Porcine Circovirus Type 2 Cap Research Articles

Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, β-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

Construction of recombinant pseudorabies virus expressing PCV2 Cap, PCV3 Cap, and IL-4: investigation of their biological characteristics and immunogenicity.

Porcine circovirus type 2 (PCV2) is a globally prevalent and recurrent pathogen that primarily causes slow growth and immunosuppression in pigs. Porcine circovirus type 3 (PCV3), a recently discovered virus, commonly leads to reproductive disorders in pigs and has been extensively disseminated worldwide. Infection with a single PCV subtype alone does not induce severe porcine circovirus-associated diseases (PCVD), whereas concurrent co-infection with PCV2 and PCV3 exacerbates the clinical manifestations. Pseudorabies (PR), a highly contagious disease in pigs, pose a significant threat to the swine industry in China. In this study, recombinant strains named rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 was constructed by using a variant strain XJ of pseudorabies virus (PRV) as the parental strain, with the TK/gE/gI genes deleted and simultaneous expression of PCV2 Cap, PCV3 Cap, and IL-4. The two recombinant strains obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster Syrian kidney-21 (BHK-21) cells and is safe to mice. rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 exhibited good safety and immunogenicity in mice, inducing high levels of antibodies, demonstrated 100% protection against the PRV challenge in mice, reduced viral loads and mitigated pathological changes in the heart, lungs, spleen, and lymph nodes during PCV2 challenge. Moreover, the recombinant viruses with the addition of IL-4 as a molecular adjuvant outperformed the non-addition group in most indicators. rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 hold promise as recombinant vaccines for the simultaneous prevention of PCV2, PCV3, and PRV, while IL-4, as a vaccine molecular adjuvant, effectively enhances the immune response of the vaccine.

Investigation of porcine circovirus type 2 and porcine circovirus type 3 infections based on dual TaqMan fluorescent quantitative PCR method and genetic evolutionary analysis of these two viruses.

Porcine circovirus type 2 (PCV2) is the pathogen of Porcine Circovirus Associated Diseases. Porcine circovirus type 3 (PCV3) is a novel porcine circovirus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCV2 is clearly pathogenic, while the pathogenicity of PCV3 remains controversial, so it is crucial to monitor the prevalence of PCV2 and PCV3 in healthy and diseased pigs to investigate the effects of PCV3 and PCV2 on the health status of pigs. Here, we developed a PCV2 and PCV3 dual TaqMan quantitative PCR (qPCR) method to test samples from healthy and diseased pigs, to clarify the differences in the positive rates and viral copy numbers of PCV2 and PCV3, and to analyze the genetic evolution and molecular characterization of the viral genomes obtained with sequence alignment and phylogenetic analysis, homology and structural analysis of Cap proteins, and selection pressure analysis. We successfully established a dual TaqMan qPCR method for PCV2 and PCV3 with good repeatability, specificity and sensitivity. In total, 1,385 samples from 15 Chinese provinces were tested with the established qPCR. The total positive rates were 37.47% for PCV3 and 57.95% for PCV2, and the coinfection rate for was 25.49%. The positive rates of PCV3 and PCV2 in 372 healthy pigs were 15.05 and 69.89%, respectively, and the coinfection rate was 12.90%. The positive rates of PCV3 and PCV2 in 246 diseased pigs were 55.69 and 83.33%, respectively, and the coinfection rate was 47.97%. Eighteen PCV3 genomes and 64 PCV2 genomes were identified, including nine each of the PCV3a-1 and PCV3b genotypes, eight of PCV2a, 16 of PCV2b, and 40 of PCV2d. The amino acid identity within the PCV3 Cap proteins was 94.00-100.0%, whereas the PCV2 Cap proteins showed an identity of 81.30-100.0%. PCV3 Cap was most variable at amino acid sites 24, 27, 77, 104 and 150, whereas PCV2 Cap had 10-13 unique sites of variation between genotypes. These results clarify the prevalence and variations of PCV2 and PCV3 in healthy and diseased pigs, which will provide a basis for the prevention and control of the two viral infections.

