Porcine Circovirus Type 2 Capsid Research Articles

Comparison of immune effects of porcine circovirus type 2d (PCV2d) capsid protein expressed by Escherichia coli and baculovirus-insect cells

Porcine circovirus type 2 (PCV2) is an important pathogen harmful to global pig production, which causes immunosuppression and serious economic losses. PCV2 capsid (Cap) protein expressed by E. coli or baculovirus-insect cells are often used in preparation of PCV2 subunit vaccines, but the latter is expensive to produce. It is therefore crucial to comparison of the immune effects of Cap protein expressed by the above two expression systems for reducing the production cost and guaranteeing PCV2 vaccine quality. In this study, the PCV2d-Cap protein lacking nuclear localization signal (NLS), designated as E. coli-Cap and Bac-Cap, was expressed by E. coli and baculovirus-Spodoptera frugiperda Sf9 (Bac-Sf9) cells, respectively. The expressed Cap proteins could self-assemble into virus-like particles (VLPs), but the Bac-Cap-assembled VLPs were more regular. The two system-expressed Cap proteins induced similar specific IgG responses in mice, but the neutralizing antibody levels of Bac-Cap-immunized mice was higher than those of E. coli-Cap. After PCV2 challenge, IL-10 in Bac-Cap immunized mice decreased significantly than that in E. coli-Cap. The lesions and PCV2 antigen positive cells in tissues of mice immunized with E. coli-Cap and Bac-Cap were significantly reduced, and Bac-Cap appeared mild lesions and fewer PCV2 antigen-positive cells compared with E. coli-Cap immunized mice. The study indicated that Cap proteins expressed by E. coli and Bac-Sf9 cells could induce specific protective immunity, but the latter induced more effective immunity, which provides valuable information for the research and development of PCV2 vaccine.

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.

A novel strategy for an anti-idiotype vaccine: nanobody mimicking neutralization epitope of porcine circovirus type 2.

Vaccination is the most effective method to protect humans and animals from diseases. Anti-idiotype vaccines are safer due to their absence of pathogens. However, the commercial production of traditional anti-idiotype vaccines using monoclonal and polyclonal antibodies (mAb and pAb) is complex and has a high failure rate. The present study designed a novel, simple, low-cost strategy for developing anti-idiotype vaccines with nanobody technology. We used porcine circovirus type 2 (PCV2) as a viral model, which can result in serious economic loss in the pig industry. The neutralizing mAb-1E7 (Ab1) against PCV2 capsid protein (PCV2-Cap) was immunized in the camel. And 12 nanobodies against mAb-1E7 were screened. Among them, Nb61 (Ab2) targeted the idiotype epitope of mAb-1E7 and blocked mAb-1E7's binding to PCV2-Cap. Additionally, a high-dose Nb61 vaccination can also protect mice and pigs from PCV2 infection. Epitope mapping showed that mAb-1E7 recognized the 75NINDFL80 of PCV2-Cap and 101NYNDFLG107 of Nb61. Subsequently, the mAb-3G4 (Ab3) against Nb61 was produced and can neutralize PCV2 infection in the PK-15 cells. Structure analysis showed that the amino acids of mAb-1E7 and mAb-3G4 respective binding to PCV2-Cap and Nb61 were also similar on the amino acids sequences and spatial conformation. Collectively, our study first provided a strategy for producing nanobody-based anti-idiotype vaccines and identified that anti-idiotype nanobodies could mimic the antigen on amino acids and structures. Importantly, as more and more neutralization mAbs against different pathogens are prepared, anti-idiotype nanobody vaccines can be easily produced against the disease with our strategy, especially for dangerous pathogens.IMPORTANCEAnti-idiotype vaccines utilize idiotype-anti-idiotype network theory, eliminating the need for external antigens as vaccine candidates. Especially for dangerous pathogens, they were safer because they did not contact the live pathogenic microorganisms. However, developing anti-idiotype vaccines with traditional monoclonal and polyclonal antibodies is complex and has a high failure rate. We present a novel, universal, simple, low-cost strategy for producing anti-idiotype vaccines with nanobody technology. Using a neutralization antibody against PCV2-Cap, a nanobody (Ab2) was successfully produced and could mimic the neutralizing epitope of PCV2-Cap. The nanobody can induce protective immune responses against PCV2 infection in mice and pigs. It highlighted that the anti-idiotype vaccine using nanobody has a very good application in the future, especially for dangerous pathogens.

Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3.

Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. Currently, there is a growing need for rapid and accurate diagnostic methods in disease monitoring. In this study, four monoclonal antibodies (mAbs) against PCV3 capsid proteins were prepared (mAbs 2F6, 2G8, 6E2, and 7E3). MAb 7E3, which had the highest binding affinity for the Cap protein, was chosen for further investigation. A novel B cell epitope 110DLDGAW115 was identified using mAb 7E3. An epitope-blocking (EB) enzyme-linked immunosorbent assay (ELISA) was successfully developed using horseradish-peroxidase-labeled mAb 7E3 to detect PCV3 antibodies in porcine sera. Moreover, the EB-ELISA showed no specific reaction with other porcine disease sera, and the cut-off value was defined as 35%. Compared with the commercial ELISA, the percentage agreement was 95.59%. Overall, we have developed a novel EB-ELISA method that accurately and conveniently detects PCV3 in serum, making it a valuable tool for the clinical detection of PCV3 infection.

Ubiquitination-dependent degradation of nucleolin mediated by porcine circovirus type 3 capsid protein.

Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes multisystem disease in pigs and poses a severe threat to the swine industry. However, the mechanisms of how PCV3 uses host proteins to regulate its own life cycle are not well understood. In this study, we found that PCV3 capsid protein interacts with nucleolin and degrades it. Degradation of nucleolin by the PCV3 capsid protein requires recruitment of the enzyme RNF34, which is transported to the nucleolus from the cytoplasm in the presence of the PCV3 capsid protein. Nucleolin also decreases PCV3 replication by promoting the release of interferon β. These findings clarify the mechanism by which nucleolin modulates PCV3 replication in cells, thereby facilitating to provide an important strategy for preventing and controlling PCV3 infection.

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.

Interaction between host cell proteins and open reading frames of porcine circovirus type 2.

Postweaning multisystemic wasting syndrome (PMWS) is caused by a systemic inflammation after porcine circovirus type 2 (PCV2) infection. It was one of the most economically important pathogens affecting pig production worldwide before PCV2 vaccine was first introduced in 2006. After the development of a vaccine against PCV2a type, pig farms gradually restored enormous economic losses from PMWS. However, vaccine against PCV2a type could not be fully effective against several different PCV2 genotypes (PCV2b - PCV2h). In addition, PCV2a vaccine itself could generate antigenic drift of PCV2 capsid. Therefore, PCV2 infection still threats pig industry worldwide. PCV2 infection was initially found in local tissues including reproductive, respiratory, and digestive tracks. However, PCV2 infection often leads to a systemic inflammation which can cause severe immunosuppression by depleting peripheral lymphocytes in secondary lymphoid tissues. Subsequently, a secondary infection with other microorganisms can cause PMWS. Eleven putative open reading frames (ORFs) have been predicted to encode PCV2 genome. Among them, gene products of six ORFs from ORF1 to ORF6 have been identified and characterized to estimate its functional role during PCV2 infection. Acquiring knowledge about the specific interaction between each PCV2 ORF protein and host protein might be a key to develop preventive or therapeutic tools to control PCV2 infection. In this article, we reviewed current understanding of how each ORF of PCV2 manipulates host cell signaling related to immune suppression caused by PCV2.

Comparison of predicted T cell epitopes in porcine circovirus type 2 isolates from 2017 to 2021 and selected vaccines (EpiCC analysis) confirms the global relevance of a bivalent vaccine approach

