C1QBP inhibits proliferation of porcine circovirus type 2 by restricting nuclear import of the capsid protein.

Complement component 1 Q subcomponent-binding protein (C1QBP) has been shown to interact with the porcine circovirus type 2 (PCV2) Cap protein. Here, using yeast two-hybrid (Y2H) and co-immunoprecipitation assays, as well as laser confocal microscopy, the interaction between C1QBP and Cap was confirmed. Furthermore, overexpression of C1QBP in cells altered the intracellular location of Cap, which was observed using confocal microscopy and verified by detection of Cap in nuclear protein extracts in a Western blot assay. By inhibiting nuclear transport of Cap, overexpression of C1QBP downregulated PCV2 proliferation in PK-15 cells, as determined by quantitative polymerase chain reaction (qPCR). As C1QBP plays a similar role in a fusion of green fluorescent protein (GFP) with the Cap nuclear localisation signal (NLS) sequence, (CapNLS-GFP), we propose that the target site for C1QBP in Cap is possibly located in the NLS region. Considering all the results together, this study demonstrated that C1QBP interacts with the Cap NLS region, resulting in changes in the intracellular localisation of the Cap protein. We confirmed that overexpression of C1QBP inhibits the proliferation of PCV2, and this is possibly related to the function of C1QBP in controlling nuclear transport of Cap.

Endothelial IL-8 induced by porcine circovirus type 2 affects dendritic cell maturation and antigen-presenting function.

BACKGROUND: Porcine circovirus (PCV) disease caused by PCV type 2 (PCV2) is mainly attributed to immunosuppression and immune damage. PCV2 can infect vascular endothelial cells and induce high expression of endothelial IL-8. Dendritic cells (DCs), as professional antigen-presenting cells, can not only present antigens but also activate naïve T-cells, causing an immune response. METHODS: To demonstrate whether endothelial IL-8 is the main factor inhibiting the maturation and related functions of dendritic cells during PCV2 infection, monocyte-derived DCs (MoDCs) and porcine iliac artery endothelial cells (PIECs) processed by different methods were co-cultured in two ways. Flow cytometry, molecular probe labeling, fluorescence quantitative PCR, and the MTS assay were used to detect the changes in related functions and molecules of MoDCs. RESULTS: Compared to those in the PIEC-DC group, the endothelial IL-8 upregulation co-culture group showed significantly lower double-positive rates for CD80/86 and MHC-II of MoDCs and significantly increased endocytosis of MoDCs. Meanwhile, the adhesion rate and average fluorescence intensity of MoDCs were significantly downregulated in migration and adhesion experiments. Furthermore, the MHC-I and LAMP7 mRNA levels in MoDCs and the proliferation of MoDC-stimulated T-cells were markedly reduced. However, the changes in MoDCs of the endothelial IL-8 downregulation co-culture group were the opposite. CONCLUSIONS: PCV2-induced endothelial IL-8 reduces the adhesion and migration ability of MoDCs, resulting in a decreased maturation rate of MoDCs, and further inhibits antigen presentation by DCs. These results may explain the immunosuppressive mechanism of PCV2 from the perspective of the interaction between endothelial cells and DCs in vitro.

Porcine circovirus type 1 was undetected in vaccine but could be cultured in the cell substrate of Lanzhou lamb rotavirus vaccine.

In 2010, Rotarix was found to be contaminated with infectious porcine circovirus type 1 (PCV1). In China, the Lanzhou lamb rotavirus (LLR) vaccine is the only vaccine used to prevent rotavirus disease. From 2006 to September 2014, more than 54 million doses of LLR vaccines have been lot released. It is a safety issue whether PCV1 is present in the LLR vaccine. Although the cell substrate of LLR, bovine kidney (BK), is different from that of Rotarix, we have investigated the cell's permissivity for PCV1 by both infectivity and full-length PCR analysis. We have assessed the LLR using a quantitative PCR (qPCR) assay. A total of 171 random batches of LLR final products over a period of 5 years were tested, and no PCV1 was detected (0/171). Infectivity studies showed that two strains of PCV1, the PCV1-prototype, which was derived from PK-15 cells, and the mutant, PCV1-GSK, which was isolated from Rotarix, were capable of replicating in BK cells over a wide m.o.i. ranging from 10 to 0.01. After culture for 6 days, copies of PCV1-prototype DNA were higher than those of PCV1-GSK on average. The genome of the virus was detected at 6 days post-infection. In summary, the LLR vaccine is free of PCV1. Nevertheless, because PCV1 can replicate in the BK cell substrate, manufacturers need to be vigilant in monitoring for this adventitious agent.