Porcine circovirus type 2 infection promotes the SUMOylation of nucleophosmin-1 to facilitate the viral circular single-stranded DNA replication.

The mechanism of genome DNA replication in circular single-stranded DNA viruses is currently a mystery, except for the fact that it undergoes rolling-circle replication. Herein, we identified SUMOylated porcine nucleophosmin-1 (pNPM1), which is previously reported to be an interacting protein of the viral capsid protein, as a key regulator that promotes the genome DNA replication of porcine single-stranded DNA circovirus. Upon porcine circovirus type 2 (PCV2) infection, SUMO2/3 were recruited and conjugated with the K263 site of pNPM1's C-terminal domain to SUMOylate pNPM1, subsequently, the SUMOylated pNPM1 were translocated in nucleoli to promote the replication of PCV2 genome DNA. The mutation of the K263 site reduced the SUMOylation levels of pNPM1 and the nucleolar localization of pNPM1, resulting in a decrease in the level of PCV2 DNA replication. Meanwhile, the mutation of the K263 site prevented the interaction of pNPM1 with PCV2 DNA, but not the interaction of pNPM1 with PCV2 Cap. Mechanistically, PCV2 infection increased the expression levels of Ubc9, the only E2 enzyme involved in SUMOylation, through the Cap-mediated activation of ERK signaling. The upregulation of Ubc9 promoted the interaction between pNPM1 and TRIM24, a potential E3 ligase for SUMOylation, thereby facilitating the SUMOylation of pNPM1. The inhibition of ERK activation could significantly reduce the SUMOylation levels and the nucleolar localization of pNPM1, as well as the PCV2 DNA replication levels. These results provide new insights into the mechanism of circular single-stranded DNA virus replication and highlight NPM1 as a potential target for inhibiting PCV2 replication.

DEAD-box RNA helicase 21 interacts with porcine circovirus type 2 Cap protein and facilitates viral replication.

Porcine circovirus type 2 (PCV2) is the etiological agent of PCV2-associated diseases that pose a serious threat to the swine industry. PCV2 capsid (Cap) protein has been shown to interact with DEAD-box RNA helicase 21 (DDX21), an important protein that regulates RNA virus replication. However, whether the interaction between DDX21 and the PCV2 Cap regulates PCV2 replication remains unclear. Herein, by using western blotting, interaction assays, and knockdown analysis, we found that PCV2 infection induced the cytoplasmic relocation of DDX21 from the nucleolus in cultured PK-15 cells. Moreover, the nuclear localization signal (NLS) of PCV2 Cap interacted directly with DDX21. The NLS of PCV2 Cap and 763GSRSNRFQNK772 residues at the C-terminal domain (CTD) of DDX21 were essential for the dual interaction. Upon shRNA-mediated DDX21 depletion in PK-15 cells, we observed impaired PCV2 replication via a lentivirus-delivered system, as evidenced by decreased levels of viral protein expression and virus production. In contrast, the replication of PCV2 increased in transiently DDX21-overexpressing cells. Our results indicate that DDX21 interacts with PCV2 Cap and plays a crucial role in virus replication. These results provide a reference for developing novel potential targets for prevention and control of PCV2 infection.

