication.
Elevation of ROS levels induced by BSO can increase PCV2replication Data presented in Figs.1and2s
uggest that elevated GSH,result-ing from incubation with antioxidant NAC,decreases the level of viral DNA copies and the number of infected cells.Therefore,we test-ed for the converse effect:does lower antioxidant activity accelerate viral replication?To address this question,intracellular ROS levels were increased by the compound BSO,which can cause irreversible inhibition of GSH biosynthesis and decrease the capacity to detoxify ROS.PK15cells were seeded at a density of1×104/well in96-plate, incubated with BSO(50μM)at different time points,and ROS levels measured by DCFH-DA oxidation.When compared with control cells,a significant increase of ROS levels was observed at different time points after50μM BSO treatment(Fig.1).After incubation with BSO at different concentrations for72h,ROS levels were elevat-ed significantly when compared with control cells(Fig.4A).After PK15cells were incubated with various concentrations of BSO for 24h before PCV2infection and then incubated with BSO for a further 72h,ROS levels were increased when compared with uninfected and infected control cells(Fig.4A).These results imply that ROS levels were increased as a result of BSO treatment.
To investigate whether elevation of ROS levels play any role in the replication of PCV2,the effects of BSO exposure on PCV2replication in PK15cells were evaluated.PK15cells were seeded in24-well cell cul-ture plates and cultured for overnight with various concentrations of BSO before PCV2infection at
MOI of1and incubated with BSO for a further72h before determination of PCV2replication and infection. The results presented in Fig.3indicate that BSO treatment led to an in-creased progression of the infection as measured by the appearance of PCV2DNA copies and infected cells.The PCV2log10DNA copies signif-icantly increased and the fold increases in numbers of PCV2infected cells were1.15,1.28and1.41after72h incubation with10,30and 50μM BSO respectively when compared with the control cells (Figs.4C,3).A high titer viral stock solution was treated with BSO at 50μM for1h at37°C to determine if BSO directly affected virus infec-tivity and replication;a mock treatment served as a control.No effect of BSO treatment on virus infectivity was observed(data not shown), showing that BSO did not directly activate the virus but may affect some aspect of viral replication.Overall,these results indicate that the elevation of ROS levels induced by BSO increases viral replication. Effect of BSO and NAC treatment on the activation of
NF-κB-dependent luciferase
It has been reported that PCV2infection can activate NF-κB via IκBαphosphorylation and degradation in PK15cells,and the activa-tion of NF-κB is required for virus replication and PCV2-induced apo-ptosis(Wei et al.,2008).Since NF-κB activity is susceptible to be regulated by alterations in the intracellular redox state,it is possible that the effect of NAC and BSO treatment on PCV2replication is a
sso-ciated with alteration in NF-κB activity induced by ROS.To address this question,we determined NF-κB activation by measuring pNF-κB-luc-reporter gene expression in transiently transfected PK15cells treated with PCV2infection,NAC or BSO.As shown in Fig.5,luciferase activity in PCV2-infected PK15cells was244%of that found in unin-fected control cells,indicating an activation of NF-κB by PCV2infec-tion.Luciferase activity in BSO-treated cells was lower,showing
a
Fig.3.Assay of infected cells by indirectfluorescence assay(IFA).PCV2-infected cells were detected by immunofluorescence(10×20magnification).(A)Non-infected control group,(B)control cells stained for PCV2,(C)5mM NAC-treated group,(D)50μM BSO-treated group and(E)10μM BAY11-7082treated groups all stained for PCV2.Cells positive for PCV2viral antigens were counted in sixfields of view and number of positive cells calculated as percentages of PCV2-positive cells compared to control cells infected with PCV2 alone(data shown in Fig.2C,Fig.4C and Fig.6B).
68X.Chen et al./Virology426(2012)66–72
rise of only 164%,173%and 201%,respectively after 10,30and 50μM BSO treatment,as compared with untreated controls.But luciferase activity in PCV2-infected cells showed an increase of 268%,290%and 304%,respectively after 10,30and 50μM BSO treatment,as compared with untreated controls.In contrast,after 1,3and 5mM NAC treat-ment,NF-κB activity in infected or non-infected PK15cells decreased signi ficantly when compared to untreated PK15cells.Furthermore,both the patterns and magnitudes of increased or decreased luciferase activity were similar to that of ROS levels in PK15cells with the same treatments.These results indicate that NF-κB activation could be in-duced by PCV2infection and is closely associated with ROS levels in host cells after BSO and NAC treatment.