Xenogenic rolling-circle replication of a synthetic beak and feather disease virus genomic clone in 293TT mammalian cells and Nicotiana benthamiana.

The preparation of infectious beak and feather disease circovirus virions (BFDV) has until now relied on the extraction of virus from whole tissue of deceased or euthanized parrots known to be infected with the virus. Extraction from diseased tissue is necessary, as the virus has yet to be grown in vitro using tissue-cultured cells from any source. While infectious DNA clones have been synthesized for porcine and duck circoviruses, and both replicate in host cells and result in active viral infection in animals, this has not been shown for BFDV. The aim of this study was to prepare an infectious BFDV genomic clone that could be used as challenge material in birds for vaccine testing. A putatively infectious BFDV genomic clone was designed and tested in mammalian cell culture, and in the plant Nicotiana benthamiana in the presence of plant-specific ssDNA geminivirus replication components. Replication was assessed using rolling-circle amplification, qPCR, replication-deficient clones and rescue plasmids. We showed that a synthetic partially dimeric BFDV genomic clone self-replicated when transfected into 293TT mammalian cells, and was also replicated in N. benthamiana in the presence of geminivirus replication elements. This is the first report of a BFDV genome replicating in any cell system, and the first report of a circovirus replicating with the aid of a geminivirus in a plant. Both of these developments could open up possibilities for making reagents and vaccines for BFDV, testing vaccine efficacy and investigating viral replication using rationally designed artificial genomes.

Proteomic alteration of PK-15 cells after infection by porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) has been identified as the essential causal agent of post-weaning multisystemic wasting syndrome, which has spread worldwide. To discover cellular protein responses of PK-15 cells to PCV2 infection, two-dimensional liquid chromatography-tandem mass spectrometry (MS) coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the proteins that were differentially expressed in PK-15 from the PCV2-infected group compared to the uninfected control group. A total of 196 cellular proteins in PK-15 that were significantly altered at different time periods post-infection were identified. These differentially expressed proteins were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc. and their interactions. Moreover, some of these proteins were further confirmed by Western blot. The high number of differentially expressed proteins identified should be very useful in elucidating the mechanism of replication and pathogenesis of PCV2 in the future.

Propagation of viruses infecting waterfowl on continuous cell lines of Muscovy duck (Cairina moschata) origin.

Duck circovirus, duck hepatitis A virus 1, goose parvovirus and goose haemorrhagic polyomavirus are economically damaging pathogens of waterfowl, and replicate poorly or not at all in established cell lines. AGE1.CR, AGE1.CR.pIX and AGE1.CS cell lines, originating from the Muscovy duck, were tested for their suitability to isolate and identify these viruses. Immunofluorescence (IF) and quantitative polymerase chain reaction investigations verified that all cell lines are permissive for all four viruses; however, AGE1.CR.pIX proved to be the most productive and most sensitive for viral infection. IF experiments revealed that the time of one infectious cycle is approximately 12 to 14 h in the AGE1.CR.pIX cells in the case of the three DNA viruses, while it is 10 to 12 h for DHAV-1. Specific viral infectivity and the limit of detection by IF varied between 55 and 1484 copies, depending on the viruses and cell lines. Despite the high sensitivity of the cell lines for viruses, their viral productivity remained relatively low for the investigated field isolates. However, optimization of virus infection and/or the adaptation of the viruses to the cells can raise viral productivity and can make these cell lines suitable for vaccine development and production.

In vitro intragenomic rearrangement of porcine circovirus type 2.

We report here for the first time the genome sequence of a rearranged porcine circovirus type 2 (PCV2) strain, CH-IVT1, isolated from PCV2-infected PK-15 cells. The complete circular genome of the CH-IVT1 is 605 nucleotides (nt) in length. The finding will help us to understand the molecular evolution of PCV2 and the relationship between PCV2 and PCV-associated diseases.

