Porcine Circovirus Type 2 Pathogenesis Research Articles

A novel PCV2 ORF5-interacting host factor YWHAB inhibits virus replication and alleviates PCV2-induced cellular response

Porcine circovirus type 2 (PCV2) infection causes porcine circovirus associated diseases (PCVAD) worldwide. Identification of host factors that interact with viral proteins is a fundamental step to understand the pathogenesis of PCV2. Our previous study reported that ORF5, a newly identified PCV2 viral protein supports PCV2 replication and interacts with multiple host factors. Here, we showed that a host factor YWHAB is an ORF5-interacting protein and plays essential roles during PCV2 infection. By using protein-protein interaction assays, we confirmed that YWHAB directly interacts with PCV2-ORF5 protein. We further showed that YWHAB expression was potently induced upon ORF5 overexpression and PCV2 infection. Remarkably, we found that the YWHAB strongly inhibited PCV2 replication, suggesting its role in defending PCV2 infection. By using the ectopic overexpression and gene knockdown approaches, we revealed that YWHAB inhibits PCV2-induced endoplasmic reticulum stress (ERS), autophagy, reactive oxygen species (ROS) production and apoptosis, suggesting its vital role in alleviating PCV2-induced cellular damage. Together, this study demonstrated that an ORF5-interacting host factor YWHAB affects PCV2 infection and PCV2-induced cellular response, which expands the current understanding of YWHAB biological function and might serves as a new therapeutic target to manage PCV2 infection-associated diseases.

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.

Porcine circovirus type 2 ORF5 protein induces endoplasmic reticulum stress and unfolded protein response in porcine alveolar macrophages.

Porcine circovirus type 2 (PCV2) is the essential infectious agent causing porcine circovirus-associated disease (PCVD) in pigs and one of the important viruses that severely jeopardize the swine husbandry industry. PCV2 elicits the unfolded protein response (UPR) via activation of the PERK pathway, and its capsid protein (Cap) has also been found to induce UPR with subsequent activation of apoptosis. The open reading frame 5 (ORF5) protein is a recently discovered non-structural protein, and its function in PCV2 pathogenesis remains unknown. The aim of this study was to determine whether the PCV2 ORF5 protein could induce endoplasmic reticulum stress (ERS) and UPR in porcine alveolar macrophages (PAMs). pEGFP-tagged ORF5 protein was transiently overexpressed in PAMs. Transmission electron microscopy (TEM) was employed to examine changes in ER morphology, and quantitative real-time PCR and western blotting analysis were used to measure UPR-related cell signaling alterations. We found that the ORF5 protein triggers swelling and degranulation of the ER and upregulates the expression of ERS markers. Further experiments demonstrated that the PCV2 ORF5 protein induces ERS and UPR via the PERK (RNA-activated protein kinase-like endoplasmic reticulum kinase), ATF6 (activating transcription factor 6) and IRE1 (inositol requiring enzyme 1) signaling pathways. Together with previous studies, we provide new information on the ERS-UPR induced by the PCV2 ORF5 protein.

Porcine circovirus type 2 capsid protein induces unfolded protein response with subsequent activation of apoptosis.

Porcine circovirus type 2 (PCV2) has recently been reported to elicit the unfolded protein response (UPR) via activation of the PERK/eIF2α (RNA-activated protein kinase-like endoplasmic reticulum (ER) kinase/eukaryotic initiation factor 2α) pathway. This study attempted to examine which viral protein might be involved in inducing UPR and whether this cellular event would lead to apoptosis of the cells expressing the viral protein. By transient expression, we found that both replicase (Rep) and capsid (Cap) proteins of PCV2 could induce ER stress as shown by increased phosphorylation of PERK with subsequent activation of the eIF2α-ATF4 (activating transcription factor 4)-CHOP (CCAAT/enhancer-binding protein homologous protein) axis. Cap expression, but not Rep, significantly reduced anti-apoptotic B-cell lymphoma-2 (Bcl-2) and increased caspase-3 cleavage, possibly due to increased expression of CHOP. Since knockdown of PERK by RNA interference clearly reduced Cap-induced CHOP expression, caspase-3 cleavage, and apoptotic cell death possibly by partially rescuing Bcl-2 expression, we propose that there is connection between Cap-induced UPR and apoptosis via the PERK/eIF2α/ATF4/CHOP/Bcl-2 pathway. This study, together with our earlier studies, provides insight into the mechanisms underlying PCV2 pathogenesis.