Porcine IGFBP3 promotes porcine circovirus type 2 replication via PERK/eIF2α mediated DNA damage

The infection of porcine circovirus type 2 (PCV2) triggers activation of the protein kinase RNA–like endoplasmic reticulum kinase (PERK) pathway and leads to DNA damage. Insulin-like growth factor-binding protein 3 (IGFBP3) may interact with the endoplasmic reticulum (ER). It remains unclear whether IGFBP3 regulates DNA damage via ER stress to mediate PCV2 replication. In this study, we observed an upregulation of porcine IGFBP3 expression during PCV2 infection, and overexpression of IGFBP3 enhanced the expression of PCV2 Cap protein, PCV2 DNA copy number, and viral titers in PK-15 B6 cells and 3D4/21 cells. Additionally, overexpression of IGFBP3 induced an increase in the DNA damage marker γH2AX by activating the PERK/eIF2α pathway without concomitant activation of ATF4, IRE1α, and ATF6α/GRP78 pathways in PK-15 B6 cells and 3D4/21 cells. Knockdown of IGFBP3 had a reverse effect on PCV2 replication in PK-15 B6 cells and 3D4/21 cells. Furthermore, treatment with etoposide enhanced PCV2 replication while KU57788 decreased it. GSK2606414 and salubrinal limited both DNA damage and viral replication. Therefore, our findings suggest that porcine IGFBP3 promotes PCV2 replication through the PERK/eIF2α pathway-mediated induction of DNA damage in PK-15 B6 cells and 3D4/21 cells. Our study provides a basis for exploring novel antiviral strategies via the extensive understanding of the relationships between host cellular proteins and viral replication.

Porcine circovirus type 2 infection inhibits macrophage M1 polarization induced by other pathogens via viral capsid protein and host gC1qR protein

Porcine circovirus type 2 (PCV2) has been proven to co-infect with a variety of pathogens and cause immunosuppression. Previously, we have reported that PCV2 infection attenuates the production of pro-inflammatory cytokines induced by other pathogens in porcine macrophages. However, whether PCV2 can affect M1-type macrophage polarization induced by other pathogens is less well reported. Herein, we found that PCV2 infection suppressed M1 macrophage production induced by porcine reproductive and respiratory syndrome virus (PRRSV) and Haemophilus parasuis (H. parasuis) in the lung and promoted the proliferation of these pathogens in the piglets. Consistently, we confirmed that PCV2 inhibits M1 macrophage production and its associated gene expression in porcine alveolar macrophages (PAMs) both ex vivo and in vitro. Meanwhile, PCV2 inhibited lipopolysaccharide (LPS)-induced pro-inflammatory cytokines in vitro in a time- and dose-dependent manner. In PCV2-infected cells, LPS-induced signal transducer and activator of transcription (STAT1) phosphorylation and its nuclear translocation were decreased. Based on these findings, we further identified a role for PCV2 capsid protein (Cap) in LPS-induced M1 macrophage-associated genes and found that PCV2 Cap can significantly reduce STAT1 phosphorylation and its nuclear translocation, as well as the production of M1 macrophage-related genes. As the binding protein of PCV2 Cap, gC1qR protein was also associated with this inhibition process. gC1qR-binding activity-deficient PCV2 Cap mutated protein (Cap RmA) appeared an attenuated inhibitory effect on other pathogen-induced polarization of M1-type macrophages, suggesting that the inhibitory effect of PCV2 infection on M1-type macrophage polarization induced by other pathogens is dependent on Cap protein and the host gC1qR protein. Altogether, our results demonstrate that PCV2 infection inhibits macrophage M1 polarization induced by other pathogens via capsid and host gC1qR protein modulating JAK/STAT signaling.

Development of a Gold Nanoparticle-Based Immunochromatographic Strip for Rapid Detection of Porcine Circovirus Type 2.