Inhibition of NF-κB activation suppresses BSO-mediated increase in PCV2replication
BAY 11-7082((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile)is widely used as an inhibitor of cytokine-induced I κB αphosphoryla-tion and consequently as an agent which results in decreased expres-sion of NF-κB (Hernandez-Garcia et al.,2010).To con firm the
relationship between NF-κB and ROS-mediated viral replication,PK15cells were treated with 10μM BAY 11-7082for 5h.After 5h of BAY 11-7082treatment,the medium was removed,and fresh basal medium containing fresh inhibitor BAY 11-7082and BSO was added to the culture.24h later,the PK15cells were then infected with PCV2and incubated with BAY 11-7082and BSO for 72h prior to determination.The results presented in Fig.6indicate that BSO-mediated increase in PCV2replication was suppressed by BAY 11-7082treatment as measured by the number of PCV2DNA copies and infected cells.After 72h incubation with 10,30and 50μM BSO alone,the percentage of PCV2infected cells increased when com-pared with the control cells,but after BAY 11-7982treatment and 72h incubation with BSO,the percentage of PCV2infected cells dropped by 62%,74%and 77%when compared to the control (Figs.6B,3).After BAY 11-7982treatment,the percentage of PCV2infected cells decreased signi ficantly when compared to cells without BAY 11-7982treatment at the same concentration of BSO.These re-sults indicate that inhibition of NF-κB activation suppresses BSO-medi
ated increase in PCV2replication.Discussion
The intracellular redox state is the result of a dynamic equilibrium between oxidant and antioxidant molecules.It has been suggested to affect viral replication within a host cell (Ciriolo et al.,1997;Garaci et al.,1997;Michalek et al.,2008;Staal et al.,1990).Results from
the
Fig.4.Elevation of ROS levels induced by BSO increases PCV2replication.(A)ROS levels in BSO-treated PK15cells at 72h were determined with DCFH-DA.Fluorescence was measured within 60min at 485nm for excitation and 530nm for emission.For the PCV2-infected groups,PK15cells were incubated with various concentration of BSO for 24h before PCV2infection and then incubated with NAC for a further 72h.Values shown are means ±SD from four independent experiments.(B/C)PK15cells were cul-tured overnight with various concentration of BSO before PCV2infection.PCV2-infected PK15cells at 72h in the presence of various concentration of BSO were assayed for the viral DNA copies by real-time PCR and the amount of PCV2viral antigen by IFA.Values shown are means ±SD mean from three independent experiments.As-terisks indicate groups statistically signi ficantly different from control by a 1-way ANOVA followed by least-signi ficant difference test (*P b 0.05,**P b
0.01).
Fig.  5.Effect of BSO and NAC treatment on ROS levels and activation of NF-kB-dependent luciferase.PK15cells were seeded at a density of 5×103/well in 96-well plates one day prior to transfection for 6h with pNF-κB-luc containing NF-κB binding motifs (GGGAATTTCC).The cells were washed and then cultured in 100μl DMEM with 5%FBS for 24h before infection with PCV2at MOI of 1and incubated with various concentrations of BSO or NAC for 72h.(A)Effect of BSO and NAC treatment on ROS levels was determined with DCFH-DA.Fluorescence was measured within 60min at 485nm for excitation and 530nm for emission.(B)Activity of NF-κB-dependent lucif-erase of cell extracts from each sample was measured using a luciferase assay kit according to the manufacturer's protocol.Activation of NF-κB-dependent luciferase was recorded as a percentage of mock infection.Values shown are means ±SD from three independent experiments.Asterisks indicate groups statistically signi ficantly dif-ferent from control by a 1-way ANOVA followed by least-signi ficant difference test (*P b 0.05,**P b 0.01).
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X.Chen et al./Virology 426(2012)66–72
present study provide evidence that PCV2infection induces ROS pro-duction in PK15cells.First,antioxidant NAC treatment of cells resulted in reduction of ROS levels and an inhibitory effect on virus growth.Second,BSO treatment of cells resulted in elevation of ROS levels and increased number of viral DNA copies and infected cells.Since NF-κB is a factor activated in response to cellular stress,includ-ing oxidative stress (Morgan and Liu,2011),these results raised the possibility that effect of NAC and BSO treatment on PCV2replication would be associated with alteration in NF-κB pathway induced by ROS.To test this hypothesis we used a reporter gene assay to deter-mine the NF-κB activity after PCV2infection,NAC and BSO treatment.NF-κB activity increased in PCV2-infected or BSO-treated PK15cells while it decreased in NAC-treated PK15cells when compared with control cells.These results indicate that NF-κB activation could be in-duced by PCV2infection and is closely associated with ROS levels in host cells after BSO and NAC treatment.The involvement of NF-κB regulation in ROS-mediated PCV2replication was supported further by the effects of the I κB αphosphorylation inhibitor BAY 11-7982.Overall these data provide the first report of an association between increased ROS production in PK15cells and PCV2infection and they demonstrate that intracellular ROS level play a role in PCV2replica-tion and this progression is associated with the alteration in activa-tion of the NF-κB pathway induced by ROS.