Porcine circovirus type 2 (PCV2) is a globally important pathogen of swine with a high capacity for genetic change, potentially including evolution of strains less susceptible to vaccine induced immunity. Starting with DNA sequences, in silico tools can be used to predict the T cell epitope content of the PCV2 capsid protein in vaccines and field strains, allowing calculation of Epitope Content Comparison (EpiCC) scores reflecting the number of T-cell epitopes held in common. Previous work has shown that a bivalent PCV2a and PCV2b vaccine gave greater T cell epitope coverage (higher EpiCC scores) than either PCV2a or PCV2b as a monovalent, with the more complete match potentially helping to preserve or enhance vaccine efficacy. This study extends the above, using refined methodology to compare the putative T-cell epitope content of 4 PCV2 vaccines (3 based on PCV2a, and one a PCV2a and PCV2b bivalent) to a larger and more contemporary global sample of PCV2 field strains. 746 PCV2, ORF2 nucleotide sequences from diagnostic submissions dating from 2017 to 2021 were included. These comprised PCV2a (129), PCV2b (109) and PCV2d (508), and originated from Asia (185), Europe (269), North America (235) and South America (57). Phylogenetic classification confirmed the predominance of PCV2d in all regions, but with continuing presence of PCV2a and PCV2b. An interesting regional divergence was noted for PCV2a, with all strains from Europe grouped within a single phylogenetic cluster (cluster 10) and all but one from North America being in a different phylogenetic cluster (cluster 6). EpiCC scores for the bivalent vaccine were significantly higher than for the monovalents for all genotypes in all regions, showing the global relevance of the bivalent approach. Calculation of the relative contributions of the PCV2a and PCV2b components showed that, while most T-cell epitopes were present in both, each also made substantial unique contributions. Of most practical relevance, given that most commercial vaccines are based on PCV2a, the addition of PCV2b increased T-cell epitope coverage by 33% and 21% for PCV2b and PCV2d respectively.

Investigation and development of transient production process for porcine circovirus Type-2 (PCV2) capsid protein in HEK293F cells

Porcine circovirus type-2 capsid protein contains a major immunodominant epitope used as a subunit vaccine. Transient expression in mammalian cells is an efficient process for producing recombinant proteins. However, there is still a lack of research on the efficient production of virus capsid proteins in mammalian cells. Here we present a comprehensive study to investigate and optimize the production process of a model “difficult-to-express” virus capsid protein, PCV2 capsid protein in HEK293F transient expression system. The study evaluated the transient expression of PCV2 capsid protein in the mammalian cell line HEK293F and investigated the subcellular distribution by confocal microscopy. In addition, the RNA sequencing (RNA-seq) was used to detect the differential expression of genes after cells transfected with pEGFP-N1-Capsid or empty vectors. The analysis revealed that the PCV2 capsid gene affected a panel of differential genes of HEK293F cells involved in protein folding, stress response, and translation process, such as SHP90β, GRP78, HSP47, and eIF4A. An integrated strategy of protein engineering combined with VPA addition was applied to promote the expression of PCV2 capsid protein in HEK293F. Moreover, this study significantly increased the production of the engineered PCV2 capsid protein in HEK293F cells, reaching a yield of 8.7 mg/L. Conclusively, this study may provide deep insight for other “difficult-to-express” virus capsid proteins in the mammalian cell system.

Surface Display of porcine circovirus type 2 antigen protein cap on the spores of bacillus subtilis 168: An effective mucosal vaccine candidate.

The oral mucosal vaccine has great potential in preventing a series of diseases caused by porcine circovirus type 2 (PCV2) infection. This study constructed a recombinant Bacillus subtilis RB with PCV2 Capsid protein (Cap) on its spore surface and cotB as a fusion partner. The immune properties of the recombinant strain were evaluated in a mouse model. IgA in intestinal contents and IgG in serum were detected by enzyme-linked immunosorbent assay (ELISA). The results demonstrated that recombinant spores could activate strong specific mucosal and humoral immune responses. In addition, spores showed good mucosal immune adjuvant function, promoting the proliferation of CD3+, CD4+ and CD8+ T cells and other immune cells. We also found that the relative expression of inflammatory cytokines such as IL-1β, IL-6, IL-10, TNF-α and IFN in the small intestinal mucosa was significantly up-regulated under the stimulation of recombinant bacteriophage. These effects are important for the balance of Th1/Th2-like responses. In summary, our results suggest that recombinant B. subtilis RB as a feed additive provides a new strategy for the development of novel and safe PCV2 mucosal subunit vaccines.