Heat shock protein 90 is essential for replication of porcine circovirus type 2 in PK-15 cells

Porcine circovirus type 2 (PCV2) is recognized as the causative agent of porcine circovirus-associated disease (PCVAD). However, the mechanism of PCV2 replication has not been understood completely. Heat shock protein 90 (Hsp90) plays an important role in viral genome replication, viral genes expression, and viral particle packaging. In this study, we firstly found that inhibition of Hsp90 by pretreatment of host cells with 17-AAG, a specific inhibitor of Hsp90, or blocking Hsp90α/Hsp90β with siRNA, resulted in significantly reduced viral replication in PK-15 cells. But inhibition of Hsp90 by 17-AAG did not affect PCV2 entry into the host cells. Meanwhile, over-expression of Hsp90α/Hsp90β enhanced PCV2 genome replication and virion production. In addition, Hsp90β was enriched in the nuclear zone in the cells infected with PCV2. But it did not interact with the viral Cap/Rep proteins. It suggested that Hsp90 is required for PCV2 production in PK-15 cells culture. It should be helpful for further evaluating the mechanism of replication and pathogenesis of PCV2 and developing novel antiviral therapies.

Porcine Circovirus Type 2 Activates CaMMKβ to Initiate Autophagy in PK-15 Cells by Increasing Cytosolic Calcium.

Porcine circovirus type 2 (PCV2) induces autophagy via the 5′ adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/tuberous sclerosis complex 2 (TSC2)/mammalian target of rapamycin (mTOR) pathway in pig kidney PK-15 cells. However, the underlying mechanisms of AMPK activation in autophagy induction remain unknown. With specific inhibitors and RNA interference (RNAi), we show that PCV2 infection upregulated calcium/calmodulin-dependent protein kinase kinase-beta (CaMKKβ) by increasing cytosolic Ca2+ via inositol 1,4,5-trisphosphate receptor (IP3R). Elevation of cytosolic calcium ion (Ca2+) did not seem to involve inositol 1,4,5-trisphosphate (IP3) release from phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide phospholipase C-gamma (PLC-γ). CaMKKβ then activated both AMPK and calcium/calmodulin-dependent protein kinase I (CaMKI). PCV2 employed CaMKI and Trp-Asp (WD) repeat domain phosphoinositide-interacting protein 1 (WIPI1) as another pathway additional to AMPK signaling in autophagy initiation. Our findings could help better understanding of the signaling pathways of autophagy induction as part of PCV2 pathogenesis. Further research is warranted to study if PCV2 interacts directly with IP3R or indirectly with the molecules that antagonize IP3R activity responsible for increased cytosolic Ca2+ both in PK-15 cells and PCV2-targeted primary cells from pigs.

The ORF4 protein of porcine circovirus type 2 antagonizes apoptosis by stabilizing the concentration of ferritin heavy chain through physical interaction.

Porcine circovirus type 2 (PCV2) is the primary aetiological agent of porcine circovirus-associated disease in swine. The mechanism of PCV2 pathogenesis remains largely unknown. A newly identified viral protein of PCV2, ORF4, has been suggested to be involved in virus-induced apoptosis. However, there is still no information regarding the molecular mechanism by which ORF4 regulates apoptosis. In this study, we reveal that a physical interaction between the PCV2 ORF4 protein and ferritin heavy chain (FHC) in the cytoplasm of host cells reduced the cellular concentration of FHC. The ORF4-mediated reduction of FHC inhibited reactive oxygen species accumulation in PCV2-infected cells. Consequently, the ORF4 protein inhibited apoptosis in host cells. This may be the first report to describe the mechanism of ORF4 cytoprotection against apoptosis during the early stages of PCV2 infection.

Identification of microRNAs in PCV2 subclinically infected pigs by high throughput sequencing.

Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of PCV2-systemic disease and has been associated with other swine diseases, all of them collectively known as porcine circovirus diseases. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. miRNAs play an increasing role in many biological processes. The study of miRNA-mediated host-pathogen interactions has emerged in the last decade due to the important role that miRNAs play in antiviral defense. The objective of this study was to identify the miRNA expression pattern in PCV2 subclinically infected and non-infected pigs. For this purpose an experimental PCV2 infection was carried out and small-RNA libraries were constructed from tonsil and mediastinal lymph node (MLN) of infected and non-infected pigs. High throughput sequencing determined differences in miRNA expression in MLN between infected and non-infected while, in tonsil, a very conserved pattern was observed. In MLN, miRNA 126-3p, miRNA 126-5p, let-7d-3p, mir-129a and mir-let-7b-3p were up-regulated whereas mir-193a-5p, mir-574-5p and mir-34a down-regulated. Prediction of functional analysis showed that these miRNAs can be involved in pathways related to immune system and in processes related to the pathogenesis of PCV2, although functional assays are needed to support these predictions. This is the first study on miRNA gene expression in pigs infected with PCV2 using a high throughput sequencing approach in which several host miRNAs were differentially expressed in response to PCV2 infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-014-0141-4) contains supplementary material, which is available to authorized users.

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.

Current understanding of genomic DNA of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in swine and is also described as the modulator of host immunity that exacerbates the clinical outcome of many bacterial and viral infections. To date, it has caused increasingly larger losses in the pig industry worldwide. The genomic DNA of PCV2 is predicted to contain 11 open reading frames (ORFs) and at least seven potential ORFs-encoding proteins larger than 5 kDa. Currently, however, only five virally encoded proteins (Rep, Rep', Cap, ORF3, and ORF4 protein) have been identified in PCV2 replication. In the present review, we strive to discuss the current understanding of the genomic DNA of PCV2 with the purpose of providing insight into the scientific basis of the pathogenesis of PCV2 and the prevention of its infection.

Heat shock protein 27 is involved in PCV2 infection in PK-15 cells

Porcine circovirus type 2 (PCV2) has been identified as the etiologic agent which causing postweaning multisystemic wasting syndrome in swine farms in the world. Some quantitative proteomic studies showed that many proteins significantly changed in PCV2-infected cells. To explore the role of cellular chaperones during PCV2 infection, cytoprotective chaperone Hsp27 was analyzed in PCV2-infected PK-15 cells in this study. The results showed that Hsp27 could up-regulate and accumulate in phosphorylated forms in the nuclear zone during PCV2 replication. Suppression of Hsp27 phosphorylation with specific chemical inhibitors or downregulation of all forms of Hsp27 via RNA interference significantly reduced the virus replication. Meanwhile, over-expression of Hsp27 enhanced PCV2 genome replication and virion production. It indicated that Hsp27 was required for PCV2 production in PK-15 cells culture. It should be helpful for understanding the mechanism of replication and pathogenesis of PCV2 and development of novel antiviral therapies in the future.

Porcine circovirus type 2 (PCV2): pathogenesis and interaction with the immune system.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD). The virus preferentially targets the lymphoid tissues, which leads to lymphoid depletion and immunosuppression in pigs. The disease is exacerbated by immunostimulation or concurrent infections with other pathogens. PCV2 resides in certain immune cells, such as macrophage and dendritic cells, and modulates their functions. Upregulation of IL-10 and proinflammatory cytokines in infected pigs may contribute to pathogenesis. Pig genetics influence host susceptibility to PCV2, but the viral genetic determinants for virulence remain unknown. PCV2 DNA and proteins interact with various cellular genes that control immune responses to regulate virus replication and pathogenesis. Both neutralizing antibodies and cell-mediated immunity are important immunological correlates of protection. Despite the availability of effective vaccines, variant strains of PCV2 continue to emerge. Although tremendous progress has been made toward understanding PCV2 pathogenesis and immune interactions, many important questions remain.

Two-dimensional liquid chromatography–tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine circovirus type 2

Porcine circovirus type 2 (PCV2) has been identified as the essential causal agent of postweaning multisystemic wasting syndrome, which has spread worldwide. Monocyte/macrophage lineage cells are the major target cells of PCV2. To discover cellular protein responses of pulmonary alveolar macrophages (PAMs) to PCV2 infection, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the proteins that were differentially expressed in PAMs from the PCV2-infected group compared to the uninfected control group. A total of 145 cellular proteins in PAMs 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. The high number of differentially expressed proteins identified should be very useful to elucidate the mechanism of replication and pathogenesis of PCV2 in the future.

Porcine circovirus type 2 induces autophagy via the AMPK/ERK/TSC2/mTOR signaling pathway in PK-15 cells.