Porcine circovirus type 2is found worldwide in pigs and has been linked to several pathological conditions collectively named porcine circovirus diseases (PCVD)(Allan and Ellis,2000;Allan et al.,1998;Gillespie et al.,2009).The most economically important PCVD is post-weaning multisystemic wasting syndrome (PMWS),which re-sults in losses of €900million per year in the European Union (Armstrong and Bishop,2004).However,not all pigs infected with
PCV-2will develop PMWS even though PCV2is recognized as an es-sential infectious agent of PMWS (Grau-Roma et al.,2011).Infection of pigs with PCV2and other unknown triggers are required for PMWS to occur (Grau-Roma et al.,2011).Evaluation of the role of these triggers is essential in order to understand the incidence of PMWS.Previous studies have demonstrated that some viruses,such as HIV,in fluenza,RSV and LCMV,can promote ROS generation during infection and ROS have also been shown to promote viral replication and immuno-suppression (Baruchel and Wainberg,1992;Casola et al.,2001;Jamaluddin et al.,2009;Korenaga et al.,2005;Seet et al.,2009;Wang et al.,2001;Waris et al.,2005).In contrast,the levels of ROS inversely correlated with bovine leukemia virus proviral loads (Bouzar et al.,2009)and glutathione is required for ef ficient production of infectious picornavirus virions (Smith and Dawson,2006).The present observations that elevation of ROS is a direct con-sequence of PCV2infection,and that PCV2replication can be affected by ROS levels after BSO and NAC treatment,
suggest that redox factors contribute to the complex interrelationship between PCV2and por-cine circovirus associated diseases.
The results presented here for PCV2are consistent with those obtained with HIV and Sendai virus where BSO treatment increased virus replication and NAC treatment decreased virus replication (Garaci et al.,1997;Macchia et al.,1999).In addition,it has been sug-gested that oxidative stress caused by elevated in flammatory cyto-kines and decreased GSH-dependent antioxidant functions in HIV infection promotes the activation of NF-κB and replication of HIV,resulting in the disease progression associated with the CD4+T-cell loss,immunode ficiency,and opportunistic infection (Nakamura et al.,2002).In fluenza replication is inhibited by GSH and is enhanced when GSH levels are reduced (Cai et al.,2003).The intracellular GSH levels decrease during in fluenza virus infection and the more oxida-tive environment established by the in fluenza virus's depletion of GSH would increase the expression and oxidation of protein disul fide isomerase in the endoplasmic reticulum,accelerating disul fide bond-ing and enhancing viral glycoprotein maturation,which is supported by the fact that BSO treatment signi ficantly decreased intracellular GSH levels and increased HA expression and viral titers in cell super-natants (Sgarbanti et al.,2011).Our results demonstrate that effect of BSO and NAC on PCV2replication is associated with the alteration in NF-κB pathway induced by ROS.
In conclusion,our results demonstrate that the PCV2infection and replication not only generate ROS but also is affected by redox state,and that this progression is associated with the alteration in NF-κB pathway induced by oxidative stress.Our research may contribute to interpret the incidence of PMWS when oxidative stress from other unknown triggers is present.Future studies targeting effect of ROS on PCV2replication in vivo and in vitro may lead to improving treatment of PCV2-associated diseases and production of PCV2vaccine.
Materials and methods Reagents and antibodies
Buthionine sulfoximine (BSO)was purchased from Sigma.N-acetyl-L -cysteine (NAC)and (E)-3-[4-methylphenylsulfonyl]-2-pro-penenitrile (BAY 11–7082)were purchased from Beyotime Institute of Biotechnology,China.The cytotoxicity of BSO,NAC and inhibitor BAY 11-7082on PK15cells was determined by trypan blue exclusion dye staining.It was noted that throughout all doses of BSO,NAC and inhibitor BAY 11-7082used in the present study,cell viability assay showed no detectable cell death in the PK15cells.Porcine anti-PCV2antibody for immuno fluorescence was purchased from Univ Biotech Co.Ltd.,China.FITC-linked secondary antibody (rabbit anti-pig IgG)was purchased from
Sigma.