Low molecular weight sulfated chitosan efficiently reduces infection capacity of porcine circovirus type 2 (PCV2) in PK15 cells

BackgroundPorcine circovirus type 2 (PCV2)-associated diseases are a major problem for the swine industry worldwide. In addition to vaccines, the availability of antiviral polymers provides an efficient and safe option for reducing the impact of these diseases. By virtue of their molecular weight and repetitious structure, polymers possess properties not found in small-molecule drugs. In this perspective, we focus on chitosan, a ubiquitous biopolymer, that adjusts the molecular weight and sulfated-mediated functionality can act as an efficient antiviral polymer by mimicking PCV2-cell receptor interactions.MethodsSulfated chitosan (Chi-S) polymers of two molecular weights were synthesized and characterized by FTIR, SEM–EDS and elemental analysis. The Chi-S solutions were tested against PCV2 infection in PK15 cells in vitro and antiviral activity was evaluated by measuring the PCV2 DNA copy number, TCID50 and capsid protein expression, upon application of different molecular weights, sulfate functionalization, and concentrations of polymer. In addition, to explore the mode of action of the Chi-S against PCV2 infection, experiments were designed to elucidate whether the antiviral activity of the Chi-S would be influenced by when it was added to the cells, relative to the time and stage of viral infection.ResultsChi-S significantly reduced genomic copies, TCID50 titers and capsid protein of PCV2, showing specific antiviral effects depending on its molecular weight, concentration, and chemical functionalization. Assays designed to explore the mode of action of the low molecular weight Chi-S revealed that it exerted antiviral activity through impeding viral attachment and penetration into cells.ConclusionsThese findings help better understanding the interactions of PCV2 and porcine cells and reinforce the idea that sulfated polymers, such as Chi-S, represent a promising candidates for use in antiviral therapies against PCV2-associated diseases. Further studies in swine are warranted.

Evaluation of a Virus-like Nanoparticle Porcine Circovirus Type-2 (PCV2) Capsid Protein Fused with the Pig Immunoglobulin Fc Fragment as a Novel Vaccine Candidate against PCV2 in Mice.

Porcine circovirus Type 2 (PCV2) is a primary etiological pathogen of post-weaning multi-systemic wasting syndrome (PMWS). The capsid protein of PCV2 is the crucial immunogenic protein which can induce antibody generation and immune responses. However, there is still a lack of efficient PCV2 vaccines with high immunogenicity. In the current study, we developed a novel engineered PCV2 capsid (∆1-41aa)-pFc fusion protein (PCFP), which comprised a truncated capsid protein of PCV2 and a porcine IgG Fc fragment, fused to the capsid protein of PCV2 at the C-terminus. We found that this novel fusion protein could auto-assemble into virus-like nanoparticles with an estimated mean diameter of 22.6 nm, characterized by transmission electron microscopy. Immunization of BALB/c mice with this fusion protein significantly increased the production levels of anti-PCV2-capsid protein antibody in serum. Besides, the virus-like nanoparticles, PCFP was demonstrated to induce efficient cellular immune responses in mice, as evident by the high specific T cell reactivity to the PCFP fusion protein and the high production of the immune cytokines IFN-γ and IL-10 in an ex vivo re-stimulation system. Collectively, these findings demonstrate that the PCV2 truncated capsid subunit Fc-fusion protein can induce both cellular and humoral immune responses, and it displays great application potential.

Selection and Characterization of a Single-Chain Variable Fragment against Porcine Circovirus Type 2 Capsid and Impedimetric Immunosensor Development.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD) that causes huge global economic losses for the swine industry. Effective strategies or rapid detection of PCV2 in pig are essential to control PCVAD. Here, single-chain variable fragments (scFvs) were selected and characterized against the PCV2 capsid using phage display technology. Phage scFv clones were selected from the human scFv phagemid library (Tomlinson I + J) for direct panning against the PCV2 capsid. Eighty-four monoclonal phage scFvs were individually tested for binding to the PCV2 capsid by ELISA. Eight scFv clones showed significant binding to the PCV2 capsid and only three clones (clone nos. 13, 37, and 81) contained both VHCDRs and VLCDRs in the sequence. Clone scFv no. 81 had the highest reactivity to the PCV2 capsid and was constructed in the pET22b (+) expression vector. The recombinant was transformed to Escherichia coli BL21(DE3) for expression and purification. The scFv showed appropriate affinity to the PCV2 capsid by western blot analysis. Kinetics of scFv and the PCV2 capsid were determined using surface plasmon resonance and showed binding affinity in the nanomolar range (KD = 57.2 nM). Our scFv was first applied in the development of an impedimetric immunosensor for PCV2 capsid detection, and results showed that impedance increased with increasing PCV2 capsid expression with limit of detection = 114 nM. Findings demonstrated that our scFv has potential for use as a receptor for biosensor devices.

