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, ß-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.

Immunoglobin G Sero-Dynamics Aided Host Specific Linear Epitope Identification and Differentiation of Infected from Vaccinated Hosts.

Differentiation of infected from vaccinated hosts (DIVH) is a critical step in virus eradication programs. DIVH-compatible vaccines, however, take years to develop, and are therefore unavailable for fighting the sudden outbreaks that typically drive pandemics. Here, we establish a protocol for the swift and efficient development of DIVH assays, and show that this approach is compatible with any type of vaccines. Using porcine circovirus 2 (PCV2) as the experimental model, the first step is to use Immunoglobin G (IgG) sero-dynamics (IsD) curves to aid epitope discovery (IsDAED): PCV2 Cap peptides were categorized into three types: null interaction, nonspecific interaction (NSI), and specific interaction (SI). We subsequently compared IsDAED approach and traditional approach, and demonstrated identifying SI peptides and excluding NSI peptides supports efficient diagnostic kit development, specifically using a protein-peptide hybrid microarray (PPHM). IsDAED directed the design of a DIVH protocol for three types of PCV2 vaccines (while using a single PPHM). Finally, the DIVH protocol successfully differentiated infected pigs from vaccinated pigs at five farms. This IsDAED approach is almost certainly extendable to other viruses and host species. IMPORTANCE Sudden outbreaks of pandemics caused by virus, such as SARS-CoV-2, has been determined as a public health emergency of international concern. However, the development of a DIVH-compatible vaccine is time-consuming and full of uncertainty, which is unsuitable for an emergent situation like the ongoing COVID-19 pandemic. Along with the development and public health implementation of new vaccines to prevent human diseases, e.g., human papillomavirus vaccines for cervical cancer; enterovirus 71 vaccines for hand, foot, and mouth disease; and most recently SARS-CoV-2, there is an increasing demand for DIVH. Here, we use the IsDAED approach to confirm SI peptides and to exclude NSI peptides, finally to direct the design of a DIVH protocol. It is plausible that our IsDAED approach is applicable for other infectious disease.

Synergistic Pathogenicity by Coinfection and Sequential Infection with NADC30-like PRRSV and PCV2 in Post-Weaned Pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCVs) are two major viruses that affect pigs. Coinfections between PRRSV and PCV2 are frequently reported in most outbreaks, with clinical presentations involving dyspnea, fever, reduced feed intake, weight loss, and death in fattening pigs. The NADC30-like PRRSV and PCV2d are the main circulating virus strains found in China. This study determines the impact of NADC30-like PRRSV and PCV2d mono-infection and coinfection on the immune system, organ pathology, and viral shedding in five-week-old post-weaned pigs. Pigs were randomly divided into six groups: PBS, PRRSV, PCV2, PRRSV-PCV2 coinfection (co), and PRRSV-PCV2 or PCV2-PRRSV sequential infections. Fever, dyspnea, decreased feed intake, weight loss, and pig deaths occurred in groups infected with PRRSV, Co-PRRSV-PCV2, and PRRSV-PCV2. The viral load was higher in Co-PRRSV-PCV2, PRRSV-PCV2, and PCV2-PRRSV than those mono-infected with PRRSV or PCV2. Additionally, cytokines (IFN-γ, TNF-α, IL-4, and IL-10) produced by pigs under Co-PRRSV-PCV2 and PRRSV-PCV2 groups were more intense than the other groups. Necropsy findings showed hemorrhage, emphysema, and pulmonary adhesions in the lungs of pigs infected with PRRSV. Smaller alveoli and widened lung interstitium were found in the Co-PRRSV-PCV2 and PRRSV-PCV2 groups. In conclusion, PRRSV and PCV2 coinfection and sequential infection significantly increased viral pathogenicity and cytokine responses, resulting in severe clinical signs, lung pathology, and death.

Harnessing the Genetic Plasticity of Porcine Circovirus Type 2 to Target Suicidal Replication.