Fig.6.Inhibition of NF-κB activation suppresses BSO-mediated increase in PCV2repli-cation.After 5h of treatment with 10μM BAY 11-7082,PK15cells were cultured over-night with various concentration of BSO in the presence of 10μM BAY 11-7082before PCV2infection.PCV2-infected PK15cells at 72h in the presence of 10μM BAY 11-7082and various concentrations of BSO were assayed for the viral DNA copies by real-time PCR (A)and the amount of infected cells by IFA (B).Values shown are means±SD from three independent experiments.Within the groups with/without BAY 11-7082treatment,bars with “*”are statistically signi ficantly different from control (0μM BSO)by oneway ANOVA followed by least-signi ficant difference test (*P b 0.05,**P b 0.01).“x ”and “xx ”denote signi ficant difference (xP b 0.05,xxP b 0.01)between treated cells and untreated cells by BAY 11-7082.
70X.Chen et al./Virology 426(2012)66–72
Virus and cells
PK-15cells(porcine kidney)were provided by China Institute of Veterinary Drug Control and free of PCV1.PK-15cells were propagat-ed at37°C in an atmosphere of5%CO2in DMEM(Invitrogen,USA) supplemented with5%FBS,penicillin(20mg/ml),streptomycin (20mg/ml),referred hereafter as the culture
medium.The wild-type PCV2(PCV2NJ2002)used in the experiment was isolated origi-nally from a kidney tissue sample of a pig with naturally occurring PMWS.The determination of PCV type was performed by sequencing (Invitrogen,USA).PCV2stocks were generated from PK-15cells infected with PCV2according to the following procedure:PK-15 cells were infected with PCV2at a multiplicity of infection(MOI)of 1,when cells had reached approximately40%–50%confluence.After 1h absorption,the inoculum was removed,and the cell monolayer was washed three times with phosphate-buffered saline(PBS). DMEM medium including5%FBS,penicillin(20mg/mL)and strepto-mycin(20mg/mL),was subsequently added,and incubation was continued at37°C with5%CO2for72h.Then the infected cells were subcultured in DMEM and the PCV2was serially passaged in PK-15cells.The virus harvested at each passage was stored at −80°C.
SYBR green real-time PCR for the quantification of PCV2DNA copies
Newly synthesized viral DNA in PK15cell culture was assayed by SYBR green real-time PCR as described elsewhere(Pan et al.,2008). Briefly,the PCV2infected PK15cells were harvested at72h post-inoculation and DNA extractions were carried out using the TaKaRa DNA Mini kit(TaKaRa,China).The purified DNA was used as tem-plates for real-time PCR amplification.A region of117bp was ampli-fied from the PCV2ORF2gene with a pair of PCV2specific primers (Forward primer:5′-TAGTATTCAAAGGG
CACAG-3′,Reverse primer: 5′-AAGGCTACCACAGTCAG-3′).Quantitative real-time PCR was car-ried out using the ABI PRISM7300Detection System(Applied Biosys-tems,USA).A recombinant pMD19plasmid vector(TaKaRa,China) containing PCV2genome insert was used as standard reference.The viral DNA quantity was detected using the TaKaRa SYBR Green qPCR Kit(TaKaRa,China).
Assay of infected cells by indirectfluorescence assay(IFA)
After72h post-inoculation,PK15cells werefixed in methanol and washed with phosphate-buffered saline(PBS).Afterfixation,the cells were blocked by PBS with2%BSA at room temperature for1h.The porcine anti-PCV2antibody(Univ Biotech,China),diluted in PBS con-taining1%BSA(PBSB)(1:50),was added to the cells and incubated at 37°C for1h.After washing with PBS containing0.1%Tween-20,an optimum dilution(1:100)of FITC-conjugated rabbit anti-pig antibody (Sigma,USA)was added and incubated for1h at37°C.After wash-ing,the cells were examined under afluorescence microscope.Cells positive for PCV2viral antigens were counted in sixfields of view. Fluorescence assay of ROS
Assay of intracellular ROS were measured with2′,7′-dichloro-fluorescein diacetate(DCFH-DA;Beyotime,China)(Hernandez-Garcia et al.,2010).DCFH-DA passively diffuses into cells and is dea-
cetylated by esterases to form nonfluorescent2′,7′-dichlorofluores-cein(DCFH)which in the presence of ROS forms thefluorescent product DCF,which is trapped inside the cells.PK15cells were seeded at a density of1×104/well in96-plate(Corning,USA).5h after seed-ing,the culture wells were infected with PCV2at MOI of1and incu-bated with various concentrations of BSO or NAC for72h.To obtain dissociated PK15cells for the ROS assay,culture medium wasfirst re-moved and the cells were washed three times with PBS.DCFH-DA,diluted to afinal concentration of10μM with a serum free DMEM, was added to cultures and incubated for30min at37°C.Thefluores-cence was measured at485nm for excitation and530nm for emis-sion with afluorescence plate reader(TECAN infinite M200,TECAN). pNF-κB-luc transfection and luciferase reporter assays
One day prior to transfection,PK15cells were seeded at a density of5×103/well in96-well plates(Corning,USA).The pNF-κB-luc re-porter gene constructs(Beyotime,China)containing NF-κB binding motifs(GGGAATTTCC)were transfected into PK15cells using Lipofec-ter reagents(Beyotime,China)in serum free DMEM.The cells were washed twice with PBS and then cultured in100μl DMEM with5% FBS for24h.PK15cells were infected by PCV2at MOI of1or incubat-ed with various concentrations of BSO or NAC for72h before deter-mination.Activity of NF-κB-dependent luciferase of cell extracts from each sample was measured using a luciferase assay kit according to th
e manufacturer's protocol.Three independent experiments were performed to assess luciferase activity.