Coreplication of the major genotype group members of porcine circovirus type 2 as a prerequisite to coevolution may explain the variable disease manifestations.

A member of the family Circoviridae, porcine circovirus type 2 (PCV2), is associated with postweaning multisystemic wasting syndrome (PMWS), a recent emerging disease worldwide. PCV2 is also found in clinically asymptomatic animals. This paradoxical finding makes the syndrome etiology challenging. We developed new assays to study PCV2 with links to syndrome etiology. For analysis, we used PCV2-infected tissues from subclinically infected and diseased piglets. We compared antigen- and PCV2 DNA-derived signals for tissue localization and intensity. Oligonucleotides were designed to the signature motif of the PCV2 capsid open reading frame to discriminate experimentally between PCV2 genotype groups by PCR, in situ hybridization (ISH), and fluorescence in situ hybridization (FISH). Unexpectedly, all PCV2-infected animals carried both PCV2a and PCV2b genotype group members. Using confocal microscopy, genotype single-cell infections and cell superinfections were visible. Additionally, we discriminated replicative DNA from total PCV2 DNA isoforms with FISH. This aided in our inquiry into cellular genotype-specific replication. Importantly, single-genotype-group replication was not observed. In infected cells with replicating virus, both genotype groups were equally present. These findings suggest PCV2 genotype group members relaxed replication regulation requirements and may even point to PCV2 replication cooperativity in vivo. These observations explain the readily seen PCV2 DNA recombinations and the high overall PCV2 genome plasticity. Hence, we suggest a novel mechanism of syndrome etiology that consists of a continuously changing PCV2 genome pool in hosts and pig herds, posing a constant challenge to the individual maturing immune system.

Instability in vitro of a PCV2 infectious clone containing an insertion between ORF1 and ORF2.

Porcine circovirus 2 (PCV2) is a small circular single-stranded DNA virus that causes postweaning multisystemic wasting syndrome in pigs. We obtained an infectious clone, derived from PCV2 strain 1147 (size 1,767 bp), with a 60 bp insertion in the cap-rep intergenic region at the 3' end of the rep. In vitro propagation and sequencing of virus showed mixed sequence for the cap-rep intergenic region. Cloning of this region of virus from the passages 2 and 20 showed deletions of various size of the 60 bp insertion. Viruses from the passage 20 showed deletions of the 60 bp insertion and had extra (+1 to +5) or less (-1 or -2) bp in the intergenic region as compared to 1,147. These findings suggest that insertion of a 60-bp DNA sequence at the 3' end of the rep is unstable in vitro. This finding can have implications for the genetic engineering of PCV2.

Characterization of porcine circovirus-like virus P1 replication and lesions in BALB/c mice.

Five-week-old male BALB/c mice were inoculated intraperitoneally with a single (sP1) or multiple doses (mP1) of porcine circovirus-like virus P1 or cell culture medium. None of the mice exhibited clinical signs or gross lesions throughout the study. However, the body weights of the mP1 mice were significantly decreased, and the mice inoculated with P1 exhibited viral replication, seroconversion, and microscopic lesions. P1 nucleic acid was detected in the heart, liver, spleen, lung, bladder, testis, brain, thymus, and pancreatic tissues. Special P1 antibody was found in the P1-inoculated mice. Microscopic lesions in the sP1 and mP1 mice were characterized by interstitial pneumonia, including edema in the connective tissue around the pulmonary vessels, mild inflammatory cell infiltrate, thickened alveolar walls, myocardial necrosis, and dissolution of Purkinje cell nuclei. The results showed that the P1 virus could infect BALB/c mice. Thus, BALB/c mice may serve as models for P1 research.

Development and validation of a SYBR green real-time PCR for the quantification of porcine circovirus type 2 in serum, buffy coat, feces, and multiple tissues.