Porcine circovirus type 2 (PCV2) uses autophagy machinery to enhance its replication in PK-15 cells. However, the underlying mechanisms are unknown. By the use of specific inhibitors, RNA interference, and coimmunoprecipitation, we show that PCV2 induces autophagy in PK-15 cells through a pathway involving the kinases AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase 1/2 (ERK1/2), the tumor suppressor protein TSC2, and the mammalian target of rapamycin (mTOR). AMPK and ERK1/2 positively regulate autophagy through negative control of the mTOR pathway by phosphorylating TSC2 in PCV2-infected PK-15 cells. Thus, PCV2 might induce autophagy via the AMPK/ERK/TSC2/mTOR signaling pathway in the host cells, representing a pivotal mechanism for PCV2 pathogenesis.

Effect of an interferon-stimulated response element (ISRE) mutant of porcine circovirus type 2 (PCV2) on PCV2-induced pathological lesions in a porcine reproductive and respiratory syndrome virus (PRRSV) co-infection model

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases (PCVAD) in swine. Coinfections of PCV2 with other swine pathogens increase the severity of PCVAD. Induction of proinflammatory cytokines by coinfecting pathogens may attribute to the exacerbation of PCVAD during coinfections. An interferon-stimulated response element (ISRE) sequence was identified in the origin of replication of PCV2 genome. To assess the role of ISRE in PCV2 pathogenesis during coinfection, an ISRE-mutant PCV2 was constructed and used to experimentally infect pigs with either ISRE mutant or wildtype PCV2 singly or in combination with porcine reproductive and respiratory syndrome virus (PRRSV). The results showed that, during early stage of infection at 14 days post-inoculation (dpi), the ISRE mutation reduced viral replication and elicited low antibody responses. However, at 28 dpi viremia in pigs infected with the ISRE-mutant was on an upward trend, and microscopic lesion scores in pigs inoculated with the ISRE-mutant were significantly more severe than in wildtype PCV2-infected pigs. Coinfection with PRRSV caused an opposite shift in the in vivo dynamics of the ISRE-mutant at 14 dpi with the lymph node histopathological lesions being significantly more severe in pigs coinfected with the ISRE-mutant PCV2 and PRRSV than in pigs coinfected with wildtype PCV2 and PRRSV. PCV2 genomic copy numbers in pigs coinfected with ISRE-mutant and PRRSV were also higher than those coinfected with wildtype PCV2 and PRRSV. The results suggested that the ISRE element in PCV2 genome may play a potential role in viral pathogenesis.

Interaction of the replication proteins and the capsid protein of porcine circovirus type 1 and 2 with host proteins

Porcine circovirus type 2 (PCV2) is an important pathogen in swine, whereas porcine circovirus type 1 (PCV1) is apathogenic. To analyze the interactions between PCV and its host, we have used a yeast two-hybrid assay to identify cellular proteins interacting with Cap and Rep proteins of both PCV genotypes. Six cellular proteins were found to interact with Cap (MKRN1, gC1qR, Par-4, NAP1, NPM1 and Hsp40) and three with Rep (ZNF265, TDG and VG5Q). These interactions were confirmed by co-immunoprecipitation. Investigation of the localisation of the proteins by immunofluorescence revealed in some cases only limited spatial overlapping with Cap, while in others a clear co-localisation and prominent protein redistribution was observed. The nine cellular proteins are associated with distinct aspects of viral lifecycle and our data is likely to support future research in the field of PCV2 pathogenesis.

Rolling-circle amplification for the detection of active porcine circovirus type 2 DNA replication in vitro

Porcine circovirus type 2 (PCV2) infections in pigs have diverse clinical presentations and are considered economically important diseases worldwide. However, despite intensive research, the early pathogenesis of PCV2 and the primary target cells for PCV2 infections and replication are still unknown. Rolling-circle amplification (RCA) is an amplification technique for small, circular DNA templates that essentially mimics rolling-circle replication in vitro. In this study, the amplification of PCV2-specific DNAs using randomly primed RCA has been demonstrated. This novel approach has circumvented the normal requirement for conventional virus isolation procedures for the characterization of PCV2 DNAs from clinical samples. In addition, the potential utility of a strand-specific derivative of RCA was further investigated. Specifically, strand-specific RCA for the detection of active virus replication following the amplification of complementary sense PCV2 DNAs, which occur as double-stranded replicative intermediates that are present only during de novo viral DNA replication both in vitro and in vivo has been demonstrated.