Porcine circovirus type 2 (PCV2), the causative agent of a wasting disease in weanling piglets, has periodically evolved into several new subtypes since its discovery, indicating that the efficacy of current vaccines can be improved. Although a DNA virus, the mutation rates of PCV2 resemble RNA viruses. The hypothesis that recoding of selected serine and leucine codons in the PCV2b capsid gene could result in stop codons due to mutations occurring during viral replication and thus result in rapid attenuation was tested. Vaccination of weanling pigs with the suicidal vaccine constructs elicited strong virus-neutralizing antibody responses. Vaccination prevented lesions, body-weight loss, and viral replication on challenge with a heterologous PCV2d strain. The suicidal PCV2 vaccine construct was not detectable in the sera of vaccinated pigs at 14 days post-vaccination, indicating that the attenuated vaccine was very safe. Exposure of the modified virus to immune selection pressure with sub-neutralizing levels of antibodies resulted in 5 of the 22 target codons mutating to a stop signal. Thus, the described approach for the rapid attenuation of PCV2 was both effective and safe. It can be readily adapted to newly emerging viruses with high mutation rates to meet the current need for improved platforms for rapid-response vaccines.

PCV2 targets cGAS to inhibit type I interferon induction to promote other DNA virus infection.

Viruses use diverse strategies to impair the antiviral immunity of host in order to promote infection and pathogenesis. Herein, we found that PCV2 infection promotes the infection of DNA viruses through inhibiting IFN-ß induction in vivo and in vitro. In the early phase of infection, PCV2 promotes the phosphorylation of cGAS at S278 via activation of PI3K/Akt signaling, which directly silences the catalytic activity of cGAS. Subsequently, phosphorylation of cGAS at S278 can facilitate the K48-linked poly-ubiquitination of cGAS at K389, which can been served as a signal for recognizing by the ubiquitin-binding domain of histone deacetylase 6 (HDAC6), to promote the translocation of K48-ubiquitinated-cGAS from cytosol to autolysosome depending on the deacetylase activity of HDAC6, thereby eventually resulting in a markedly increased cGAS degradation in PCV2 infection-induced autophagic cells relative to Earle's Balanced Salt Solution (EBSS)-induced autophagic cells (a typical starving autophagy). Importantly, we found that PCV2 Cap and its binding protein gC1qR act as predominant regulators to promote porcine cGAS phosphorylation and HDAC6 activation through mediating PI3K/AKT signaling and PKCδ signaling activation. Based on this finding, gC1qR-binding activity deficient PCV2 mutant (PCV2RmA) indeed shows a weakened inhibitory effect on IFN-ß induction and a weaker boost effect for other DNA viruses infection compared to wild-type PCV2. Collectively, our findings illuminate a systematic regulation mechanism by which porcine circovirus counteracts the cGAS-STING signaling pathway to inhibit the type I interferon induction and promote DNA virus infection, and identify gC1qR as an important regulator for the immunosuppression induced by PCV2.

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.

Pathogenic Characterization of a Porcine Circovirus Type 3 Isolate from Heilongjiang, China.

The clinical outcome of porcine circovirus 3 (PCV3) infection is still controversial. Herein, a novel PCV3 isolate (PCV3-China/DB-1/2017) with the molecular characterization of 24A and 27K in the Cap protein was used to inoculate three-week-old cesarean-derived, colostrum-deprived piglets. The nine PCV3 DB-1 inoculated piglets exhibited no obvious clinical symptoms or macroscopic lesions. PCV3 displayed a broad histotropism, including the heart, liver, spleen, lung, kidney, brain, lymph nodes, and tonsil, and the lungs and lymph nodes contained a higher quantity of viral genomes compared to that of the other organs. From 7 days after PCV3 DB-1 inoculation, the piglets showed obvious IgG antibody responses against PCV3 rCap-VLPs. The cumulative results demonstrated that PCV3 trend to low pathogenicity.

Relevance of antibodies against the Chicken Anaemia Virus.

Chicken Infectious Anaemia (CIA) Virus (CAV) inhibits the function of multiple immune compartments. Mortality due to clinical infection is controlled in broilers by passive immunization derived from vaccinated breeders. Therefore, serological tests are often used in chicks to determine maternally-derived antibodies (MDA). We used a vaccine overdose-induced model of CIA. The model replicated the most common features of the disease. This model was used to determine the role of MDA in the protection of chicks. Hatchlings were tested for anti-CAV titers by ELISA and were sorted into groups based on antibody levels. SPF chicks were used as a no-antibody control. Lower specific antibody levels seemed to facilitate viral entry into the thymus, but viral levels, CD4+ and CD8+ counts, thymus architecture, and haematocrit were preserved by MDA, regardless of its levels. Levels of MDA are not correlated with protection from CIA, but are important for the progression CAV infection.

