当前位置：文档库 › PCV2a和PCV2b对2个不同遗传特征的PRRSV在商品猪感染动力学和致病性的影响

PCV2a和PCV2b对2个不同遗传特征的PRRSV在商品猪感染动力学和致病性的影响

Effect of porcine circovirus type 2a or 2b on infection kinetics and

pathogenicity of two genetically divergent strains of porcine reproductive and respiratory syndrome virus in the conventional pig model

Tanja Opriessnig a ,*,Phillip C.Gauger a ,Kay S.Faaberg b ,Huigang Shen a ,Nathan M.Beach c ,Xiang-Jin Meng c ,Chong Wang a ,d ,Patrick G.Halbur a

Department of Veterinary Diagnostic and Production Animal Medicine,College of Veterinary Medicine,Iowa State University,Ames,IA,USA b

Virus and Prion Research Unit,National Animal Disease Center,USDA-Agricultural Research Service,Ames,IA,USA c

Department of Biomedical Sciences and Pathobiology,Center for Molecular Medicine and Infectious Diseases,College of Veterinary Medicine,Virginia Polytechnic Institute and State University,Blacksburg,VA,USA d

Department of Statistics,College of Liberal Arts &Sciences,Iowa State University,Ames,IA,USA

1.Introduction

Porcine reproductive and respiratory syndrome virus (PRRSV),a single-stranded,positive-sense RNA virus,is

characterized by a high mutation rate with the potential of genetically diverse strains evolving over time (Forsberg et al.,2001;Hanada et al.,2005;Pirzadeh et al.,1998;Rowland et al.,1999).In the past,PRRSV isolates have emerged within the swine population with varying degrees of virulence (Fang et al.,2007;Han et al.,2006;Nelsen et al.,1999)possibly due to a high degree of mutation and recombination (Yuan et al.,1999,2000,2001,2004).More recently,attention has focused on the occurrence of high mortality in Chinese swine herds which

Veterinary Microbiology 158(2012)69–81

A R T I C L E I N F O

Article history:

Received 12December 2011

Received in revised form 3February 2012Accepted 9February 2012

Keywords:Coinfection Interaction

Porcine circovirus type 2(PCV2)

Porcine reproductive and respiratory syn-drome virus (PRRSV)

A B S T R A C T

To determine differences in infection kinetics of two temporally and genetically different type 2porcine reproductive and respiratory syndrome virus (PRRSV)isolates in vivo with and without concurrent porcine circovirus (PCV)type 2a or 2b infection,62pigs were randomly assigned to one of seven groups:negative controls (n =8);pigs coinfected with a 1992PRRSV strain (VR-2385)and PCV2a (CoI-92-2a;n =9),pigs coinfected with VR-2385and PCV2b (CoI-92-2b;n =9),pigs coinfected with a 2006PRRSV strain (NC16845b)and PCV2a (CoI-06-2a;n =9),pigs coinfected with NC16845b and PCV2b (CoI-06-2b;n =9),pigs infected with VR-2385(n =9),and pigs infected with NC16845b (n =9).Blood samples were collected before inoculation and at day post-inoculation (dpi)3,6,9and 12and tested for the presence of PRRSV antibody and RNA,PCV2antibody and DNA,complete blood counts,and interferon gamma (IFN-g )levels.Regardless of concurrent PCV2infection,VR-2385initially replicated at higher levels and reached peak replication levels at dpi 6.Pigs infected with VR-2385had signi?cantly higher amounts of viral RNA in serum on both dpi 3and dpi 6,compared to pigs infected with NC16845b.The peak of NC16845b virus replication occurred between dpi 9and dpi 12and was associated with a delayed anti-PRRSV antibody response in these pigs.PCV2coinfection resulted in signi?cantly more severe macroscopic and microscopic lung lesions and a stronger anti-PRRSV IgG response compared to pigs infected with PRRSV alone.This work further emphasizes in vivo replication differences among PRRSV strains and the importance of coinfecting pathogens.

*Corresponding author at:Department of Veterinary Diagnostic and Production Animal Medicine,College of Veterinary Medicine,Iowa State University,Ames,IA 50011,USA.Tel.:+15152941137;fax:+15152943564.

E-mail address:tanjaopr@https://www.wendangku.net/doc/da17399974.html, (T.Opriessnig).

Contents lists available at SciVerse ScienceDirect

Veterinary Microbiology

j o u rn a l ho m e pa g e :w w w.e l s e v i e r.c o m /l o c a t e /v e t m i c

was associated with novel PRRSV isolates and described as porcine high fever disease in 2006(Tian et al.,2007;Tong et al.,2007;Wu et al.,2009).The PRRSV isolates involved in porcine high fever disease contained unique nucleotide differences compared to other isolates.Speci?cally,a discontinuous,30amino acid deletion was identi?ed within the nsp2region which was initially suggested to be correlated with the pathogenicity of the virus (Tian et al.,2007;Wu et al.,2009).However,more recent reports have concluded that this deletion is unrelated to virulence (Zhou et al.,2009)in spite of the high mortality that was initially associated with this PRRSV variant (Tian et al.,2007;Tong et al.,2007).Interestingly,analysis of 582samples from affected pigs resulted in the identi?cation of PRRSV,porcine circovirus type 2(PCV2)and classical swine fever virus as the most common co-infection pathogens,suggesting that a potential synergistic interaction among these viruses may account for the unusually high mortality (Lv et al.,2008;Wu et al.,2009).

PCV2is a small,circular,non-enveloped DNA virus belonging to the Circoviridae family in the genus Circovirus .PCV2can be further divided into several subtypes of which PCV2a and PCV2b are prevalent worldwide (Patterson and Opriessnig,2010).To date,experimental infections com-paring PCV2a and PCV2b in gnotobiotic and conventional pigs have not demonstrated major differences in virulence (Beach et al.,2010;Fort et al.,2008;Lager et al.,2007;Opriessnig et al.,2008b ).PCV2is the cause of porcine circovirus associated disease (PCVAD)with multiple clinical manifestations including respiratory disease (Harms et al.,2002).PRRSV has become an important component of the porcine respiratory disease complex (PRDC)with major economic impact on the swine industry (Chae,2005).Retrospective studies identi?ed PRRSV as the

most common cofactor in cases of PCVAD (Pallare

′s et al.,2002).Experimental coinfection with PRRSV and PCV2has yielded mixed results.One study completed in 2002reported minimal clinical disease or death loss in conven-tional pigs coinfected with PCV2and PRRSV (Rovira et al.,2002).In contrast,in another study,severe clinical disease and death in 10of 11pigs between 10and 21days post-infection (dpi)was reported in dually infected,caesarian-derived and colostrum-deprived (CDCD)pigs (Harms et al.,2001).Despite differences in severity of clinical presenta-tion,experimental coinfection of pigs with PCV2and PRRSV has consistently resulted in up-regulation of PCV2

replication (enhanced viremia and PCV2tissue load)and increased severity of PRRSV-induced lesions in lung tissues (Allan et al.,2000;Harms et al.,2001;Rovira et al.,2002).

In North America,both PRRSV and PCV2b have been identi?ed in PCVAD outbreaks characterized by excessive mortality suggesting a synergistic relationship between these two viruses (Cheung et al.,2007;Gagnon et al.,2007;Horlen et al.,2007).The objective of this study was to characterize the infection dynamics and pathogenicity of two different type 2PRRSV isolates in a conventional pig model under the in?uence of concurrent PCV2a or PCV2b infection.The severity of clinical disease,macroscopic and microscopic lesions,amount of PRRSV and PCV2antibodies and nucleic acids in sera,amount of PRRSV and PCV2antigen associated with lesions,and interferon gamma (IFN-g )concentrations in serum were measured and compared between groups.

2.Materials and methods

2.1.Animals,housing,and experimental design

Fifty-three colostrum-fed,crossbred pigs were derived from sows known to be free of PCV2,PRRSV and Mycoplasma hyopneumoniae in two separate batches,44pigs in batch 1(B1)and 9pigs in batch 2(B2).In addition,batch 3(B3)consisted of 9colostrum-fed crossbred pigs derived from sows free of PRRSV and M.hyopneumoniae but seropositive for PCV2.B2and B3pigs were challenged at the same age as B1pigs but the experiment was conducted approximately 24months after B1pigs.Insuf?-cient numbers of pigs were available from the source herd for singularly PRRSV-infected groups to be included with the original experiment.The experimental design and group designations are summarized in Table 1.All pigs were housed under the same conditions and treated in a similar way.All pigs were weaned at three weeks of age and transported to the Livestock Infectious Disease Isolation Facility at Iowa State University,Ames,Iowa.On the day of arrival,all B1pigs were comingled and randomly assigned to one of ?ve rooms each containing 8or 9pigs:negative controls (n =8);pigs coinfected with a 1992isolate of PRRSV (VR-2385)and PCV2a (CoI-92-2a;n =9),pigs coinfected with PRRSV VR-2385and PCV2b (CoI-92-2b;n =9),pigs coinfected with a 2006isolate of PRRSV (NC16845b)and PCV2a (CoI-06-2a;n =9)and pigs

Table 1

Experimental design and group designations.

Group designation

Batch a Number of pigs

PCV2inoculum

PRRSV inoculum

Negative controls 18None None CoI-92-2a 19PCV2a VR-2385CoI-92-2b 19PCV2b VR-2385CoI-06-2a 19PCV2a NC16845b CoI-06-2b 19PCV2b NC16845b PRRSV-I-9224None VR-2385PRRSV-I-06

25None

NC16845b B3-PRRSV-I-9235None VR-2385B3-PRRSV-I-06

None

NC16845b

Batch 1and 2pigs were derived from the same source herd free of PRRSV and PCV2whereas batch 3pigs were derived from a different source herd seropositive for PCV2.