Porcine circovirus type 2 (PCV2) is an important swine infectious pathogen that seriously threatens the global swine industry. PCV2 Cap protein is the only structural and the main immunogenic protein constituting the viral capsid. In this study, a gold nanoparticle-based immunochromatographic strip with high sensitivity and specificity was developed which could be used for rapid detection of PCV2 virions or Cap protein in research. The visual detection limit of the strip was 103.18 50% tissue culture infective does (TCID50)/mL for PCV2, and 2.03 μg/mL for PCV2 Cap protein. No cross-reactivity was observed with the PCV1 and PCV3 Cap proteins and other common swine pathogens such as porcine reproductive and respiratory syndrome virus, classical swine fever virus, pseudorabies virus, porcine epidemic diarrhea virus, porcine parvovirus, and swine influenza virus. The repeatability of the strip was good. The stability of the strip was perfect for 12 months in a dry state at room temperature. Visual results could be obtained within 5 min by simply inserting the strip into the diluted sample. The strip is a time-saving, labor-saving, and reliable tool for testing of PCV2 virions or Cap protein in research. The idea of this study might open a new perspective for the application of the strip. IMPORTANCE Porcine circovirus type 2 (PCV2) Cap protein is the only structural and the main immunogenic protein constituting the viral capsid. Although many methods can be used to identify PCV2 or PCV2 Cap protein in vaccine research, they usually require high workload and time. The developed strip can specifically detect PCV2 virions or Cap protein, and visual qualitative results can be obtained within 5 min by simply diluting the sample and inserting the strip into the sample. The final value of the strip is providing a simple and time-saving method for real-time monitoring of PCV2 antigen in vaccine research with reliable results, such as the different stages of PCV2 Cap protein expression and purification, as well as the different stages of PCV2 reproduction and purification.

Immunogenicity and Immunoprotection of PCV2 Virus-like Particles Incorporating Dominant T and B Cell Antigenic Epitopes Paired with CD154 Molecules in Piglets and Mice.

Porcine circovirus type 2 (PCV2) is capable of causing porcine circovirus-associated disease (PCVAD) and is one of the major threats to the global pig industry. The nucleocapsid protein Cap encoded by the PCV2 ORF2 gene is an ideal antigen for the development of PCV2 subunit vaccines, and its N-terminal nuclear localization sequence (NLS) structural domain is essential for the formation of self-assembling VLPs. In the present study, we systematically expressed and characterized full-length PCV2 Cap proteins fused to dominant T and B cell antigenic epitopes and porcine-derived CD154 molecules using baculovirus and found that the Cap proteins fusing epitopes were still capable of forming virus-like particles (VLPs). Both piglet and mice experiments showed that the Cap proteins fusing epitopes or paired with the molecular adjuvant CD154 were able to induce higher levels of humoral and cellular responses, particularly the secretion of PCV2-specific IFN-γ and IL-4. In addition, vaccination significantly reduced clinical signs and the viral load of PCV2 in the blood and tissues of challenged piglets. The results of the study provide new ideas for the development of a more efficient, safe and broad-spectrum next-generation PCV2 subunit vaccine.

PDZK1 upregulates nitric oxide production through the PI3K/ERK2 pathway to inhibit porcine circovirus type 2 replication

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated disease. Changes in host cell gene expression are induced by PCV2 infection. Here, we showed that porcine PDZ Domain-Containing 1 (PDZK1) expression was enhanced during PCV2 infection and that overexpression of PDZK1 inhibited the expression of PCV2 Cap protein. PCV2 genomic DNA copy number and viral titers were decreased in PDZK1-overexpressing PK-15B6 cells. PDZK1 knockdown enhanced the replication of PCV2. Overexpression of PDZK1 activated the phosphoinositide 3-kinase (PI3K)/ERK2 signaling pathway to enhance nitric oxide (NO) levels, while PDZK1 knockdown had the opposite effects. A PI3K inhibitor (LY294002) and a NO synthase inhibitor (L-NAME hydrochloride) decreased the activity of PDZK1 in restricting PCV2 replication. ERK2 knockdown enhanced the proliferation of PCV2 by decreasing levels of NO. Levels of interleukin (IL)− 4 mRNA were reduced in PDZK1 knockdown and ERK2 knockdown PK-15B6 cells. Increased IL-4 mRNA levels were unable to decrease NO production in PDZK1-overexpressing cells. Thus, we conclude that PDZK1 affected PCV2 replication by regulating NO production via PI3K/ERK2 signaling. PDZK1 affected IL-4 expression through the PI3K/ERK2 pathway, but PDZK1 modulation of PCV2 replication occurred independently of IL-4. Our results contribute to understanding the biological functions of PDZK1 and provide a theoretical basis for the pathogenic mechanisms of PCV2.