The Mink Circovirus Capsid Subunit Expressed by Recombinant Baculovirus Protects Minks against Refractory Diarrhea in Field.

Mink refractory diarrhea is a seasonal disease that occurs in many mink farms in China. Mink circovirus (MiCV) has been recognized as the causative agent of the disease. The aim of the study was to develop a subunit vaccine against mink refractory diarrhea. A recombinant baculovirus strain expressing the capsid protein was constructed using the baculovirus expression vector system (BEVS). A subunit vaccine was developed based on the capsid protein with appropriate adjuvant. Then, a field trial was carried out in two districts in order to evaluate the efficiency of the subunit vaccine. The field trial indicated that in total, only 1.8% of the minks developed typical diarrhea in the vaccinated group compared with 74.5% in the control group. The vaccination could significantly reduce the infection rate of MiCV among the mink herds and could restrain the virus' shedding from feces. Furthermore, the vaccinated group had a higher average litter size in the following year compared to the control group. Collectively, the results indicated that the subunit vaccine based on the capsid protein can provide reliable protection against MiCV infection.

A nanobody-horseradish peroxidase fusion protein-based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2.

BACKGROUND: The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)­horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum. RESULTS: Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15­HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect anti­PCV2 antibodies. The cut­off value of the cELISA is 20.72 %. Three hundreds and sixty porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68 % and 95.92 %, respectively. The coincidence rate of the two methods was 99.17 %. When detecting 620 clinical porcine serum samples, a good consistent (kappa value = 0.954) was found between the results of the cELISA and those of commercial kits. CONCLUSIONS: In brief, the newly developed cELISA based PCV2-Nb15­HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccine efficacy and the indirect diagnosis of PCV2 infection.

A 14 bp indel polymorphism in the promoter region is associated with different responses to porcine circovirus type 2 infection by regulating MRC1 transcription.

Mannose receptor, C type 1 (MRC1) is a key factor in regulating the body's immune response to resist pathogen invasions. In this study, mRNA expressions of MRC1 gene in nine porcine organs/tissues were compared between Laiwu (LW) and Yorkshire × Landrace crossbred (YL) pigs prior to and post PCV2 infection. We found that, for pigs uninfected with PCV2, MRC1 mRNA expressions in the lung, spleen, large intestine, small intestine and mesenteric lymph node tissues of LW were significantly higher than those of YL pigs (P < 0.05). After PCV2 infection, MRC1 mRNA levels in the liver, kidney and mesenteric lymph node were significantly increased in LW pigs (P < 0.05); while, significantly decreased in the heart and lung tissues of YL pigs (P < 0.05). The transcriptional activity of porcine MRC1 promoter was further analyzed to investigate the molecular mechanism underlying these expressional differences in response to PCV2 infection. Luciferase assay indicated that a 14 bp indel polymorphism "GTTTTTTTTTTTTT" at the site -864 of MRC1 promoter contributed to the transcriptional activity. The frequency of 14 bp insertion in LW and Dapulian pigs, generally resistant to PCV2 infection, was higher than that in Duroc, Landrace and Yorkshire pigs, which were sensitive to PCV2 infection. The promoter with 14 bp insertion displayed higher MRC1 transcription level both prior to and post PCV2 infection compared with that carrying no insertion in PK15 cells (P < 0.01). The results suggest that this 14 bp indel polymorphism is associated with different responses to PCV2 infection by regulating MRC1 transcription.

Pathogenicity and immune response against porcine circovirus type 3 infection in caesarean-derived, colostrum-deprived pigs.