T.Opriessnig et al./Veterinary Microbiology 158(2012)69–81

coinfected with PRRSV NC16845b and PCV2b(CoI-06-2b; n=9).B2pigs were randomly assigned to one of two rooms each containing4or5pigs which were infected with PRRSV VR-2385(PRRSV-I-92)and PRRSV NC16845b (PRRSV-I-06),respectively.Similarly,B3pigs were comingled and randomly assigned to one of two rooms each containing4or5pigs that were infected with PRRSV VR-2385(B3-PRRSV-I-92)and PRRSV NC16845b(B3-PRRSV-I-06),respectively.

The pigs from the different batches were kept in different but identical rooms.Each room had18m2of solid concrete?oor space,separate ventilation systems and one nipple drinker.Inoculation was conducted at approxi-mately23days of age.Blood samples were collected from all pigs prior to inoculation and at dpi3,6,9and12in serum separator tubes(8.5ml BD Vacutainer,Benton Dixon,Franklin Lakes,NJ,USA).The blood was centrifuged at2000?g for10min at48C and serum was stored at à808C until testing.Serum samples were analyzed for levels of anti-PCV2IgG antibody,anti-PRRSV-IgG antibody, IFN-g,PCV2DNA,and PRRSV RNA.In addition,EDTA tubes (8.5mL MONOJECT TM15%EDTA liquid,Tyco Healthcare Group LP,Mans?eld,MA,USA)were collected at dpi3,6,9, and12,stored at room temperature and used within12h after collection to determine blood cell counts.All pigs were necropsied on dpi12and tissues collected during necropsy were analyzed by immunohistochemistry(IHC) for the presence of PCV2and PRRSV antigens.The experimental protocol was approved by Iowa State University Institutional Animal Care and Use Committee (IACUC approval#7-08-6595-S).

2.2.Inocula and inoculation

2.2.1.PRRSV

PRRSV isolate VR-2385with a RFLP pattern1-3-4was recovered in1992from pig tissues obtained from a160sow herd in southwestern Iowa affected by severe respiratory disease in3–16-week-old pigs and high numbers of late term abortions(Halbur et al.,1995b;Meng et al.,1994).The passage5virus of the original VR-2385isolate was used to inoculate pigs in2001as described previously(Opriessnig et al.,2002).Serum from the pigs infected with VR-2385in 2001was used to re-isolate the virus followed by two subsequent passages in MARC-145cells to produce the virus stock of VR-2385for this study.PRRSV isolate NC16845b with a RFLP pattern1-18-2was isolated in2006from a clinically affected9-week-old pig with systemic PCVAD from a group of pigs from North Carolina with a history of severe respiratory disease in50%of the pigs and approx-imatley20%mortality in the group(Gauger et al.,2012).The passage2virus of the original isolate was used to experimentally infect a set of PRRSV-free conventional pigs (data not shown)and the lung tissues from these pigs collected two weeks after infection were used for re-isolation of the NC16845b virus followed by two subsequent passages in MARC-145cells to produce the NC16845b virus stock for this study.The two inocula were separated in different aliquots,stored atà808C,and virus of the same lot was used for all batches of pigs.On dpi0,CoI-92-2a,CoI-92-2b,PRRSV-I-92,and B3-PRRSV-I-92groups received2ml of PRRSV isolate VR-2385at a dose of105.0median tissue culture infective dose(TCID50)per ml.All pigs in groups CoI-06-2a,CoI-06-2b,PRRSV-I-06,and B3-PRRSV-I-06received 2ml of PRRSV isolate NC16845b at a dose of105.0TCID50per ml.Inoculation was intranasal by holding the pig in the upright position and slowly dripping1ml of the inoculum into each nostril using a3ml syringe(Fisher Scienti?c,Inc.).

2.2.2.PCV2

Two different PCV2subtypes were used for the inoculation of pigs.Pigs in groups CoI-92-2a and CoI-06-2a were inoculated with the PCV2a isolate40895,which was recovered from an Iowa farm in1998(Fenaux et al., 2000)and has been well characterized genetically(Fenaux et al.,2000)and in the conventional speci?c pathogen free (SPF)pig model(Opriessnig et al.,2003,2004a).Both PCV2a and PCV2b viruses were produced as described previously(Opriessnig et al.,2008b)and used for inocula-tion in this study at a titer of104.0TCID50per ml.Pigs in groups CoI-92-2b and CoI-06-2b were inoculated with PCV2b isolate NC16845which was isolated in2006from a pig farm in North Carolina(Opriessnig et al.,2008b).Both, PCV2b NC16845and PRRSV NC16845b originated from the same tissues.The PCV2groups were inoculated intrana-sally(3ml)and intramuscularly(2ml)with their respec-tive PCV2subtype by injecting2ml of the inoculum intramuscularly into the right neck area and3ml(1.5ml per nostril)intranasally by holding the pig in the upright position and slowly dripping1.5ml of the inoculum into each nostril using a3ml syringe(Fisher Scienti?c,Inc.).

2.3.Leukogram

EDTA-treated blood samples were analyzed for white blood cells using a multispecies hematology instrument (Hemavet HV950FS,Drew Scienti?c,Inc.).The white blood cell(WBC)count was reported as actual numbers of neutrophils,lymphocytes and total WBC per m l of whole blood.In addition to WBC,a ratio was determined between the total neutrophil count and the total lymphocyte count reported as the N/L ratio.Values from negative control pigs were considered as baseline for the infected pigs on each dpi.

2.4.Serology

2.4.1.PRRSV

Serum samples from all pigs were also tested for the presence of anti-PRRSV antibodies by a commercial PRRSV ELISA(HerdChek PRRS virus antibody test kit2XR,IDEXX Laboratories Inc.Westbrook,MA,USA),according to the instructions of the manufacturer.Samples were considered positive if the calculated S/P ratio was equal to0.4or greater.

2.4.2.PCV2

All serum samples were tested for the presence of anti-PCV2IgG antibodies based on an open reading frame2 (ORF2)ELISA(Nawagitgul et al.,2002).Samples were considered positive if the calculated sample-to-positive(S/ P)ratio was equal to0.2or greater.

T.Opriessnig et al./Veterinary Microbiology158(2012)69–8171

2.4.

3.Other viruses

On dpi12,three samples were randomly chosen from each group and room and tested for the presence of swine in?uenza virus(SIV)antibodies by an in house nucleopro-tein NS1ELISA(Richt et al.,2006)and for the presence of antibodies to porcine parvovirus(PPV)by hemagglutina-tion inhibition(HI)assay(Mengeling et al.,1988).

2.5.Interferon gamma(IFN-g)

A commercial ELISA kit(Swine IFN-g;Invitrogen, Camarillo,CA,USA)was used to detect and quantify IFN-g concentrations in serum according to the instruc-tions of the manufacturer.

2.6.Clinical evaluation

Following PRRSV/PCV2coinfection,the pigs were monitored daily for respiratory disease(dyspnea,sneezing, coughing,nasal discharge).Rectal temperatures and behavioral changes such as lethargy and inappetence/ anorexia were also recorded daily.The observers were aware(not blinded)to the treatment status.

2.7.Quantitative real-time PCR

2.7.1.PRRSV RT-PCR

RNA extraction on serum collected at dpi0,3,6,9,and 12was performed using a QIAamp viral RNA mini kit (Qiagen,Valencia,CA,USA).The AgPATH-ID PRRSV multi-plex reagent kit(Applied Biosystems,Foster City,CA,USA) was used for the real-time,reverse transcriptase PCR(RT-PCR)on each extracted RNA sample.All samples were run in duplicate.Each PCR consisted of8m l template RNA and 17m l of PCR master mix.The PCR master mix contained 12.5m l of2?RT-PCR buffer,2.5m l10?PRRSV primer probe mix,1.25m l20?multiplex RT-PCR enzyme mix, 0.25m l of ZenoRNA-01internal control RNA and0.5m l nuclease-free water.Each reaction included eight pro-gressive1:10dilutions of a known copy number of PRRSV to generate a standard curve for quanti?cation.Each plate was run in the sequence detection system(GeneAmp5700 Sequence Detection System,Applied Biosystems)using the AgPATH-ID company speci?c conditions(10min at458C, 10min at958C,followed by40cycles of2s at978C and 40s at608C).Samples were considered negative when no signal was observed within the40ampli?cation cycles.

2.7.2.PCV2ORF1-based PCR

DNA extraction on serum collected on dpi0,3,6,9,and 12days was performed using the QIAamp DNA blood mini kit(Qiagen,Valencia,CA,USA)and subsequently used for detection of PCV2DNA by quantitative real-time PCR utilizing primers and a probe designed for PCV2ORF1as described(Opriessnig et al.,2003).The real-time PCR reaction consisted of a25m l PCR mixture containing 12.5m l commercially available master mix(TaqMan1 Universal PCR master mix,Applied Biosystems by Life Technologies),2.5m l DNA,1m l(0.4m M)of each primer, and0.5m l(0.2m M)probe.The reaction was run in a7500 Fast Real-Time PCR system(ABI,Foster City,CA,USA)under the following conditions:508C for2min,958C for 10min,followed by40cycles of958C for15s and608C for 1min.All samples were run in duplicate.Serial dilutions of a recombinant PCV2DNA clone were included on each plate to generate a standard curve.Viral concentrations were expressed as the DNA copy numbers per ml of sample.Samples were considered negative when no signal was observed within the40ampli?cation cycles.

2.7.