The emergence of multiple genotypes of PCV2, as demonstrated by phylogenetic analysis of whole genome or capsid sequences, makes it necessary to have quantitative diagnostic assays that perform equally well on all strains. The objectives of this study were to develop and validate a novel real-time polymerase chain reaction (PCR) assay targeting the highly conserved rep gene (ORF1) and investigate the effects of diagnostic specimen choice on its performance. The assay was tested in naturally infected conventional pigs, experimentally infected gnotobiotic pigs, and plasmid-spiked negative serum, lung tissue, and feces and found to have a linear detection range of 2.2x10(3) to 2.2x10(10) copies of PCV2 per mL. The assay successfully detected and quantified PCV2 DNA in serum, buffy coat, feces, and multiple lymphoid (bronchial, mesenteric, and superficial inguinal lymph nodes; thymus; tonsil; ileal Peyer's patches; and spleen), and non-lymphoid (myocardium; lung; kidney; liver; and gluteal muscle) tissues from naturally infected pigs. Across all tissues and sera of naturally infected pigs, the mean PCV2 concentration was 3.0logs higher in wasting versus non-wasting pigs. PCV2 concentration measured by tissue culture and immunohistochemical staining in homogenized liver samples of experimentally infected gnotobiotic pigs were compared to the concentrations estimated by quantitative PCR. Similar trends were noted with increasing PCV2 concentration detected in subclinically infected to severely PMWS-affected pigs across all assays. Our diagnostic assay was developed with a conserved target sequence, and performed efficiently in quantification of PCV2 in a variety of tissues from naturally and experimentally infected pigs.

Porcine circovirus type 2 induces a strong cytopathic effect in the serum-free culture cell line CPK-NS.

When the adherent stable serum-free porcine kidney cell line CPK-NS were inoculated with porcine circovirus type 2 (PCV2) and passaged, viral titre concentration-dependent cell detachment was observed. The copy number of viral genes in supernatants of the infected CPK-NS cells decreased as cell detachment progressed. Furthermore, cell detachment was completely inhibited via neutralisation of the virus using antisera collected from PCV2-infected specific pathogen-free pigs. These results indicated that detachment of CPK-NS cells is a cytopathic effect (CPE) caused via infection with PCV2. Only a single round of cell passaging was required to observe clear a CPE when the inoculated viral titre was significantly high [≥104.5 median tissue culture infectious dose (TCID50)/mL]. Our study confirms that PCV2, which is normally non-cytopathogenic, is capable of inducing a distinct CPE in CPK-NS cells. Application of CPK-NS cells for detection of viruses may contribute towards the diagnosis and control of PCV2-mediated infectious diseases.

Human cells are permissive for the productive infection of porcine circovirus type 2 in vitro.

Porcine circovirus 2 (PCV2) is the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases, which are widespread in swine-producing countries. However, there is controversy regarding the susceptibility of human cells to PCV2 infection. In this study, human cell lines were infected with PCV2 and blind passaged several times. PCV2 entered and replicated in human cells, and infectious virions were generated, indicating that human cell lines were permissive to PCV2 replication. Furthermore, PCV2 replication in human cell lines was enhanced by D-glucosamine or concanavalin A (ConA). However, the infection efficiency of PCV2 was lower in human cells than in PK-15 cells, suggesting that PCV2 infection was limited in human cells. Our study reveals that human cells are permissive for the productive infection of porcine circovirus type 2 in vitro.

Generation of PK-15 cell lines highly permissive to porcine circovirus 2 infection by transposon-mediated interferon-gamma gene transfer.

Porcine circovirus 2 (PCV2)-associated diseases affect the swine industry worldwide. Vaccination is the major tool for the disease control, but the vaccine production is hindered by lower propagation rate of PCV2 in vitro. Previous studies showed that interferons (IFNs) can increase PCV2 yield in PK-15 cells. In the present study, we constructed a Sleepy Beauty (SB) transposon vector expressing porcine IFNg gene fused with the coding sequence for immunoglobulin G Fc domain. After dilution cloning, the transposon and transposase vectors were co-transfected into PK-15 cell clones with higher permissivity to PCV2 infection. Two transgenic PK-15 cell lines, namely PK15-IFNgRan and PK15-IFNgSB which contained randomly integrated transfer vector or SB cassette without selection marker, were screened by PCR analysis. The characterization results demonstrated that the two transgenic cell lines can stably express IFNg-Fc fusion protein with potent antiviral activities. Both viral titration and quantitative PCR analyses showed that the two transgenic cell lines are highly permissive to PCV2 infection with significantly increased viral yields. These results indicate that the two transgenic PK-15 cell lines, PK15-IFNgSB in particular, can be used for PCV2 vaccine development.