Macrophage Polarization Modulated by Porcine Circovirus Type 2 Facilitates Bacterial Coinfection.

Polarization of macrophages to different functional states is important for mounting responses against pathogen infections. Macrophages are the major target cells of porcine circovirus type 2 (PCV2), which is the primary causative agent of porcine circovirus–associated disease (PCVAD) leading to immense economic losses in the global swine industry. Clinically, PCV2 is often found to increase risk of other pathogenic infections yet the underlying mechanisms remain to be elusive. Here we found that PCV2 infection skewed macrophages toward a M1 status through reprogramming expression of a subset of M1-associated genes and M2-associated genes. Mechanistically, induction of M1-associated genes by PCV2 infection is dependent on activation of nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK) signaling pathways whereas suppression of M2-associated genes by PCV2 is via inhibiting expression of jumonji domain containing-3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase that regulates M2 activation of macrophages. Finally, we identified that PCV2 capsid protein (Cap) directly inhibits JMJD3 transcription to restrain expression of interferon regulatory factor (IRF4) that controls M2 macrophage polarization. Consequently, sustained infection of PCV2 facilitates bacterial infection in vitro. In summary, these findings showed that PCV2 infection functionally modulated M1 macrophage polarization via targeting canonical signals and epigenetic histone modification, which contributes to bacterial coinfection and virial pathogenesis.

Oral Immunization with Lactobacillus casei Expressing the Porcine Circovirus Type 2 Cap and LTB Induces Mucosal and Systemic Antibody Responses in Mice.

Porcine circovirus type 2 (PCV2) causes many diseases in weaned piglets, leading to serious economic losses to the pig industry. This study investigated the immune response following oral administration of Lactobacillus casei ATCC393 (L. casei 393) expressing PCV2 capsid protein (Cap) fusion with the Escherichia coli heat-labile toxin B subunit (LTB) in mice. Recombinant L. casei strains were constructed using plasmids pPG611.1 and pPG612.1. The expression and localization of proteins from recombinant pPG611.1-Cap-LTB (pPG-1-Cap-LTB)/L. casei 393 and pPG612.1-Cap-LTB (pPG-2-Cap-LTB)/L. casei 393 were detected. All recombinant strains were found to be immunogenic by oral administration in mice and developed mucosal and systemic immune responses against PCV2. The titers of specific antibodies in mice administered pPG-2-Cap-LTB/L. casei 393 were higher than those in mice administered pPG-1-Cap-LTB/L. casei 393 in serum and the mucosal samples. The mucosal immune response was not only limited to the gastrointestinal tract but was also generated in other mucosal parts. Thus, the application of recombinant L. casei could aid in vaccine development for PCV2.

AlphaB-crystallin promotes porcine circovirus type 2 replication in a cell proliferation-dependent manner

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) and causes heavy economic losses to the porcine industry worldwide. In this study, PK-15 cells were infected with PCV2 for 48 h, then harvested and subjected to label-free quantitative proteomic mass spectrometry. In total, 1212 proteins were differentially expressed in PCV2-infected cells compared with mock-infected cells, including 796 upregulated and 416 downregulated proteins. Gene ontology analysis showed that these differentially expressed proteins were involved in biological processes, cellular components and molecular functions, and these categories included cellular processes, environmental information processing, genetic information processing, disease, metabolism, and body systems. Enrichment analysis of the KEGG pathway showed that innate immune responses were significantly enriched. AlphaB-crystallin (CRYAB) interacts with desmin and cytoplasmic actin to prevent protein misfolding and aggregation, helping to maintain cytoskeletal integrity and promoting cell proliferation. In this study, CRYAB was found to effect the replication of PCV2, as verified by qRT-PCR, TCID50 determination and western blot analysis. Overexpression of CRYAB significantly upregulated PCV2 capsid protein and increased viral titers in both PK-15 cells and culture supernatants, whereas the opposite results were obtained in CRYAB knockdown cells. Furthermore, we revealed that the promotion of PCV2 replication by CRYAB was dependent on cell proliferation. To our knowledge, this is the first report of the effect of CRYAB on PCV2 replication and our findings contribute to a greater understanding of the mechanism of PCV2 replication and pathogenesis, as well as the host’s response to PCV2 infection.