Cap Is the Protease of the Porcine Circovirus 2.

Circoviruses are the smallest single-stranded DNA viruses that infect mammalian species, avian species, fish, and insects. The infections of circoviruses are known to be associated with a series of fatal diseases, but the protease of circovirus still remains unknown. In this research, we identified viral capsid protein (Cap) as the protease of porcine circovirus type 2 (PCV2), to our knowledge the first circoviruses protease to be reported. First, we found that the expression of host proteins is affected due to PCV2 infection in the porcine kidney (PK-15) cells. Then, by proteomic analysis, 253 host proteins that were down regulated were identified due to direct or indirect effects of PCV2. Further, Cap expression, but not other ORFs of PCV2, significantly reduced both JMJD6 (bifunctional arginine demethylase and lysyl-hydroxylase) and CCT5 (the chaperonin containing TCP1 subunit 5) in PK-15 cells. Finally, the results in vitro hydrolysis assays demonstrated that Cap could directly degraded either JMJD6 or CCT5 with different catalytic efficiency. In summary, our study expands repertoire of PCV2 Cap and promotes the development of inhibitors toward the anti-PCV2.

Fusion Expression and Immune Effect of PCV2 Cap Protein Tandem Multiantigen Epitopes with CD154/GM-CSF.

Porcine circovirus associated diseases (PCVAD) is a contagious disease of swine caused by porcine circovirus type 2 (PCV2). The capsid protein (Cap) is the sole structural protein and the main antigen of PCV2. Cap is the principal immunogenic protein and induces humoral and cellular immunity. CD154 and GM-CSF are immune adjuvants that enhance responses to vaccines. However, whether these two cellular molecules could produce an enhanced effect in PCV2 vaccines still needs to be further studied. The results of PCR and restriction enzyme showed that the recombinant lentiviral plasmids pCDH-TB-Cap, pCDH-TB-Cap-CD154 and pCDH-TB-Cap were successfully constructed. Western blot and IFA showed that the three fusion proteins TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF were stably expressed in CHO-K1 cells. Indirect ELISA assay showed that mice immunized with TB-Cap-CD154 and TB-Cap-GM-CSF fusion proteins produced higher PCV2-specific antibodies than mice immunized with the TB-Cap and a commercial vaccine (p < 0.0001). Moreover, lymphocyte proliferation and flow cytometry showed that the cellular immune response of each immune group was significantly enhanced (p < 0.0001). After PCV2 challenge, the results revealed that the viral loads in serum, lung and kidney of all vaccinated groups were significantly lower than the PBS group (p < 0.0001). The transcription levels of IL-2, IFN-gamma, IL-4 and IL-10 cytokines in the TB-Cap, TB-Cap-CD154 and TB-Cap-GM-CSF groups were significantly higher than those in the PBS and recombinant vaccine groups (p < 0.0001). These results indicated that CD154 and GM-CSF could enhance the ability of TB-Cap protein to induce the body to produce PCV2 specific antibodies and increase the transcription level of cytokines. Thus, CD154 and GM-CSF molecules were a powerful immunoadjuvant for PCV2 subunit vaccines. The novel TB-Cap-CD154 and TB-Cap-GM-CSF subunit vaccine has the potential to be used for the prevention and control of PCVAD.