3.PCV2a/b ORF2-based differential PCR

All DNA extracts were also tested for presence of PCV2a and PCV2b DNA by utilizing a forward primer(50-GCAGGGCCAGAATTCAACC-30),a reverse primer(50-GGCGGTGGACATGATGAGA-30),a probe speci?c for PCV2a (50-Cal Fluor Orange560-GGGGACCAACAAAATCTCTA-TACCCTTT-BHQ-30),and a probe speci?c for PCV2b(50-Quasar670-CTCAAACCCCCGCTCTGTGCCC-BHQ-30),which were designed in the PCV2ORF2as described(Opriessnig et al.,2010).The multiplex real-time PCR reaction consisted of a total volume of25m l containing12.5m l of the commercially available master mix(Applied Biosystems),5m l DNA,0.4m M of each primer,and 0.2m M of each probe.All samples were run in duplicate. The reactions were carried out under the following conditions:508C for2min,958C for10min,followed by 40cycles of958C for15s and608C for1min.The sensitivity and speci?city of the real-time PCR reaction was evaluated using known PCV2a and PCV2b isolates as well as PPV,PRRSV,and PCV type1(PCV1)isolates.Samples were considered negative when no signal was observed within the40ampli?cation cycles.

2.8.Sequencing

Open reading frame(ORF)5of one PRRSV RT-PCR positive pig in each group was sequenced on dpi12as previously described(Gauger et al.,2012).

2.9.Necropsy

On dpi12,all pigs were humanely euthanized by intravenous pentobarbital overdose(Fatal-Plus1Vortech Pharmaceutical,Ltd.,Dearborn,MI,USA).Macroscopic lung lesions were estimated based on the percentage of the lung surface affected by pneumonia ranging from0to100% (Halbur et al.,1995b).The scoring system was based on the approximate volume that each lung lobe contributes to the entire lung:the right cranial lobe,cranial part of the left cranial lobe,and the caudal part of the left cranial lobe contribute10%each to the total lung volume,the accessory lobe contributes5%,and the right and left caudal lobes contribute27.5%each(Halbur et al.,1995b).Additionally, lymph node size was scored ranging from0(normal)to3 (four times the normal size)(Opriessnig et al.,2006).Lungs were insuf?ated with?xative as previously described (Halbur et al.,1995b).Sections of lymph nodes(tracheo-bronchial,mesenteric,mediastinal,super?cial inguinal, and external iliac),tonsil,thymus,ileum,kidney,colon, spleen,heart,liver,and brain were collected at necropsy and?xed in10%neutral-buffered formalin and routinely processed for histological examination.

T.Opriessnig et al./Veterinary Microbiology158(2012)69–81 72

2.10.Histopathology

Microscopic lesions were evaluated independently by two veterinary pathologists(TO,PCG)blinded to the treatment status.Sections of lung were scored for the presence and severity of interstitial pneumonia ranging from 0(normal)to6(severe,diffuse)(Halbur et al.,1995b). Sections of heart,liver,kidney,ileum,colon and brain were evaluated for the presence of lymphohistiocytic in?amma-tion and scored from0(none)to3(severe).Lymphoid tissues including lymph nodes(trachea-bronchial,mediastinal, mesenteric,external iliac and super?cial inguinal),tonsil, spleen and thymus were evaluated for the presence of lymphoid depletion ranging from0(normal)to3(severe) and histiocytic in?ammation and replacement of follicles ranging from0(normal)to3(severe)(Opriessnig et al.,2006).

2.11.Immunohistochemistry

2.11.1.PRRSV

Detection of PRRSV-speci?c antigen was performed by IHC staining on lung sections as previously described (Halbur et al.,1995a).Sections were scored for presence of PRRSV antigen independently by two veterinary pathol-ogists(TO,PCG)blinded to the treatment groups.

2.11.2.PCV2

IHC for detection of PCV2-speci?c antigen was per-formed on sections of lung,lymph nodes(tracheobronchial, mediastinal,mesenteric,super?cial inguinal and external iliac),tonsil,spleen,thymus and small intestine using a rabbit polyclonal antiserum(Sorden et al.,1999).Sections were scored for presence and amount of PCV2antigen independently by two veterinary pathologists(TO,PCG) blinded to the treatment groups.If the results obtained by the two pathologists on a certain tissue differed,the mean of the two scores was used.PCV2scores ranged from0(no antigen)to3(more than50%of the lymphoid follicles contain cells with PCV2-antigen staining)(Opriessnig et al., 2006).Any tissue or tissue pool with detectable staining was given at least a score of1.For the purpose of determining prevalence rates,a score of0was considered negative and scores of1,2and3were considered positive.

2.12.Statistical analysis

For data analysis,JMP1software version8.0.1and SAS1 software version9.2.0(both SAS Institute,Cary,NC,USA) were used.Summary statistics were calculated for groups to assess the distributional property and data that were not distributed normally(PCR data)were log transformed prior to analysis.As log transformation can only be applied to numbers above0,a constant number(1)was added to each number in the data set prior to log transformation.A linear mixed model with the random effects‘‘Source’’(Source A:B1and B2and Source B:B3)and‘‘Batch’’(B1,B2, B3,nested within‘‘Source’’)and the?xed effects‘‘PRRSV strain’’(none,VR-2385,NC16845b)and‘‘PCV2subtype’’(none,PCV2a,PCV2b)was used?rst on all outcomes.From this,it was determined that the random effect‘‘Source’’contributed to the overall variation whereas‘‘Batch’’did not.To decrease the heterogeneity of the animals in the analysis,all data obtained from the second source,B3,were removed from the analysis but were provided as supple-mental information throughout the‘‘Results’’and Tables. The?nal model to analyze continuous data collected over time(rectal temperatures,blood cell counts,log trans-formed PCV2and PRRSV genomic copies,and ELISA S/P ratios)was a repeated measures analysis of variance (ANOVA),where PRRSV strain,PCV2subtype,DPI and their interactions were the?xed effects and pig was the subject of repeated https://www.wendangku.net/doc/da17399974.html,pound symmetry variance-covariance structure was used to model the within pig correlation.A one-way ANOVA was used to analyze cross-sectional data(macroscopic and microscopic lung lesions) where PRRSV strain,PCV2subtype,and their interaction were the?xed effects.Differences among the interacting groups(PRRSV strain?PCV2subtype)in the repeated measures ANOVA or the one-way ANOVA were assessed using Tukey’s t-test.A p-value of less than0.05was considered signi?cant.Differences in prevalence of PRRSV and PCV2antigen between groups(IHC staining)were determined by Fisher’s exact test.

3.Results

3.1.Clinical presentation

Mild,transient lethargy and inappetence were observed in all inoculated groups,although coughing or sneezing was not a feature.Pigs in all inoculated groups regardless of coinfection status developed a transient to persistent fever ranging from40.08C to41.88C between dpi3and dpi12.The mean rectal temperature time by group interaction after inoculation was signi?cant(P<0.05).All six inoculated groups had rectal temperatures signi?cantly higher than the negative controls at dpi6and dpi9.By dpi12,the mean group rectal temperatures in the PRRSV-I-92,PRRSV-I-06, CoI-92-2a,CoI-92-2b and CoI-06-2a groups were signi?-cantly(P<0.05)higher compared to the negative controls. When the effect of‘‘PRRSV strain’’was evaluated across groups,no differences were https://www.wendangku.net/doc/da17399974.html,pared to pigs infected with PRRSV alone,coinfected pigs had higher mean rectal temperatures at dpi3,6and9.When the effect‘‘PCV2 subtype’’was evaluated among coinfected groups,PCV2a pigs had signi?cantly(P<0.01)higher rectal temperatures on dpi9compared to PCV2b pigs(data not shown).B3pigs (B3-PRRSV-I-92and B3-PRRSV-I-06)had similar rectal temperatures as B2(PRRSV-I-92and PRRSV-I-06)pigs.

3.2.Whole blood counts

Hematology results are summarized in Table2.There was an effect of‘‘PRRSV strain’’on white blood cell counts at dpi3with pigs infected with NC16845b having signi?cantly(P=0.03)higher levels of white blood cells compared to pigs infected with VR-2385(10.5?0.9versus 8.1?0.5).Also,there was a signi?cant effect of‘‘PCV2’’(P<0.05):PCV2-infected pigs had higher levels of white blood cells at dpi9and12compared to non-PCV2-infected pigs(17.1?0.9versus9.4?0.6and20.3?1.0versus 14.5?1.2).There was no effect of‘‘PRRSV strain’’on numbers

T.Opriessnig et al./Veterinary Microbiology158(2012)69–8173

of neutrophils;however,there was a signi?cant effect of ‘‘PCV2’’on mean group neutrophil counts at dpi 6,9,and 12with PCV2-infected pigs having elevated levels compared to pigs not infected with PCV2(5.3?0.4versus 3.1?0.3,10.0?0.6versus 3.4?0.3,and 11.9?6.1versus 6.1?0.9,respectively).Differences in mean group lymphocyte counts were only observed on dpi 9(Table 2)and there was an effect of PRRSV strain (P =0.048):pigs infected with NC16845b had lower levels of lymphocytes compared to pigs infected with VR-2385(3.9?0.3versus 5.1?0.5).Additionally,pigs coinfected with PCV2had higher levels of lymphocytes (P =0.016)compared to pigs infected with PRRSV alone (4.8?0.3versus 3.0?0.3)suggesting an effect of ‘‘PCV2’’.

3.3.Antibody levels

3.3.1.Anti-PRRSV-IgG antibody levels

All pigs in all groups were negative for PRRSV-speci?c antibodies at 0dpi and negative control pigs remained

negative for anti-PRRSV antibody throughout the study.Prevalence and group mean S/P ratios are summarized in Table 3.Overall,there was a signi?cant effect of ‘‘PRRSV strain’’and ‘‘PCV2’’on the anti-PRRSV antibody response at dpi 9.Speci?cally,pigs infected with VR-2385had a signi?cantly (P =0.017)higher anti-PRRSV antibody response compared to those infected with NC16845b.Similarly,coinfected pigs had signi?cantly (P =0.028)higher anti-PRRSV S/P ratios compared to pigs singularly infected with PRRSV.There was no effect of ‘‘PCV2subtype’’on the magnitude of the anti-PRRSV-antibody response among coinfected groups.