Porcine HMGCR Inhibits Porcine Circovirus Type 2 Infection by Directly Interacting with the Viral Proteins.

Porcine circovirus type 2 (PCV2) is the etiological agent of porcine circovirus diseases and porcine circovirus-associated diseases (PCVDs/PCVADs). However, the pathogenesis of PCV2 is not fully understood. We previously found that 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is negatively associated with PCV2 infection in vitro and in vivo. HMGCR inhibits the early stages of PCV2 infection, while PCV2 infection induces the phosphorylation of HMGCR to inactivate the protein. In this study, we investigated the possibility that adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), and protein phosphatase 2 (PP2A) participate in HMGCR-mediated inhibition of PCV2 infection and the interaction of porcine HMGCR with PCV2 proteins. The results showed that AMPK activity fluctuated in cells during the early stage of PCV2 infection, while PP2A had little effect on PCV2 infection and HMGCR activity. Furthermore, PCV2 infection may enhance or maintain the level of phosphorylated HMGCR by directly interacting with the protein in PK-15 cells. These findings may provide a better understanding of PCV2 pathogenesis, and HMGCR may be a novel PCV2 antiviral target.

Adenovirus-mediated shRNA interference against porcine circovirus type 2 replication both in vitro and in vivo.

Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome (PMWS), which is responsible for the heavy economic losses in stockbreeding. There are no specific antiviral drugs for treatment of the virus infection. We have now constructed two recombinant adenoviruses expressing short-hairpin RNAs (shRNAs) directed against either ORF1 (rAdS1) or ORF2 (rAdS2) of PCV2 and measured the inhibition of PCV2 replication. The results showed that delivery of these shRNAs by recombinant adenovirus into PK15 cells could induce a significant inhibition of viral RNA and DNA replication and protein synthesis level in cells subsequently infected with PCV2. The antiviral effect was dose-dependent and could sustain at least for 120h and the inhibition of virus replication could be significantly strengthened by combination of rAdS1 with rAdS2. Mice injected with shRNA before PCV2 infection showed substantial and low level of PCV2 DNA replication in the spleen during the period of 21-28 days post-PCV2 infection. These results indicated that shRNAs generated by adenovirus could sufficiently and continuously inhibit PCV2 infection in vitro as well as in vivo. The adenovirus based shRNA targeting ORF1 and ORF2 of PCV2 might be a new potential alternative strategy for controlling PCV2 infection.

Characterization of porcine circovirus type 2 (PCV2) infection in swine lymphocytes using mitogen-stimulated peripheral blood lymphocytes from healthy PCV2-carrier pigs.

Information regarding the susceptibility of swine lymphocytes to PCV2 is rather limited. To further explore and characterize the PCV2 infection in swine lymphocytes, an in vitro model using concanavalin A (Con A)-stimulated peripheral blood lymphocytes (PBLs) obtained from clinically healthy PCV2-carrier pigs was introduced. It was found that the PCV2 antigen-containing rate was below 2% in PBLs from healthy PCV2-free pigs following treated simultaneously with Con A and PCV2. However, significantly higher PCV2 antigen- and nucleic acid-containing rates could be seen in Con A-stimulated PBLs from clinically healthy PCV2-carrier pigs. Prior to Con A treatment, both of the PCV2 antigen- and nucleic acid-containing rates in PBLs from healthy PCV2-carrier pigs were less than 1%; however, they reached 22.1+/-5.7% by flow cytometry and 27.1+/-6.5% by in situ hybridization, respectively, at 4-day post-incubation with Con A. Phenotyping of PCV2 antigen-containing cells revealed that PCV2-positive cells could be detected in both T and B lymphocyte populations within which IgM-positive B lymphocytes appeared to have a relatively higher positive rate. The Con A-stimulated PBLs also displayed a significantly higher viral load by the measurement of either PCV2 DNA copy number or viral titer when compared with the non-treated PBLs from healthy PCV2-carrier pigs. The results indicate that PBLs, especially IgM-bearing B lymphocytes, are indeed susceptible to PCV2 infection and PCV2 is capable of replicating in dividing lymphocytes. This activation-induced replication may explain in part the pathogenesis of lymphoid depletion in PMWS-affected pigs.