Displaying epitope B and epitope 7 of porcine reproductive and respiratory syndrome virus on virus like particles of porcine circovirus type 2 provides partial protection to pigs

The Cap of porcine circovirus type 2 (PCV2) can be assembled into virus like particles (VLPs) in vitro that have multiple loops located on the particle surface. This would make it a good vehicle for displaying exogenous proteins or epitopes. We derived two epitopes, epitope B (EpB, S37HIQLIYNL45) and epitope 7 (Ep7, Q196WGRL200) from Gp5 of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). We replaced the core region of Loop CD (L75PPGGGSN82) and the carboxyl terminus (K222DPPL226) of PCV2 Cap, respectively, to construct a bi-epitope chimeric PCV2 Cap. Its immunogenicity and protective effects were evaluated as one PRRSV subunit vaccine. The chimeric PCV2 Cap was soluble, efficiently expressed in an Escherichia coli expression system, and could be self-assembled into chimeric virus like particles (cVLPs) with a diameter of 12–15 nm. Western blotting confirmed that the cVLPs could be specifically recognized by anti-PCV2, anti-EpB and anti-Ep7 antibodies. The cVLPs vaccine could alleviate the clinical symptoms and reduce the viral loads after HP-PRRSV challenge in 100–120 days old pigs. These data suggest that the cVLPs vaccine could provide pigs with partial protection against homologous PRRSV strains, and it provides a new design for additional PRRSV subunit vaccines.

Identification of porcine circovirus type 2 (PCV2), type 3 (PCV3) and porcine parvovirus (PPV) in swine by multiplex PCR test

This study aimed to simultaneously detect three important viruses reported to be involved in the reproductive problems of sows. A multiplex PCR (mPCR) test was developed to provide rapid diagnosis of porcine circovirus type 2 and 3 (PCV2, PCV3) and to illustrate parvovirus (PPV) prevalence in sow herds. Three pairs of specific primers were designed to target PCV2 Cap gene, PCV3 Cap gene and PPV NS1 gene, with predicted mPCR products of 702 bp, 267 bp and 380 bp, respectively. The detection limit of mPCR was 100 copies/reaction per target gene. The mPCR was run against a panel of 94 swine serum samples whose infection status had been pre-determined by commercial real-time PCR kits. Sequencing of mPCR products performed with clinical serum samples accurately confirmed the results. Overall, the results indicated that the mPCR functioned accurately and specifically and matched 100% with the single-target real-time PCRs. The mPCR was developed successfully and can be used in routine diagnosis of PCV2, PCV3 and PPV.

Displaying epitope B and epitope 7 of porcine reproductive and respiratory syndrome virus on virus like particles of porcine circovirus type 2 provides partial protection to pigs.

The Cap of porcine circovirus type 2 (PCV2) can be assembled into virus like particles (VLPs) in vitro that have multiple loops located on the particle surface. This would make it a good vehicle for displaying exogenous proteins or epitopes. We derived two epitopes, epitope B (EpB, S37HIQLIYNL45) and epitope 7 (Ep7, Q196WGRL200) from Gp5 of the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). We replaced the core region of Loop CD (L75PPGGGSN82) and the carboxyl terminus (K222DPPL226) of PCV2 Cap, respectively, to construct a bi-epitope chimeric PCV2 Cap. Its immunogenicity and protective effects were evaluated as one PRRSV subunit vaccine. The chimeric PCV2 Cap was soluble, efficiently expressed in an Escherichia coli expression system, and could be self-assembled into chimeric virus like particles (cVLPs) with a diameter of 12-15 nm. Western blotting confirmed that the cVLPs could be specifically recognized by anti-PCV2, anti-EpB and anti-Ep7 antibodies. The cVLPs vaccine could alleviate the clinical symptoms and reduce the viral loads after HP-PRRSV challenge in 100-120 days old pigs. These data suggest that the cVLPs vaccine could provide pigs with partial protection against homologous PRRSV strains, and it provides a new design for additional PRRSV subunit vaccines.

Efficient application of a baculovirus-silkworm larvae expression system for obtaining porcine circovirus type 2 virus-like particles for a vaccine.