3.3.2.Anti-PCV2-IgG antibody levels

All pigs in B1and B2were negative for PCV2-speci?c anti-IgG antibodies at 0dpi and the negative controls and B2pigs remained PCV2seronegative throughout the trial.In the PCV2coinfected groups,seroconversion was observed at dpi 9.The prevalence and mean group anti-PCV2-IgG S/P

ratios

Table 2

Mean group leukocyte values (?1000/m l of whole blood except for ratios)in the different treatment groups on days post-inoculation (dpi)0,3,6,9and 12.Data obtained from B3-PRRSV-I-92and B3-PRRSV-I-06pigs (gray shaded area)were not included in the analysis.

Group

Hematology a

Negative controls (n =8)

WBC

8.6?1.0 6.0?0.7A 12.0?0.819.8?2.8A 17.8?0.9A,B Neutrophils 2.7?0.3 2.7?0.3 4.9?0.39.1?1.5A,B 7.7?0.7A,B Lymphocytes 4.1?0.3 2.4?0.3 4.9?0.6 6.4?0.4A,B 7.2?0.3N/L ratio

0.6?0.3

1.1?0.1

1.1?0.1A

1.5?0.2A,B

1.1?0.2A

CoI-92-2a (n =9)

WBC

13.6?2.18.3?0.8A,B 8.4?1.217.5?1.7A.B 19.5?1.3A,C Neutrophils 5.5?1.3 4.6?0.8 4.5?0.710.2?1.0A 12.0?0.8B,C Lymphocytes 6.0?0.9 2.5?0.3 2.7?0.6 4.7?0.6A,B,C 5.0?0.6N/L ratio

1.0?0.2

2.1?0.5

1.9?0.3A,B

2.3?0.2A

2.6?0.3B

CoI-92-2b (n =9)

WBC

12.1?1.68.6?0.8A,B 10.9?1.120.2?1.4A 23.5?2.0A Neutrophils 4.6?1.1 4.7?0.7 6.2?0.711.3?0.8A 14.1?1.1C Lymphocytes 5.7?0.7 2.7?0.2 3.4?0.4 6.6?0.9A 6.9?1.2N/L ratio

0.9?0.2

1.8?0.3

1.9?0.2A,B

2.4?0.3B,C

CoI-06-2a (n =9)

WBC

10.8?0.98.7?1.0A,B 9.8?0.714.8?2.1A,B 18.7?1.7A,B Neutrophils 3.4?0.3 3.9?0.5 4.0?0.3 4.7?0.6A,B,C 9.9?0.8A,B,C Lymphocytes 5.3?0.6 3.4?0.5 3.4?0.4 4.0?0.5B,C 6.4?0.8N/L ratio

0.7?0.4

1.2?0.2

1.3?0.1A,B

2.3?0.3A

1.7?0.2A,B,C

CoI-06-2b (n =9)

WBC

12.8?2.410.4?1.5A,B 11.7?1.716.0?1.6A,B 19.5?2.0A,B Neutrophils 6.0?1.8 5.3?0.8 6.6?1.19.8?1.2A,B 11.7?1.6C,B Lymphocytes 5.1?0.7 2.8?0.3 3.4?0.5 4.1?0.5A,B,C 6.1?0.8N/L ratio

0.9?0.5

2.0?0.2

2.1?0.3B

2.5?0.1A

2.0?0.2A,B,C

PRRSV-I-92(n =4)

WBC

7.8?0.9 6.6?0.6A 6.9?1.09.1?0.8B 16.4?1.3A,B Neutrophils 3.0?0.6 1.8?0.2 2.6?0.4 3.8?0.6B,C 7.4?1.3A,B Lymphocytes 4.1?0.6 2.7?0.3 2.5?0.6 2.6?0.2C 6.0?1.1N/L ratio

0.7?0.1

0.7?0.0

1.3?0.5A,B

1.4?0.1A,B

1.3?0.2A,C

PRRSV-I-06(n =5)

WBC

10.6?0.413.9?2.3B 10.4?1.59.5?0.9B 13.3?1.7B Neutrophils 3.4?0.3 5.6?1.7 3.4?0.5 3.1?0.2C 5.1?1.1A Lymphocytes 4.8?0.6 2.6?0.4 4.2?1.0 3.3?0.4C 4.5?0.4N/L ratio

0.7?0.1

2.1?0.5

1.0?0.3A,B

1.0?0.1B

1.1?0.2A

Different superscripts (A,B,C)within columns indicate signi?cant (P <0.05)differences among groups.a

WBC:white blood cells;N/L ratio:neutrophil to lymphocyte ratio.

T.Opriessnig et al./Veterinary Microbiology 158(2012)69–81

are summarized in Table 4.There was no effect of ‘‘PRRSV strain’’or ‘‘PCV2subtype’’on the magnitude of the anti-PCV2-antibody response.B3pigs were seropositive for PCV2at the time of arrival (mean PCV2ELISA S/P ratio:1.58?0.09)and the S/P ratios remained at a similar level for the duration of the study (data not shown).

3.3.3.Other viruses

At termination of the study,pigs randomly selected from each batch tested negative for antibodies against PPV,SIV H1N1and SIV H3N2(data not shown).

3.4.IFN-g levels

Prevalence of IFN-g positive samples and mean group IFN-g concentrations are summarized in Table 5.There was no effect of ‘‘PRRSV strain’’or ‘‘PCV2’’on the IFN-g

levels and no differences were found among groups;however,analysis of an effect of ‘‘PCV2subtype’’among coinfected groups revealed that on dpi 6,PCV2b-inocu-lated pigs had signi?cantly (P =0.028)higher levels of IFN-g compared to PCV2a-inoculated pigs (0.46?0.17pg/ml versus 0.06?0.05pg/ml).

3.5.PRRSV and PCV2viremia

3.5.1.Prevalence and amount of PRRSV RNA

All pigs were negative for PRRSV-RNA in serum at 0dpi and the negative controls remained negative for PRRSV RNA throughout the study.The prevalence of PRRSV RNA positive pigs and group mean genomic copy numbers/ml are summarized in Table 6.Sequencing of the ORF5gene of PRRSV and comparison with the original inocula con?rmed that the correct PRRSV isolates were present in

the

Table 3

Prevalence of anti-PRRSV antibodies and mean group sample-to-positive (S/P)ratios in the different treatment groups on days post-inoculation (dpi)0,3,6,9and 12.Data presented as prevalence (mean S/P ratio ?SE).Data obtained from B3-PRRSV-I-92and B3-PRRSV-I-06pigs (gray shaded area)were not included in the analysis.

Group

Negative controls 0/8(0.01?0.00)0/8(0.02?0.00)0/8(0.02?0.00)0/8(0.02?0.01)A 0/9(0.02?0.00)A CoI-92-2a 0/9(0.01?0.00)0/9(0.01?0.00)0/9(0.03?0.01)7/9(0.74?0.19)B 9/9(0.84?0.09)B CoI-92-2b 0/9(0.02?0.01)0/9(0.01?0.00)0/9(0.02?0.01)6/9(0.82?0.26)C 9/9(0.98?0.24)B CoI-06-2a 0/9(0.08?0.07)0/9(0.08?0.07)0/9(0.08?0.06)4/9(0.40?0.08)A,B 7/9(0.70?0.11)B CoI-06-2b 0/9(0.01?0.00)0/9(0.01?0.01)0/9(0.01?0.00)6/9(0.52?0.08)A,B 6/9(0.68?0.12)B PRRSV-I-920/4(0.01?0.00)0/4(0.01?0.00)0/4(0.00?0.00)2/4(0.35?0.06)A,B 4/4(0.83?0.13)B PRRSV-I-06

0/5

(0.02?0.00)

0/5

(0.01?0.00)

0/5

(0.00?0.00)

0/5

(0.08?0.03)A,B

2/5

(0.36?0.19)B

Different superscripts (A,B,C)within columns indicate signi?cant (P <0.05)differences in mean group S/P ratios among

groups.

Table 4

Prevalence of anti-PCV2IgG antibodies and mean group sample-to-positive (S/P)ratios in the different treatment groups except PRRSV-I-92and PRRSV-I-06on day post-inoculation (dpi)0,3,6,9and 12.Data presented as prevalence (mean S/P ratio ?SE).Grey shaded areas indicate the presence of PCV2seropositive pigs (S/P ratio >0.2)within a treatment group.

Group

A A Different superscripts (A,B)within columns indicate signi?cant (P <0.05)differences in mean group S/P ratios among

groups.

Table 5

Prevalence of IFN-g and mean group concentration (pg/ml)in the different treatment groups on day post-inoculation (dpi)0,3,6,9and 12.Data presented as prevalence (mean log 10group concentration ?SE).Data obtained from B3-PRRSV-I-92and B3-PRRSV-I-06pigs (gray shaded area)were not included in the analysis.

Group

Negative controls 0/81/8(0.09?0.09)0/8(0.00?0.00)1/8(0.05?0.05)CoI-92-2a 0/91/9(0.03?0.03)7/9(1.16?0.30)1/9(0.27?0.27)CoI-92-2b 0/92/9(0.39?0.26)6/9(0.77?0.33)2/9(0.39?0.28)CoI-06-2a 0/91/9(0.09?0.09)3/9(0.46?0.24)1/9(0.17?0.17)CoI-06-2b 0/94/9(0.53?0.22)

6/9(1.01?0.28)

2/9(0.31?0.21)PRRSV-I-920/4

0/40/41/4(0.35?0.35)PRRSV-I-06

1/5(0.25?0.25)

1/5

(0.24?0.24)

1/5

(0.32?0.32)

1/5

(0.40?0.40)

T.Opriessnig et al./Veterinary Microbiology 158(2012)69–81

respective groups and rooms.When results were analyzed for a possible effect of ‘‘PRRSV strain’’,a signi?cantly (P <0.05)higher amount of PRRSV RNA was detected in pigs infected with VR-2385at dpi 3and 6compared to pigs infected with NC16845b (Fig.1).When the pigs infected with PRRSV alone were removed from the analysis,coinfected pigs with NC16845b had signi?cantly higher amounts of PRRSV RNA in serum compared to pigs infected with VR-2385on dpi 9(6.49?0.10versus 5.99?0.11)and dpi 12(6.24?0.08versus 5.55?0.12),respectively.An effect of ‘‘PCV2’’or ‘‘PCV2subtype’’on PRRSV replication was not evident.