Recently, a novel PCV species (PCV3) has been detected in cases associated with sow mortality, lesions consistent with porcine dermatitis and nephropathy syndrome, reproductive failure and multisystemic inflammation. The pathogenesis and clinical significance of PCV3 is still unclear. In this study, we investigated the immunopathogenesis of PCV3 in CD/CD pigs. Four treatment groups, PCV3 (n=6), PCV3-KLH (n=6), control (n=3) and control-KLH (n=3), were included with PCV3-positive tissue homogenate (gc=3.38×1012 ml-1 and gc=1.04×1011 ml-1), confirmed by quantitative PCR (qPCR) and next-generation sequencing. Clinical signs, viremia, viral shedding, systemic cytokines, humoral (IgG) and T-cellular response were evaluated for 42 days. At necropsy, tissues were collected for histological evaluation and PCV3 detection by qPCR and in situ hybridization. No significant clinical signs were observed through the study. Viremia was detected in both PCV3-inoculated groups from 3 days post-inoculation (p.i.) until the end of the study. Nasal shedding was detected from 3 to 28 days p.i. and faecal shedding was transient. PCV3 induced an early (7 days p.i.) and sustained (42 days p.i.) IgG response. No significant T-cell response was observed. Histological evaluation demonstrated lesions consistent with multisystemic inflammation and perivasculitis. All tissues evaluated were positive by qPCR and virus replication was confirmed by positive in situ hybridization. This study demonstrated the potential role of PCV3 in subclinical infection, producing a mild, multisystemic inflammatory response, prolonged viremia detectable for 42 days p.i., presence of IgG humoral response and viral shedding in nasal secretions. More research is required to understand and elucidate potential co-factors necessary in the manifestation and severity of clinical disease.

Probiotic supplementation containing Bacillus velezensis enhances expression of immune regulatory genes against pigeon circovirus in pigeons (Columba livia).

AIMS: In this study, we aimed to isolate and evaluate the efficacy of Bacillus velezensis as a probiotic and to assess its activity towards pigeons infected with pigeon circovirus (PiCV). METHODS AND RESULTS: Bacillus velezensis, isolated from pigeon faeces, was orally administered to pigeons for 60 days. After pigeons were challenged with PiCV, the PiCV viral load and expression of indicator genes for innate immunity were detected in spleen tissue and faeces of pigeons. Bacillus velezensis significantly reduced the PiCV viral load in the faeces and spleen of pigeons 5 days post-challenge (dpc). The mRNA expression levels of treated pigeons showed that interferon-gamma (IFN-γ), myxovirus resistance 1 (Mx1), and signal transducers and activators of transcription 1 (STAT1) genes were upregulated, whereas no expression of interleukin-4 (IL-4) was detected. Moreover, toll-like receptor 2 (TLR2) and 4 (TLR4) were significantly upregulated in probiotic-treated pigeons (P < 0·05). CONCLUSIONS: This is the first report showing that probiotic supplementation can effectively enhance the T-helper type 1 immune response and decrease the PiCV viral loads in pigeons. SIGNIFICANCE AND IMPACT OF THE STUDY: This study proposes that the administration of a probiotic strain, B. velezensis, to pigeons can protect against PiCV infection.

Integrated System for Purification and Assembly of PCV Cap Nano Vaccine Based on Targeting Peptide Ligand.

PURPOSE: The vaccine design has shifted from attenuated or inactivated whole pathogen vaccines to more pure and defined subunit vaccines. The purification of antigen proteins, especially the precise display of antigen regions, has become a key step affecting the effectiveness of subunit vaccines. MATERIALS AND METHODS: This work presents the application of molecular docking for a peptide ligand designed for PCV2 Cap purification and assembly in one step. Based on the PCV2 Cap protein affinity peptide (L11-DYWWQSWE), the amino terminal of PCV2 Cap was covalently coupled with the polylactic acid-glycolic acid copolymer (PLGA) carboxyl terminal through the EDC/NHS method. RESULTS: The PLGA had an average diameter of 106 nm. The average diameter increased to 122 nm after the PCV2 Cap protein conjugation, and the Zeta potential shifted from -13.7 mV to -9.6 mV, indicating that the PCV2 Cap protein stably binds to the PLGA. Compared with the free PCV2 Cap protein group, the neutralizing antibody titer was significantly increased on the 14th day after the PLGA-Cap immunization (P < 0.05). The neutralizing antibody level was extremely significant on the 28th day (P < 0.001). The CCK-8 analysis showed that PLGA-Cap had an obvious cytotoxic effect on RAW264.7 cells at the PLGA nanoparticle concentration up to 200 µg/mL but had no obvious cytotoxic effect on DC2.4 cells. Compared with the Cap protein group, the antigen-presenting cells had a stronger antigen uptake capacity and a higher fluorescence in the PLGA-Cap group. The immune effect showed that the level of the neutralizing antibody produced by this structure is much better than that of purified protein and helps improve the immune system response. CONCLUSION: This technology provides a potential new perspective for the rapid enrichment of the antigen protein with the affinity peptide ligand.