In vitro and in silico studies reveal capsid-mutant Porcine circovirus 2b with novel cytopathogenic and structural characteristics.

Porcine circovirus 2 (PCV2) is an icosahedral, non-enveloped, and single-stranded circular DNA virus that belongs to the family Circoviridae, genus Circovirus, and is responsible for a complex of different diseases defined as porcine circovirus diseases (PCVDs). These diseases - including postweaning multisystemic wasting syndrome (PMWS), enteric disease, respiratory disease, porcine dermatitis and nephropathy syndrome (PDNS), and reproductive failure - are responsible for large economic losses in the pig industry. After serial passages in swine testicle (ST) cells of a wild-type virus isolated from an animal with PMWS, we identified three PCV2b viruses with capsid protein (known as Cap protein) cumulative mutations, including two novel mutants. The mutant viruses were introduced into new ST cell cultures for reisolation and showed, in comparison to the wild-type PCV2b, remarkable viral replication efficiency (> 1011 DNA copies/ml) and cell death via necrosis, which were clearly related to the accretion of capsid protein mutations. The analysis of a Cap protein/capsid model showed that the mutated residues were located in solvent-accessible positions on the external PCV2b surface. Additionally, the mutated residues were found in linear epitopes and participated in pockets on the capsid surface, indicating that these residues could also be involved in antibody recognition. Taking into account the likely natural emergence of PCV2b variants, it is possible to consider that the results of this work increase knowledge of Circovirus biology and could help to prevent future serious cases of vaccine failure that could lead to heavy losses to the swine industry.

Taurine attenuates OTA-promoted PCV2 replication through blocking ROS-dependent autophagy via inhibiting AMPK/mTOR signaling pathway.

Previous research found that ochratoxin A (OTA) could promote PCV2 replication by inducing autophagy. The aim of this study is to evaluate the effect of dietary amino acid derivative taurine on OTA-promoted PCV2 replication and explore the underlying mechanism. The results showed that taurine could inhibit OTA-promoted PCV2 replication in PK-15 cells. The effect of taurine could be mediated by its ability to attenuate ROS level and block OTA-promoted autophagy. Indeed, induction of autophagy by rapamycin could suppress the inhibitory effect of taurine on OTA-promoted PCV2 replication. Furthermore, taurine supplementation inhibited 5'AMP-activated protein kinase (AMPK) and activated mammalian target of rapamycin (mTOR). Activation of AMPK by acadesine (AICAR) could suppress the effect of taurine. In conclusion, taurine treatment suppresses autophagy by regulating the ROS/AMPK/mTOR signaling axis, thereby inhibiting OTA-promoted PCV2 replication. These findings provide the rationale for the use of taurine as an intervention against PCV2 infection.

Quantification of PCV2 capsid transcript in peripheral blood mononuclear cells (PBMCs) in vitro.

The presence of PCV2 DNA or spliced capsid mRNA (Cap mRNA) for viral replication was assessed following addition of PCV2 to resting or concanavalin A (ConA) stimulated peripheral blood mononuclear cells (PBMCs). Real-time PCR or real-time RT-PCR assays were used to measure viral DNA or Cap mRNA, respectively. The study demonstrated that PCV2 replication increased in infected PBMCs over time. Replication within infected PBMCs was significantly (P<0.05) increased when PBMCs were stimulated with ConA, compared to unstimulated PBMCs. The data showed a strong correlation between the level of PCV2 Cap mRNA and the level of viral DNA in the ConA stimulated PBMCs. Replication of PCV2 was also assessed in T lymphocyte- and monocyte/macrophage-enriched or monocyte/macrophage-depleted PBMC populations which had been stimulated with ConA for 3 days. It was demonstrated that the enriched T lymphocytes and the monocyte/macrophage-depleted PBMCs had significantly higher Cap mRNA and viral DNA levels (P<0.05) compared to the monocyte/macrophage-enriched population, indicating that in addition to monocytes/macrophages, PCV2 replicates in lymphocytes, particularly T lymphocytes following stimulation. These results suggest that the presence of activated T lymphocytes may play an important role in PCV2 replication and potentially the development of clinical disease.