3.5.2.Prevalence and amount of PCV2DNA

All pigs were negative for PCV2-DNA in serum at 0dpi and the negative controls and B2and B3pigs remained PCV2DNA negative throughout the study (data not shown).At dpi 3,28/36PCV2-inoculated pigs were positive for PCV2-DNA,and all PCV2-inoculated pigs were positive

for PCV2-DNA by 6dpi and remained positive until dpi 12.The log 10group mean PCV2DNA amounts are summarized in Fig.2.When results were analyzed for a possible effect of ‘‘PRRSV strain’’it was found that there was a signi?cantly higher amount of PCV2DNA in pigs infected with VR-2385(7.99?0.19)compared to pigs infected with NC16845b (7.01?0.21)on dpi 12.There was a signi?cant effect of ‘‘PCV2subtype’’on dpi 3;groups infected with PCV2b had signi?cantly higher amounts of PCV2DNA in serum compared to groups infected with PCV2a (4.63?0.40versus 2.96?0.51,respectively).An effect of ‘‘PCV2subtype’’was not evident in the later stages of infection.

3.5.3.PCV2subtypes

All pigs in the PCV2a or PCV2b groups were correctly infected with their respective subtype as determined by multiplex real-time PCR (data not shown)and cross-contamination between groups and rooms was not

detected.

Table 6

Prevalence of PRRSV and group mean log 10PRRSV genomic copies per ml in the different treatment groups on days post-inoculation (dpi)3,6,9and 12.Data presented as prevalence (log 10PRRSV RNA ?SE).Data obtained from B3-PRRSV-I-92and B3-PRRSV-I-06pigs (gray shaded area)were not included into the analysis.

Group

Negative controls 0/8(0.00?0.00)A 0/8(0.00?0.00)A 0/8(0.00?0.00)A 0/8(0.00?0.00)A CoI-92-2a 9/9(5.44?0.23)B,D 9/9(6.74?0.14)B 9/9(6.63?0.05)B 9/9(6.17?0.08)B CoI-92-2b 9/9(5.42?0.22)B,D 9/9(6.93?0.16)B 9/9(6.23?0.18)B 9/9(5.76?0.22)B CoI-06-2a 9/9(4.66?0.16)B,C 9/9(6.35?0.17)B,C 9/9(6.93?0.12)B 9/9(6.56?0.08)B CoI-06-2b 9/9(5.35?0.36)B,D 9/9(6.53?0.28)B 9/9(6.90?0.16)B 9/9(6.76?0.14)B PRRSV-I-924/4(6.36?0.25)D 4/4(7.45?0.18)B 4/4(7.11?0.09)B 4/4(7.11?0.16)B PRRSV-I-06

3/5

(2.99?1.26)C

4/5

(4.82?1.47)C

5/5

(6.00?0.92)B

5/5

(6.25?0.02)B

Different superscripts (A,B,C,D)within columns indicate signi?cant (P <0.05)differences in mean group S/P ratios among

groups.

Fig.1.Log 10transformed mean PRRSV RNA genomic copies (?SE)in VR-2385and NC16845b infected pigs regardless of coinfection status on day post-inoculation (dpi)0,3,6,9and 12.Signi?cant (P <0.05)differences between groups within a dpi are indicated by asterisks.The lines indicate the linear trend for pigs infected with VR-2385(gray,dashed)or NC16845b (black,solid).

T.Opriessnig et al./Veterinary Microbiology 158(2012)69–81

3.6.Macroscopic lesions

Macroscopic lesions were characterized by mild-to-moderate enlargement of lymph nodes (especially tra-cheobrochiolar lymph nodes and mediastinal lymph nodes)and mottled-tan lungs with varying degrees of the lung surface affected by visible pneumonia lesions.The group mean lung lesion severity scores are summarized in Table 7and were signi?cantly (P <0.05)lower for negative controls compared to all coinfected groups.There were no signi?cant differences in lung lesions severity between the negative controls and the pigs infected with PRRSV alone.There was an effect of ‘‘PCV2’’on the mean group macroscopic lung lesion scores as evidenced by the coinfected pigs having more severe macroscopic lung

lesions compared to pigs infected with PRRSV alone.However,there was no effect of ‘‘PRRSV strain’’or ‘‘PCV2subtype’’on the severity of the observed macroscopic lung lesions.

3.7.Microscopic lesions

Lung tissues had multifocal-to-diffuse,mild-to-severe,lymphohistiocytic interstitial pneumonia.The mean microscopic lung lesion scores,which are summarized in Table 7,were signi?cantly (P <0.0001)lower in the negative controls compared to the four coinfected groups;however,the scores in the negative controls were not signi?cantly lower than observed in the groups singularly infected with PRRSV.There was a signi?cant effect of ‘‘PCV2’’(P <0.001)on microscopic lung lesions but there was no effect of ‘‘PRRSV strain’’or ‘‘PCV2subtype’’on the severity of the observed microscopic lung lesions.

Lymphoid lesions were either not observed or were characterized by mild depletion of follicles and minimal granulomatous lymphadenitis in all coinfected groups.Signi?cant differences in lymphoid lesion scores were not observed among the groups (data not shown).

3.8.Prevalence of PRRSV and PCV2antigens in tissues

3.8.1.PRRSV

All control pigs were negative for PRRSV antigen by IHC on sections of lung.The prevalence of PRRSV antigen in lung sections was 16/23pigs in the NC16845b-inoculated group (B3:4/4pigs)compared to 21/22pigs in the VR-2385-inoculated group (B3:5/5pigs).There were no signi?cant differences in the prevalence rates of

PRRSV

Fig.2.Log 10transformed group mean PCV2DNA amounts (?SE)in the PCV2-PRRSV coinfected groups on day post-inoculation (dpi)0,3,6,9and 12.Signi?cant (P <0.05)differences between groups within a dpi are indicated by different superscripts (A,

B).

Table 7

Mean group macroscopic (percentage of lung surface affected by lesions)and microscopic (interstitial pneumonia ranging from 0=normal to 6=severe,diffuse)lung lesions (mean group amount ?SE).Data obtained from B3-PRRSV-I-92and B3-PRRSV-I-06pigs (gray shaded area)were not included in the analysis.Signi?cant (P <0.05)differences between groups are indicated by different superscripts (A,B,C).

Group

Macroscopic lung lesions (0–100%)

Microscopic lung lesions (0–6)

Negative controls 0.1?0.1A 0.75?0.25A CoI-92-2a 54.8?4.3B 4.44?0.24B CoI-92-2b 56.3?4.5B 4.67?0.17B CoI-06-2a 52.8?6.4B 4.78?0.32B CoI-06-2b 48.7?4.7B,C 4.44?0.24B PRRSV-I-9231.8?8.3B,C 2.50?0.87A PRRSV-I-06

4.3?2.0A,C

1.80?0.80A

T.Opriessnig et al./Veterinary Microbiology 158(2012)69–81

antigen in lungs between the virus-inoculated groups.The prevalence of PRRSV antigen in lungs was independent of ‘‘PRRSV strain’’or‘‘PCV2subtype’’.

3.8.2.PCV2

All control pigs and all B2and B3pigs were negative for PCV2antigen by IHC.Low-to-high amounts of PCV2-antigen in lung sections were detected in8/9CoI-92-2a pigs,7/9CoI-92-2b pigs,8/9CoI-06-2a pigs and in4/9CoI-06-2b pigs which corresponds to16/18PCV2a-inoculated pigs and11/18PCV2b-inoculated pigs.Moreover,PCV2 antigen was detected in15/18VR-2385-inoculated pigs and in12/18NC16845b-inoculated pigs.The prevalence of PCV2antigen in lung tissues was independent of‘‘PRRSV strain’’or‘‘PCV2subtype’’.In lymphoid tissues,low-to-high amounts of PCV2antigen were detected in8/9CoI-92-2a pigs,7/9CoI-92-2b pigs,8/9CoI-06-2a pigs and in5/9 CoI-06-2b pigs which corresponds to16/18pigs inoculated with PCV2a and12/18pigs inoculated with PCV2b,as well as13/18pigs inoculated with NC16845b and15/18pigs inoculated with VR-2385.The prevalence of PCV2antigen in lymphoid tissues was independent of‘‘PRRSV strain’’or ‘‘PCV2subtype’’.

4.Discussion

The objective of this study was to characterize the infection dynamics and pathogenicity of two different type 2PRRSV isolates recovered from pigs in1992and2006in a conventional pig model.To mimic?eld conditions where coinfections frequently occur,the pigs were concurrently infected with either PCV2a or PCV2b.The effect of each PRRSV isolate was also evaluated in singularly inoculated pigs.However,due to limitations in numbers of available pigs from the source herd,the experiments with singularly PRRSV-inoculated groups were conducted separately but under the same study conditions,using the same inocula and assays to analyze the samples.