Construction of polycistronic baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of its immunogenicity in mice and swine.

To increase expression levels of the PCV2 Cap(d41) protein, novel baculovirus surface display vectors with multiple expression cassettes were constructed to create recombinant baculoviruses BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41). Our results reveal that the recombinant baculovirus BacDD-4Cap(d41) was able to express the highest levels of Cap(d41) protein. Optimum conditions for expressing the PCV2 Cap(d41) protein were determined, and our results show that 107 of Sf-9 infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 5 for 3 days showed the highest level of protein expression. Mice immunized with the 4Cap(d41) vaccine which was prepared from the recombinant baculovirus-infected cells (107) elicited higher ELISA titers compared to the Cap (d41) vaccine. The 4Cap(d41) vaccine could elicit anti-PCV2 neutralizing antibodies and IFN-γ in mice, as confirmed by virus neutralization test and IFN-γ ELISA. Moreover, the swine lymphocyte proliferative responses indicated that the 4Cap(d41) vaccine was able to induce a clear cellular immune response. Flow cytometry analysis showed that the percentage of CD4+ T cells and CD4+/CD8+ ratio was increased significantly in SPF pigs immunized with the 4Cap(d41) vaccine. Importantly, the 4Cap(d41) vaccine induced an IFN-γ response, further confirming that its effect is through cellular immunity in SPF pigs. An in vivo challenge study revealed that the 4Cap(d41) and the commercial vaccine groups significantly reduce the viral load of vaccinated pigs as compared with the CE negative control group. Taken together, we have successfully developed a 4Cap(d41) vaccine that may be a potential subunit vaccine for preventing the disease associated with PCV2 infections.

Basigin-CyP elevated porcine circovirus type2 replication.

Porcine circovirus type2 (PCV2) is a member of the circoviridae family. PCV2 was identified as the main pathogen of postweaning multisystemic wasting syndrome (PMWS) in weaned piglets and causes massive economic loss. Basigin, is a transmembrane glycoprotein belonging to the immunoglobulin superfamily; which is also a receptor for cyclophilins. CyP belongs to the immunophilin family that has peptidyl-prolyl cis-trans isomerase activity. Basigin-CyP interaction affects the replication stages of several viruses. In this study, we found that Basigin could elevate the replication of PCV2, and the Basigin only affected the replication stage rather than adsorption or endocytosis stages. In addition, the ligands of Basigin, CyPA and CyPB also elevated the replication of PCV2. Basigin-CyP interation was necessary for elevating PCV2 replication; At last, CyPs were proved to promote the replication of PCV2 by activating ERK signaling.

Coinfection with Porcine Circovirus Type 2 (PCV2) and Streptococcus suis Serotype 2 (SS2) Enhances the Survival of SS2 in Swine Tracheal Epithelial Cells by Decreasing Reactive Oxygen Species Production.