Porcine circovirus 2 capsid protein produced in N. benthamiana forms virus-like particles that elicit production of virus-neutralizing antibodies in guinea pigs

Porcine circovirus type 2 (PCV2) is a non-enveloped, icosahedral virus of the Circoviridae family, with a small, circular, single-stranded DNA genome. PCV2 infections cause substantial economic losses in the pig industry worldwide. Currently, commercially produced PCV2 vaccines are expensive, whereas plant-based expression systems can produce recombinant proteins at low cost for use as vaccines. In this study, recombinant PCV2 capsid protein (rCap) was transiently expressed in Nicotiana benthamiana and purified by metal affinity chromatography, with a yield of 102 mg from 1 kg plant leaves. Electron microscopy confirmed that purified rCap self-assembled into virus-like particles (VLPs) at neutral pH. It was shown to provoke a strong immune response in guinea pigs. The results indicate that plant systems can enable production of large amounts of proteins to serve as candidates for subunit vaccines.

Porcine circovirus genotypes and their copathogens in pigs with respiratory disease in southern provinces of Vietnam.

This study was conducted to investigate the genetic diversity of porcine circovirus type 2 (PCV2) and its coinfecting pathogens in pigs with respiratory disease in Vietnam. Samples from 127 clinical cases were obtained from different southern provinces of Vietnam from January 2018 to January 2020 for PCR and sequence analysis. The infection rate of PCV2 was 78.8%, and the major pathogens found in coinfections with PCV2 were porcine reproductive and respiratory syndrome virus, Mycoplasma hyopneumoniae, and Haemophilus parasuis. Forty-three PCV2-positive clinical samples were selected for amplification and sequencing of the ORF2 region. Phylogenetic analysis of PCV2 ORF2 showed that five of the sequences belonged to PCV2b (11.6%) and 38 belonged to PCV2d (88.4%), indicating that PCV2d strains were predominant in southern provinces of Vietnam. Alignment of the predicted amino acid sequences of the PCV2 capsid protein revealed polymorphic sites in the antibody recognition regions. This study demonstrates the prevalence of the PCV2d genotype in southern Vietnam and presents a comprehensive overview of the coinfecting pathogens associated with PCV2 in young pigs with respiratory disease.

High repetitive arginine in the anterior of PCV3 capsid protein is a severe obstacle for its expression in E. coli

Porcine circovirus type 3 (PCV3) is a novel circovirus identified in sows with PDNS-like clinical signs and reproductive failure. The capsid protein (CAP) of PCV3 is expected to be an effective vaccine candidate. Here, we expressed the original capsid protein, truncated capsid protein without anterior highly repetitive arginine (ΔCAP) and their codon-optimized counterparts in E. coli. These results showed that lots of repeated arginine could severely lower the expression of PCV3 capsid protein in E. coli. At the same time, the recombined truncated PCV3 capsid protein forms typic virions. The efficient expression of capsid protein is expected to serve the development of PCV3 vaccines and other studies of PCV3 capsid protein.

Genotyping of PCV3 based on reassembled viral gene sequences.

Porcine circovirus type 3 (PCV3) has been reported in many countries such as USA, China, Korea and many European countries during 2015–2018. The six PCV3 strains named IH, SJ, N5, N10, N13 and N62 were detected out of 220 samples by PCR methods while the prevalence our study was conducted in 2017 to 2018. The six detected strains were hard to genotype with reference viruses due to their diverse phylogenetic relationship. PCV3 capsid, ORF3 and replicase protein coding genes were reassembled at the nucleotide sequence level, then 16 new reassembled PCV3 sequences were generated. Based on the maximum likelihood mapping analysis of 303 PCV3 sequences a model with a combination of replicase, ORF3 and capsid protein coding genes was selected as the most appropriate target for genotyping, which provided the best support for the clade classification into three genotypes and several subtypes (genotype 1, genotype 2; subtype: a and b, genotype 3; subtype a, b, c, d, e, f, g, h). This study, the IH_Korea_2017 and N62_Korea_2018 strains belong to genogroup 3 (subtype a) the SJ_Korea_2017 strain genogroup 3 (subtype g) and the N5, N10, N13 Korea_ 2018 strains genogroup 3 (subtype f), respectively. In conclusion, this study may provide insights to classification of PCV3 genotypes around the world.