The PRRSV isolate VR-2385used in this experiment has been well-characterized in the CDCD and the conventional pig models and is considered a relatively highly pathogenic PRRSV isolate from the1990s(Halbur et al.,1995b,1996; Meng et al.,1996).In contrast,NC16845b represents a more recent PRRSV isolated from an outbreak of respira-tory disease on a farm characterized by high morbidity and mortality in2006(Gauger et al.,2012).The ORF2-7 sequence homology between VR-2385(GenBank accession PRU20788and PRU03040)and NC16845b(GenBank accession HQ699067)was approximately90.4%.The ORF5region demonstrated the least nucleotide and amino acid homology at87.4%and87.1%,respectively(Gauger et al.,2012).

In the past,dual infections with PRRSV and porcine respiratory coronavirus(PRCV)or PRRSV and SIV were studied using conventional pigs(Van Reeth et al.,1996) and gnotobiotic pigs(Van Reeth and Nauwynck,2000)and in general disease was found to be more pronounced in dually inoculated pigs.Interestingly,in gnotobiotic pigs the effect of the coinfection appeared additive rather than synergistic(Van Reeth and Nauwynck,2000).More recent studies have shown that PRRSV modi?es the innate immune response,induces immunosuppression and enhances the in?ammatory response to PRCV in pigs(Jung et al.,2009;Renukaradhya et al.,2010).In another study, dual infection of speci?c pathogen-free pigs with PRRSV and pseudorabies virus(PRV)resulted in more severe clinical signs and increased pneumonia in pigs inoculated with both viruses compared to pigs infected with PRRSV or PRV alone(Shibata et al.,2003).It is also well recognized that PCV2replication is enhanced by concurrent PRRSV infection in both CD and conventional pigs compared to singularly inoculated pigs(Allan et al.,2000;Rovira et al., 2002).To the authors’knowledge,the pathogenicity of genetically different PRRSV isolates in the presence of concurrent viral infection has not been evaluated in vivo.

The combination of PRRSV and PCV2is one of the most common coinfections associated with swine respiratory disease under?eld conditions(Dorr et al.,2007;Pallare′s et al.,2002).Both PRRSV isolates used in the current study were isolated from?eld cases of high mortality and experimental infection of pigs with PRRSV VR-2385has resulted in severe lesions and high levels of viremia (Halbur et al.,1995b,1996).The two PCV2isolates were initially recovered from typical?eld cases of PCVAD in Iowa and North Carolina and have been characterized in the conventional pig model side by side without identi?-able differences between PCV2subtypes(Opriessnig et al., 2008b;Sinha et al.,2011).In the current study,clinical disease in the treatment groups was characterized by variable numbers of infected pigs experiencing transient, mild lethargy,mild respiratory disease and inappetence. Coinfected groups had signi?cantly higher mean rectal temperatures compared to pigs infected with PRRSV alone and the negative controls.Interestingly,when organized by coinfection status and analyzed by PCV2subtype,pigs inoculated with PCV2a had signi?cantly higher mean group rectal temperatures compared to pigs inoculated with PCV2b on dpi9which was associated with an anti-PCV2-antibody response in38.9%(7/18)of the PCV2a-inoculated pigs on dpi9whereas a delayed antibody response was seen in PCV2b-inoculated pigs(11.1%;2/18).

It is well documented that pathogenic differences between type2PRRSV isolates exist(Halbur et al., 1995b,1996).The uniqueness of the current study is the utilization of two temporally and genetically different PRRSV isolates both from cases of high morbidity and mortality in the?eld but isolated15years apart.In a separate in vitro study comparing phenotypic traits of the two PRRS viruses,NC16845b demonstrated reduced growth characteristics compared to VR-2385(Gauger et al.,2012).NC16845b plaque sizes were slightly smaller than VR-2385and the peak viral titer demonstrated by NC16845b was approximately13-fold lower than the VR-2385peak titer.This is in contrast to the in vivo growth characteristics demonstrated in this report.There were clear differences in initial replication between the two PRRSV isolates used in this study.The VR-2385-inoculated pigs had signi?cantly higher levels of PRRSV RNA in serum on dpi3and6.Moreover,NC16845b replicated at higher levels at dpi9and dpi12compared to VR-2385which was associated with signi?cantly lower levels of lymphocytes at dpi9and a signi?cantly lower N/L ratio at dpi12.These

T.Opriessnig et al./Veterinary Microbiology158(2012)69–81 78

results suggest that highly pathogenic PRRSVs may replicate more ef?ciently in vivo in contrast to their decreased growth properties in vitro as previously suggested(Johnson et al.,2004;Wang et al.,2008).This is further supported by the data obtained from the pigs infected with PRRSV alone(B2and B3)which clearly show an increase in replication in pigs infected with NC16845b in the later stages of the in vivo study.

Similar to other PCV2-PRRSV coinfection studies(Allan et al.,2000;Harms et al.,2001),macroscopic and microscopic lesions in coinfected groups were enhanced compared to pigs singularly infected with PRRSV.Recently, it has been shown that pigs infected with VR-2385had signi?cantly prolonged(until70DPI)PCV2viremia and shedding in PRRSV-PCV2coinfected pigs(Sinha et al., 2011).A similar approach using PRRSV NC16845b,which replicated differently from VR-2385in the early stages of infection,could potentially offer new insights on viral interactions in pigs.In the current study,PCV2b replication was signi?cantly up-regulated shortly after initiation of the study at dpi3compared to PCV2a.Furthermore,the CoI-92-2b group had signi?cantly higher quantities of PCV2b in the serum compared to CoI-06-2a(dpi3and12) and CoI-06-2b(dpi12)which was associated with a higher prevalence of PCV2antigen in tissues(93.8%versus75.0%) indicating a synergistic relationship between PRRSV-1992 (VR-2385)and PCV2.Unlike previous studies where the average trial length ranged from21to32days(Allan et al., 2000;Rovira et al.,2002),this trial was terminated at dpi 12to evaluate PRRSV-induced lung lesions which tend to be most severe between dpi10and dpi14.It remains unknown if the observed trend would have resulted in a difference in clinical disease in the later stages of infection. As expected,and similar to a previous study(Yu et al., 2007),the pathological lesions associated with PCV2were either not present or they were mild;however,PCV2 antigen was detected in most tissues in coinfected pigs.In this study,PCV2na?¨ve pigs were utilized,thus the relevance of the model to actual?eld situations is limited considering the majority of young pigs have high levels of passively acquired anti-PCV2antibodies(Opriessnig et al., 2004b).Therefore,the impact of anti-PCV2immunity on the PCV2infection could not be ascertained in the experiment;however,this was not a major concern as we know from several experiments that pigs with passively derived antibodies,although protected from clinical PCV2associated disease,can still be infected with PCV2(McKeown et al.,2005;Opriessnig et al.,2008a). Therefore,we believe that a PCV2na?¨ve pig model increases the ability to identify trends and associations between PRRSV and PCV2.

In the current study,PRRSV-PCV2coinfection was administered intranasally on the same day.This model of simultaneous dual inoculation does not fully mimic the population dynamics due to the variability in timing of exposure to these two pathogens within and between herds in?eld situations.On many conventional farms, endemic exposure and seroconversion to PRRSV often occurs earlier than exposure to PCV2.Infection of pigs with PRRSV prior to PCV2may contribute to the manifestation of more severe PCV2-induced clinical disease and lesions.PRRSV is immunosuppressive,primarily infecting porcine alveolar macrophages(Drew,2000),which decreases the pig’s ability to clear subsequent infections.In contrast, prior PRRSV infection may induce an immunostimulatory effect on the host immune response that serves to enhance PCV2replication and lesions(Krakowka et al.,2001).

It is possible that amino acid mutations acquired during serial passaging of PRRSV on MARC-145cells could result in attenuation as reported previously (Allende et al.,2000;An et al.,2011).While this is also applicable to the current study,we attempted to minimize this risk,by using a relatively low passage of both viruses with a pig passage followed by only two in vitro passages in MARC-145cells.Inoculation was completed two days after weaning and transport of the pigs to the research facility.It is also possible that the stress from weaning,transport,new socialization,and adjusting to a new environment may have affected the ability of the pigs to respond to concurrent PRRSV-PCV2 infection and in?uenced the level of PRRSV replication in the pigs.However,the data obtained from pigs infected with PRRSV alone indicate that this was not the case and that the ability of the pigs to develop a humoral immune response was normal.

5.Conclusions

Overall,the data indicate no signi?cant differences between the two PRRSV isolates based on clinical signs, gross pathology,histology or hematology even though the PRRSV isolates we utilized in this study were isolated from geographically separated herds(VR-2385from Iowa and NC16845b from North Carolina)over a period of15years. Differences in in vivo replication kinetics were identi?ed. VR-2385initially replicated more quickly and to higher levels and peaked at dpi6and the amount of VR-2385RNA steadily declined thereafter.In contrast,pigs infected with NC16845b had lower levels of PRRSV RNA in serum initially and this steadily increased through termination of the study at dpi12.Concurrent PCV2viremia was enhanced by PRRSV VR-2385infection but not by concurrent PRRSV NC16845b infection.A higher preva-lence of PCV2antigen was demonstrated in the lungs of pigs coinfected with VR-2385(83.3%)compared to pigs coinfected with PRRSV NC16845b(66.7%).This work further emphasizes in vivo replication differences among PRRSV strains and the importance of coinfecting pathogens on PRRSV kinetics.Additional investigations are necessary to further elucidate the speci?c mechanisms of the PCV2-PRRSV interaction in pigs.

Acknowledgment

The authors thank the Iowa Livestock Health Advisory Council for funding of this study.

References

An,T.Q.,Tian,Z.J.,Zhou,Y.J.,Xiao,Y.,Peng,J.M.,Chen,J.,Jiang,Y.F.,Hao, X.F.,Tong,G.Z.,https://www.wendangku.net/doc/da17399974.html,parative genomic analysis of?ve pairs of virulent parental/attenuated vaccine strains of PRRSV.Vet.Microbiol.

149,104–112.