Porcine circovirus type 2 (PCV2) is a major pathogen associated with swine diseases. It is the smallest single-stranded DNA virus, and its genome contains four major open reading frames (ORFs). ORF2 encodes the major structural protein Cap, which can self-assemble into virus-like particles (VLPs) in vitro and contains the primary antigenic determinants. In this study, we developed a high-efficiency method for obtaining VLPs and optimized the purification conditions. In this method, we expressed the protein Cap with a 6× His tag using baculovirus-infected silkworm larvae as well as the E. coli BL21(DE3) prokaryotic expression system. The PCV2 Cap proteins produced by the silkworm larvae and E. coli BL21(DE3) were purified. Cap proteins purified from silkworm larvae self-assembled into VLPs in vitro, while the Cap proteins purified from bacteria were unable to self-assemble. Transmission electron microscopy confirmed the self-assembly of VLPs. The immunogenicity of the VLPs produced using the baculovirus system was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Furthermore, the purification process was optimized. The results demonstrated that the expression system using baculovirus-infected silkworm larvae is a good choice for obtaining VLPs of PCV2 and has potential for the development of a low-cost and efficient vaccine.

NAP1L4 inhibits porcine circovirus type 2 replication via IFN-β signaling pathway

Porcine circovirus type 2 (PCV2) capsid protein (Cap) was previously reported to interact with nucleosome assembly protein 1-like 4 (NAP1L4). The biological function of Cap-NAP1L4 interaction is unknown. Here, we demonstrated that PCV2 Cap could directly interact with NAP1L4, which the amino acid residues 124–279 of NAP1L4 were responsible for the interaction. Furthermore, over-expression of NAP1L4 down-regulated the expression of PCV2 Cap and Rep. DNA copies and virus titers were also decreased in NAP1L4 over-expressed PK15 cells. While, knockout of NAP1L4 through CRISPR/Cas9 technology in PK15 cells could up-regulate the mRNA and protein levels of PCV2 Cap and Rep. PCV2 genomic DNA copies and virus titers were also increased in NAP1L4-knockdown/-knockout PK15 cells compared with wild type PK15 cells. In addition, NAP1L4 depletion was demonstrated to facilitate cytosolic carboxypeptidase-like protein 5 (CCP5) and cytosolic carboxypeptidase 6 (CCP6) expression, which could activate cGAS to promote IFN-β production. Indeed, knockout of NAP1L4 was also confirmed to increase IFN-β expression. And IFN-β stimulation could promote PCV2 replication in PK15 cells. Taken together, our findings suggest that NAP1L4 interacts with PCV2 Cap and inhibits PCV2 replication through regulating IFN-β production. Our study provides theoretical basis for further study of PCV2.

Porcine DNAJB6 promotes PCV2 replication via enhancing the formation of autophagy in host cells

Hsp40/DnaJ family proteins play important roles in the infection process of various viruses. Porcine DNAJB6 (pDNAJB6) is a major member of this family, but its role in modulating the replication of porcine circovirus type 2 (PCV2) is still unclear. In the present study, pDNAJB6 was found to be significantly upregulated by PCV2 infection, and confirmed to be interacted with PCV2 capsid (Cap) protein and co-localized at both cytoplasm and nucleus in the PCV2-infected cells. Knockout of pDNAJB6 significantly reduced the formation of autophagosomes in PCV2-infected cells or in the cells expressing Cap protein, whereas overexpression of pDNAJB6 showed an opposite effect. In addition, the domain mapping assay showed that the J domain of pDNAJB6 (amino acids (aa) 1–99) and the C terminus of Cap (162-234 aa) were required for the interaction of pDNAJB6 with Cap. Notably, the interaction of pDNAJB6 with Cap was very important to promoting the formation of autophagosomes induced by PCV2 infection or Cap expression and enhancing the replication of PCV2. Taken together, the results presented here show a novel function of pDNAJB6 in regulation of porcine circovirus replication that pDNAJB6 enhances the formation of autophagy to promote viral replication through interacting with viral capsid protein during PCV2 infection.