Statistical analysis
Statistical analysis of the experimental data was performed using the statistical package(SPSS)V17.0for windows.Data were analyzed for establishing their significance using one-way analysis of variance (ANOVA)followed by least-significant difference test.Data are expressed as means±SD.Differences were regarded as significant at P b0.05.
Acknowledgments
This work was funded by the National Natural Science Foundation of China(NFSC)(Grant numbers30871892,31011130155),an interna-tional collaboration grant to JEH and KH from The Royal Society and NSFC,the Research Fund for the Doctoral Program of Higher Education of China(Grant number20110097110014)and the Priority Academic Program Development of Jiangsu Higher Education Institutions. References
Allan,G.M.,Ellis,J.A.,2000.Porcine circoviruses:a review.J.Vet.Diagn.Investig.12, 3–14.
Allan,G.M.,McNeilly,F.,Cassidy,J.P.,Reilly,G.A.,Adair,B.,Ellis,W.A.,McNulty,M.S., 1995.Pathogenesis of porcine circovirus;experimental infections of colostrum deprived piglets and examination of pig foetal material.Vet.Microbiol.44,49–64. Allan,G.M.,McNeilly,F.,Kennedy,S.,Daft,B.,Clarke,E.G.,Ellis,J.A.,Haines,D.M., Meehan,B.M.,Adair,B.M.,1998.Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe.J.Vet.Diagn.Investig.10,3–10. Armstrong,D.,Bishop,S.,2004.Does genetics or litter effect influence mortality in PMWS?Proceedings of the18th International Pig Veterinary Society Congress, Hamburg,Germany,p.809.
Baruchel,S.,Wainberg,M.A.,1992.The role of oxidative stress in disease progression in individuals infected by the human immunodeficiency virus.J.Leukoc.Biol.52, 111–114.
Bouzar,A.B.,Boxus,M.,Florins,A.,Francois,C.,Reichert,M.,Willems,L.,2009.Reduced levels of reactive oxygen species correlate with inhibition of apoptosis,rise in thioredoxin expression and increased bovine leukemia virus proviral loads.Retro-virology6,102.
Cai,J.,Chen,Y.,Seth,S.,Furukawa,S.,Compans,R.W.,Jones,D.P.,2003.Inhibition of influenza infection by glutathione.Free Radic.Biol.Med.34,928–936.
Casola,A.,Burger,N.,Liu,T.,Jamaluddin,M.,Brasier,A.R.,Garofalo,R.P.,2001.Oxidant tone regulates RANTES gene expression in airway epithelial cells infected with respiratory syncytial virus.Role in viral-induced interferon regulatory factor acti-vation.J.Biol.Chem.276,19715–19722.
Choi,C.,Chae,C.,Clark,E.G.,2000.Porcine postweaning multisystemic wasting syn-drome in Korean pig:detection of porcine circovirus2infection by immunohisto-chemistry and polymerase chain reaction.J.Vet.Diagn.Investig.12,151–153. Ciriolo,M.R.,Palamara,A.T.,Incerpi,S.,Lafavia,E.,Bue,M.C.,De Vito,P.,Garaci,E., Rotilio,G.,1997.Loss of GSH,oxidative stress,and decrease of intracellular pH as sequential steps in viral infection.J.Biol.Chem.272,2700–2708.
Clark,E.,1997.Postweaning multisystemic wasting syndrome.Proceedings of the Annual Meeting of the American Association of Swine Practitioners,Quebec City, Canada,pp.499–501.
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X.Chen et al./Virology426(2012)66–72

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