T.Opriessnig et al./Veterinary Microbiology158(2012)69–8179

Allan,G.M.,McNeilly,F.,Ellis,J.,Krakowka,S.,Meehan,B.,McNair,I., Walker,I.,Kennedy,S.,2000.Experimental infection of colostrum deprived piglets with porcine circovirus2(PCV2)and porcine repro-ductive and respiratory syndrome virus(PRRSV)potentiates PCV2 replication.Arch.Virol.145,2421–2429.

Allende,R.,Kutish,G.F.,Laegreid,W.,Lu,Z.,Lewis,T.L.,Rock,D.L.,Friesen, J.,Galeota,J.A.,Doster, A.R.,Osorio, F.A.,2000.Mutations in the genome of porcine reproductive and respiratory syndrome virus responsible for the attenuation phenotype.Arch.Virol145,1149–1161.

Beach,N.M.,Ramamoorthy,S.,Opriessnig,T.,Wu,S.Q.,Meng,X.J.,2010.

Novel chimeric porcine circovirus(PCV)with the capsid gene of the emerging PCV2b subtype cloned in the genomic backbone of the non-pathogenic PCV1is attenuated in vivo and induces protective and cross-protective immunity against PCV2b and PCV2a subtypes in pigs.Vaccine29,221–232.

Chae,C.,2005.A review of porcine circovirus2-associated syndromes and diseases.Vet.J.169,326–336.

Cheung,A.K.,Lager,K.M.,Kohutyuk,O.I.,Vincent,A.L.,Henry,S.C.,Baker, R.B.,Rowland,R.R.,Dunham,A.G.,2007.Detection of two porcine circovirus type2genotypic groups in United States swine herds.Arch.

Virol.152,1035–1044.

Dorr,P.M.,Baker,R.B.,Almond,G.W.,Wayne,S.R.,Gebreyes,W.A.,2007.

Epidemiologic assessment of porcine circovirus type2coinfection with other pathogens in swine.J.Am.Vet.Med.Assoc.230,244–250. Drew,T.W.,2000.A review of evidence for immunosuppression due to porcine reproductive and respiratory syndrome virus.Vet.Res.31, 27–39.

Fang,Y.,Schneider,P.,Zhang,W.P.,Faaberg,K.S.,Nelson,E.A.,Rowland, R.R.,2007.Diversity and evolution of a newly emerged North Amer-ican Type1porcine arterivirus:analysis of isolates collected between 1999and2004.Arch.Virol.152,1009–1017.

Fenaux,M.,Halbur,P.G.,Gill,M.,Toth,T.E.,Meng,X.J.,2000.Genetic characterization of type2porcine circovirus(PCV-2)from pigs with postweaning multisystemic wasting syndrome in different geo-graphic regions of North America and development of a differential PCR-restriction fragment length polymorphism assay to detect and differentiate between infections with PCV-1and PCV-2.J.Clin.Micro-biol.38,2494–2503.

Forsberg,R.,Oleksiewicz,M.B.,Petersen,A.M.,Hein,J.,Botner,A.,Stor-gaard,T.,2001.A molecular clock dates the common ancestor of European-type porcine reproductive and respiratory syndrome virus at more than10years before the emergence of disease.Virology289, 174–179.

Fort,M.,Sibila,M.,Allepuz,A.,Mateu,E.,Roerink,F.,Segales,J.,2008.

Porcine circovirus type2(PCV2)vaccination of conventional pigs prevents viremia against PCV2isolates of different genotypes and geographic origins.Vaccine26,1063–1071.

Gagnon,C.A.,Tremblay,D.,Tijssen,P.,Venne,M.H.,Houde,A.,Elahi,S.M., 2007.The emergence of porcine circovirus2b genotype(PCV-2b)in swine in Canada.Can.Vet.J.48,811–819.

Gauger,P.,Faaberg,K.S.,Guo, B.,Kappes,M.A.,Opriessnig,T.,2012.

Genetic and phenotypic characterization of a2006United States porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the?eld.Virus Res.163,98–107. Halbur,P.G.,Miller,L.D.,Paul,P.S.,Meng,X.J.,Huffman,E.L.,Andrews,J.J., 1995a.Immunohistochemical identi?cation of porcine reproductive and respiratory syndrome virus(PRRSV)antigen in the heart and lymphoid system of three-week-old colostrum-deprived pigs.Vet.

Pathol.32,200–204.

Halbur,P.G.,Paul,P.S.,Frey,M.L.,Landgraf,J.,Eernisse,K.,Meng,X.J.,Lum, M.A.,Andrews,J.J.,Rathje,J.A.,https://www.wendangku.net/doc/da17399974.html,parison of the pathogeni-city of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus.Vet.Pathol.32,648–660. Halbur,P.G.,Paul,P.S.,Meng,X.J.,Lum,M.A.,Andrews,J.J.,Rathje,J.A., https://www.wendangku.net/doc/da17399974.html,parative pathogenicity of nine US porcine reproductive and respiratory syndrome virus(PRRSV)isolates in a?ve-week-old cesar-ean-derived,colostrum-deprived pig model.J.Vet.Diagn.Invest.8, 11–20.

Han,J.,Burkhart,K.M.,Vaughn, E.M.,Roof,M.B.,Faaberg,K.S.,2006.

Replication and expression analysis of PRRSV defective RNA.Adv.

Exp.Med.Biol.581,445–448.

Hanada,K.,Suzuki,Y.,Nakane,T.,Hirose,O.,Gojobori,T.,2005.The origin and evolution of porcine reproductive and respiratory syndrome viruses.Mol.Biol.Evol.22,1024–1031.

Harms,P.A.,Halbur,P.G.,Sorden,S.D.,2002.Three cases of porcine respiratory disease complex associated with porcine circovirus type 2infection.J.Swine Health Prod.10,27–30.

Harms,P.A.,Sorden,S.D.,Halbur,P.G.,Bolin,S.R.,Lager,K.M.,Morozov,I., Paul,P.S.,2001.Experimental reproduction of severe disease in CD/CD

pigs concurrently infected with type2porcine circovirus and porcine reproductive and respiratory syndrome virus.Vet.Pathol.38,528–539.

Horlen,K.P.,Schneider,P.,Anderson,J.,Nietfeld,J.C.,Henry,S.C.,Tokach, L.M.,Rowland,R.R.,2007.A cluster of farms experiencing severe porcine circovirus associated disease:clinical features and associa-tion with the PCV2b genotype.J.Swine Health Prod.15,270–278. Johnson,W.,Roof,M.,Vaughn,E.,Christopher-Hennings,J.,Johnson,C.R., Murtaugh,M.P.,2004.Pathogenic and humoral immune responses to porcine reproductive and respiratory syndrome virus(PRRSV)are related to viral load in acute infection.Vet.Immunol.Immunopathol.

102,233–247.

Jung,K.,Renukaradhya,G.J.,Alekseev,K.P.,Fang,Y.,Tang,Y.,Saif,L.J., 2009.Porcine reproductive and respiratory syndrome virus modi?es innate immunity and alters disease outcome in pigs subsequently infected with porcine respiratory coronavirus:implications for respiratory viral co-infections.J.Gen.Virol.90,2713–2723. Krakowka,S.,Ellis,J.A.,McNeilly,F.,Ringler,S.,Rings,D.M.,Allan,G.,2001.

Activation of the immune system is the pivotal event in the produc-tion of wasting disease in pigs infected with porcine circovirus-2 (PCV-2).Vet.Pathol.38,31–42.

Lager,K.M.,Gauger,P.C.,Vincent,A.L.,Opriessnig,T.,Kehrli Jr.,M.E., Cheung,A.K.,2007.Mortality in pigs given porcine circovirus type 2subgroup1and2viruses derived from DNA clones.Vet.Rec.161, 428–429.

Lv,J.,Zhang,J.,Sun,Z.,Liu,W.,Yuan,S.,2008.An infectious cDNA clone of a highly pathogenic porcine reproductive and respiratory syndrome virus variant associated with porcine high fever syndrome.J.Gen.

Virol.89,2075–2079.

McKeown,N.E.,Opriessnig,T.,Thomas,P.,Guenette,D.K.,Elvinger,F., Fenaux,M.,Halbur,P.G.,Meng,X.J.,2005.Effects of porcine circovirus type2(PCV2)maternal antibodies on experimental infection of piglets with https://www.wendangku.net/doc/da17399974.html,b.Immunol.12,1347–1351. Meng,X.J.,Paul,P.S.,Halbur,P.G.,1994.Molecular cloning and nucleotide sequencing of the30-terminal genomic RNA of the porcine reproduc-tive and respiratory syndrome virus.J.Gen.Virol.75,1795–1801. Meng,X.J.,Paul,P.S.,Halbur,P.G.,Lum,M.A.,1996.Characterization of a high-virulence US isolate of porcine reproductive and respiratory syndrome virus in a continuous cell line,ATCC CRL11171.J.Vet.

Diagn.Invest.8,374–381.

Mengeling,W.L.,Ridpath,J.F.,Vorwald,A.C.,1988.Size and antigenic comparisons among the structural proteins of selected autonomous parvoviruses.J.Gen.Virol.69,825–837.

Nawagitgul,P.,Harms,P.A.,Morozov,I.,Thacker,B.J.,Sorden,S.D.,Lek-charoensuk,C.,Paul,P.S.,2002.Modi?ed indirect porcine circovirus (PCV)type2-based and recombinant capsid protein(ORF2)-based enzyme-linked immunosorbent assays for detection of antibodies to https://www.wendangku.net/doc/da17399974.html,b.Immunol.9,33–40.

Nelsen,C.J.,Murtaugh,M.P.,Faaberg,K.S.,1999.Porcine reproductive and respiratory syndrome virus comparison:divergent evolution on two continents.J.Virol.73,270–280.