Quantitative protein detection using single molecule imaging enzyme-linked immunosorbent assay (iELISA)

Protein detection is a key step in molecular biology research and is required for pathogen and protein marker testing for disease diagnostics. Here, single molecule imaging enzyme-linked immunosorbent assay (iELISA) is proposed to quantitatively measure the porcine circovirus type 2 (PCV2) Cap protein. The monoclonal antibody against PCV2 Cap protein indirectly immobilized on a polyethylene glycol (PEG) passivated slide by biotin-streptavidin interaction is used to capture the PCV2 Cap protein, and the PCV2 Cap protein can be detected in single molecule level according to the fluorescein isothiocyanate (FITC)-labeled secondary antibody using total internal reflection fluorescence microscopy. The single molecule iELISA measurements can be finished within 1 h skipping the time-consuming sample preparation procedures; moreover, it also exhibits excellent protein selectivity and anti-interference capability. With the proposed single molecule iELISA, linear relation between the fluorescent signals and logarithm of target protein concentrations is obtained with the detection limit of 7 ng/mL. Considering its high accuracy in target protein detection with simple procedures and fast speed, it is believed single molecule iELISA can be potentially adopted in fast trace protein detection.

Porcine Circovirus Type 2 Induces ORF3-Independent Mitochondrial Apoptosis via PERK Activation and Elevation of Cytosolic Calcium.

Our previous studies demonstrated that porcine circovirus type 2 (PCV2) triggers an unfolded protein response (UPR) in porcine kidney PK-15 cells by activating the protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) pathway of endoplasmic reticulum (ER) stress, which in turn facilitates viral replication (Y. Zhou et al., Viruses 8:e56, 2016, https://doi.org/10.3390/v8020056; Y. Zhou et al., J Zhejiang Univ Sci B 18:316-323, 2017, https://doi.org/10.1631/jzus.B1600208). PCV2 is found to cause oxidative stress and upregulation of cytoplasmic Ca2+ levels. The virus is reported to employ its open reading frame 3 (ORF3) to induce apoptosis. We wondered whether and how PCV2-induced UPR would lead to apoptosis independent of ORF3. Using an ORF3-deficient PCV2 mutant (ΔORF3), apoptotic responses in infected PK-15 and porcine alveolar macrophage (PAM) cells were still apparent, although lower than in the parental PCV2 strain. We hypothesized that apoptosis induced by ΔORF3 might result from the UPR. We found that ΔORF3-induced apoptosis was significantly reduced when the infected cells were treated with the selective PERK blocker GSK2606414 (GSK) or the general ER stress attenuator 4-phenylbutyrate (4-PBA). Such treatments also ameliorated elevation of cytoplasmic Ca2+ and reactive oxygen species (ROS) levels in PK-15 and PAM cells, two predisposing factors for apoptosis via disruption of the ER-mitochondrion units. Treatment of ΔORF3-infected cells with GSK and 4-PBA also decreased the mitochondrial Ca2+ load and increased the mitochondrial membrane potential (MMP). With transient expression of the structural protein capsid (Cap) in combination with PERK silencing, we found that Cap induced MMP collapse and mitochondrial apoptosis could result from the UPR and elevation of Ca2+ and ROS levels, which were inhibitable by downregulation of PERK. We propose that PCV2-driven ER stress is Cap dependent and could lead to mitochondrial apoptotic responses independent of ORF3 via perturbation of intracellular Ca2+ homeostasis and accumulation of ROS.IMPORTANCE PCV2 encodes protein ORF3, a putative protein with proapoptotic activity. Our early studies showed that PCV2 infection triggers ER stress via selective activation of the PERK pathway, a branch of the ER stress pathways, in permissive cells for enhanced replication and infection increased cytosolic Ca2+ and ROS levels. Here we clearly show that PCV2 infection or Cap expression induces ORF3-independent apoptosis via increased cytosolic and mitochondrial Ca2+ levels and cellular ROS levels as a result of activation of the PERK pathway.