Opriessnig,T.,Halbur,P.G.,Yoon,K.J.,Pogranichniy,R.M.,Harmon,K.M., Evans,R.,Key,K.F.,Pallares,F.J.,Thomas,P.,Meng,X.J.,https://www.wendangku.net/doc/da17399974.html,-parison of molecular and biological characteristics of a modi?ed live porcine reproductive and respiratory syndrome virus(PRRSV)vaccine (Ingelvac PRRS MLV),the parent strain of the vaccine(ATCC VR2332), ATCC VR2385,and two recent?eld isolates of PRRSV.J.Virol.76, 11837–11844.

Opriessnig,T.,Halbur,P.G.,Yu,S.,Thacker,E.L.,Fenaux,M.,Meng,X.J., 2006.Effects of the timing of the administration of Mycoplasma hyopneumoniae bacterin on the development of lesions associated with porcine circovirus type2.Vet.Rec.158,149–154. Opriessnig,T.,Patterson,A.R.,Elsener,J.,Meng,X.J.,Halbur,P.G.,2008a.

In?uence of maternal antibodies on ef?cacy of porcine circovirus type 2(PCV2)vaccination to protect pigs from experimental infection with PCV2.Clin.Vaccine Immunol.15,397–401.

Opriessnig,T.,Prickett,J.R.,Madson, D.M.,Shen,H.-G.,Juhan,N.M., Pogranichniy,R.R.,Meng,X.-J.,Halbur,P.G.,2010.Porcine circovirus type2(PCV2)-infection and re-inoculation with homologous or het-erologous strains:virological,serological,pathological and clinical effects in growing pigs.Vet.Res.41,31.

Opriessnig,T.,Ramamoorthy,S.,Madson,D.M.,Patterson,A.R.,Pal,N., Carman,S.,Meng,X.J.,Halbur,P.G.,2008b.Differences in virulence among porcine circovirus type2isolates are unrelated to cluster type 2a or2b and prior infection provides heterologous protection.J.Gen.

Virol.89,2482–2491.

Opriessnig,T.,Thacker,E.L.,Yu,S.,Fenaux,M.,Meng,X.J.,Halbur,P.G., 2004a.Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyop-neumoniae and porcine circovirus type2.Vet.Pathol.41,624–640.

T.Opriessnig et al./Veterinary Microbiology158(2012)69–81 80

Opriessnig,T.,Yu,S.,Gallup,J.M.,Evans,R.B.,Fenaux,M.,Pallare′s,F.J., Thacker,E.L.,Brockus,C.W.,Ackermann,M.R.,Thomas,P.,Meng,X.J., Halbur,P.G.,2003.Effect of vaccination with selective bacterins on conventional pigs infected with type2porcine circovirus.Vet.Pathol.

40,521–529.

Opriessnig,T.,Yu,S.,Thacker,E.,Halbur,P.G.,2004b.Derivation of porcine circovirus type2-negative pigs from positive breeding herds.J.Swine Health Prod.12,186–191.

Pallare′s,F.J.,Halbur,P.G.,Opriessnig,T.,Sorden,S.D.,Villar,D.,Janke,B.H., Yaeger,M.J.,Larson,D.J.,Schwartz,K.J.,Yoon,K.J.,Hoffman,L.J.,2002.

Porcine circovirus type2(PCV-2)coinfections in US?eld cases of postweaning multisystemic wasting syndrome(PMWS).J.Vet.Diagn.

Invest.14,515–519.

Patterson,A.R.,Opriessnig,T.,2010.Epidemiology and horizontal trans-mission of porcine circovirus type2(PCV2).Anim.Health Res.Rev.11, 217–234.

Pirzadeh,B.,Gagnon,C.A.,Dea,S.,1998.Genomic and antigenic variations of porcine reproductive and respiratory syndrome virus major envel-ope GP5glycoprotein.Can.J.Vet.Res.62,170–177. Renukaradhya,G.J.,Alekseev,K.,Jung,K.,Fang,Y.,Saif,L.J.,2010.Porcine reproductive and respiratory syndrome virus-induced immunosup-pression exacerbates the in?ammatory response to porcine respira-tory coronavirus in pigs.Viral Immunol.23,457–466.

Richt,J.A.,Lekcharoensuk,P.,Lager,K.M.,Vincent,A.L.,Loiacono,C.M., Janke,B.H.,Wu,W.H.,Yoon,K.J.,Webby,R.J.,Solorzano,A.,Garcia-Sastre,A.,2006.Vaccination of pigs against swine in?uenza viruses by using an NS1-truncated modi?ed live-virus vaccine.J.Virol.80, 11009–11018.

Rovira,A.,Balasch,M.,Segale′s,J.,Garcia,L.,Plana-Du′ran,J.,Rosell,C., Ellerbrok,H.,Mankertz,A.,Domingo,M.,2002.Experimental inocu-lation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus2.J.Virol.76,3232–3239. Rowland,R.R.,Steffen,M.,Ackerman,T.,Ben?eld,D.A.,1999.The evolu-tion of porcine reproductive and respiratory syndrome virus:qua-sispecies and emergence of a virus subpopulation during infection of pigs with VR-2332.Virology259,262–266.

Shibata,I.,Yazawa,S.,Ono,M.,Okuda,Y.,2003.Experimental dual infection of speci?c pathogen-free pigs with porcine reproductive and respiratory syndrome virus and pseudorabies virus.J.Vet.Med.B: Infect.Dis.Vet.Public Health50,14–19.

Sinha,A.,Shen,H.G.,Schalk,S.,Beach,N.M.,Huang,Y.W.,Meng,X.J., Halbur,P.G.,Opriessnig,T.,2011.Porcine reproductive and respira-tory syndrome virus(PRRSV)in?uences infection dynamics of por-cine circovirus type2(PCV2)subtypes PCV2a and PCV2b by prolonging PCV2viremia and shedding.Vet.Microbiol.152,235–246. Sorden,S.D.,Harms,P.A.,Nawagitgul,P.,Cavanaugh,D.,Paul,P.S.,1999.

Development of a polyclonal-antibody-based immunohistochemical

method for the detection of type2porcine circovirus in formalin-?xed,paraf?n-embedded tissue.J.Vet.Diagn.Invest.11,528–530.

Tian,K.,Yu,X.,Zhao,T.,Feng,Y.,Cao,Z.,Wang,C.,Hu,Y.,Chen,X.,Hu,D., Tian,X.,Liu,D.,Zhang,S.,Deng,X.,Ding,Y.,Yang,L.,Zhang,Y.,Xiao,H., Qiao,M.,Wang,B.,Hou,L.,Wang,X.,Yang,X.,Kang,L.,Sun,M.,Jin,P., Wang,S.,Kitamura,Y.,Yan,J.,Gao,G.F.,2007.Emergence of fatal PRRSV variants:unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark.PLoS ONE2,e526. Tong,G.Z.,Zhou,Y.J.,Hao,X.F.,Tian,Z.J.,An,T.Q.,Qiu,H.J.,2007.Highly pathogenic porcine reproductive and respiratory syndrome.China.

Emerg.Infect Dis13,1434–1436.

Van Reeth,K.,Nauwynck,H.,2000.Proin?ammatory cytokines and viral respiratory disease in pigs.Vet.Res.31,187–213.

Van Reeth,K.,Nauwynck,H.,Pensaert,M.,1996.Dual infections of feeder pigs with porcine reproductive and respiratory syndrome virus fol-lowed by porcine respiratory coronavirus or swine in?uenza virus:a clinical and virological study.Vet.Microbiol.48,325–335.

Wang,Y.,Liang,Y.,Han,J.,Burkhart,K.M.,Vaughn,E.M.,Roof,M.B., Faaberg,K.S.,2008.Attenuation of porcine reproductive and respira-tory syndrome virus strain MN184using chimeric construction with vaccine sequence.Virology371,418–429.

Wu,J.,Li,J.,Tian,F.,Ren,S.,Yu,M.,Chen,J.,Lan,Z.,Zhang,X.,Yoo,D.,Wang, J.,2009.Genetic variation and pathogenicity of highly virulent por-cine reproductive and respiratory syndrome virus emerging in China.

Arch.Virol.154,1589–1597.

Yu,S.,Opriessnig,T.,Kitikoon,P.,Nilubol,D.,Halbur,P.G.,Thacker,E., 2007.Porcine circovirus type2(PCV2)distribution and replication in tissues and immune cells in early infected pigs.Vet.Immunol.

Immunopathol.115,261–272.

Yuan,S.,Mickelson,D.,Murtaugh,M.P.,Faaberg,K.S.,https://www.wendangku.net/doc/da17399974.html,plete genome comparison of porcine reproductive and respiratory syn-drome virus parental and attenuated strains.Virus Res.79,189–200. Yuan,S.,Murtaugh,M.P.,Faaberg,K.S.,2000.Heteroclite subgenomic RNAs are produced in porcine reproductive and respiratory syndrome virus infection.Virology275,158–169.

Yuan,S.,Murtaugh,M.P.,Schumann,F.A.,Mickelson,D.,Faaberg,K.S., 2004.Characterization of heteroclite subgenomic RNAs associated with PRRSV infection.Virus Res.105,75–87.

Yuan,S.,Nelsen,C.J.,Murtaugh,M.P.,Schmitt,B.J.,Faaberg,K.S.,1999.

Recombination between North American strains of porcine repro-ductive and respiratory syndrome virus.Virus Res.61,87–98. Zhou,L.,Zhang,J.,Zeng,J.,Yin,S.,Li,Y.,Zheng,L.,Guo,X.,Ge,X.,Yang,H., 2009.The30-amino-acid deletion in the Nsp2of highly pathogenic porcine reproductive and respiratory syndrome virus emerging in China is not related to its virulence.J.Virol.83,5156–5167.

T.Opriessnig et al./Veterinary Microbiology158(2012)69–8181