Porcine circovirus type 2 (PCV2) and Streptococcus suis serotype 2 (SS2) clinical coinfection cases have been frequently detected. The respiratory epithelium plays a crucial role in host defense against a variety of inhaled pathogens. Reactive oxygen species (ROS) are involved in killing of bacteria and host immune response. The aim of this study is to assess whether PCV2 and SS2 coinfection in swine tracheal epithelial cells (STEC) affects ROS production and investigate the roles of ROS in bacterial survival and the inflammatory response. Compared to SS2 infection, PCV2/SS2 coinfection inhibited the activity of NADPH oxidase, resulting in lower ROS levels. Bacterial intracellular survival experiments showed that coinfection with PCV2 and SS2 enhanced SS2 survival in STEC. Pretreatment of STEC with N-acetylcysteine (NAC) also helps SS2 intracellular survival, indicating that PCV2/SS2 coinfection enhances the survival of SS2 in STEC through a decrease in ROS production. In addition, compared to SS2-infected STEC, PCV2/SS2 coinfection and pretreatment of STEC with NAC prior to SS2 infection both downregulated the expression of the inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and IL-1ß. Further research found that activation of p38/MAPK promoted the expression of inflammatory cytokines in SS2-infected STEC; however, PCV2/SS2 coinfection or NAC pretreatment of STEC inhibited p38 phosphorylation, suggesting that coinfection of STEC with PCV2 and SS2 weakens the inflammatory response to SS2 infection through reduced ROS production. Collectively, coinfection of STEC with PCV2 and SS2 enhances the intracellular survival of SS2 and weakens the inflammatory response through decreased ROS production, which might exacerbate SS2 infection in the host.

PCV cap proteins fused with calreticulin expressed into polymers in Escherichia coli with high immunogenicity in mice.

BACKGROUND: Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus diseases (PCVDs) which causes huge yearly economic losses in the swine industry. Capsid protein (Cap) is the major structural protein of PCV2 that can induce a protective immune response. Therefore, developing a novel and safe subunit vaccine against PCV2 infection is needed. RESULTS: In this study, the Cap gene was bound to the truncated calreticulin (CRT) (120-250 aa/120-308 aa) at the N/C terminal, and then the CRT-Cap fusion genes were expressed in Escherichia coli (E.coli). The size-exclusion chromatography and dynamic light scattering (DLS) data showed that the purified recombinant CRT-Cap fusion protein (rP5F) existed in the form of polymers. Immunization with rP5F stimulated high levels of PCV2 specific antibody and neutralization antibody in mice, which were almost identical to those induced by the commercial subunit and inactivated vaccines. The lymphocyte proliferation and cytokine secretion were also detected in rP5F immunized mice. According to the results of PCV2-challenge experiment, the virus loads significantly decreased in mice immunized with rP5F. The data obtained in the current study revealed that rP5F had the potential to be a subunit vaccine candidate against PCV2 in the future. CONCLUSIONS: We have successfully expressed Cap-CRT fusion proteins in E.coli and optimized rP5F could form into immunogenic polymers. Mice immunized with rP5F efficiently induced humoral and part of cellular immune responses and decreased the virus content against PCV2-challenge, which suggested that rF5P could be a potential subunit vaccine candidate.

A molecular survey and phylogenetic analysis of porcine circovirus type 3 using oral fluid, faeces and serum.

BACKGROUND: Porcine circovirus type 3 is the most recently discovered porcine circovirus, and an emerging pathogen. In this study the status of its presence on some Slovenian farms is reported. The effectiveness of the vaccine against porcine circovirus type 2 was assessed against porcine circovirus type 3. Group samples of oral fluid, faeces and individual serum samples were taken from six different pig categories and tested for presence of viral DNA, using both real time and conventional PCR. Positive samples were subjected to direct Sanger sequencing. Nucleotide sequences were analyzed and compared to GenBank PCV3 sequences. RESULTS: Positive samples were sent for genome sequencing, which confirmed the presence of virus in all different pig categories on five farms. A high to moderate correlation of strong statistical significance was found between individual serum samples, oral fluid and faeces. Slovenian PCV3 was found to be distributed in a way similar to that of other countries. Slovenian PCV3 nt sequences are highly related, sharing more than 99.5% nt identity. On one farm a commercially available vaccine against porcine circovirus type 2 was used on 3-week-old pigs. It did not affect the presence of porcine circovirus type 3 in oral fluid or sera of any of the seven age groups of pigs, each with two control groups. CONCLUSIONS: The results constitute the first discovery of the virus in Slovenia. Genome sequencing has revealed a high degree of similarity between Slovenian and GenBank isolates.

Establishment of a Rep' protein antibody detection method to distinguish natural infection with PCV2 from subunit vaccine immunization.

Introduction. PCV2 is a DNA virus that exists widely in pigs and has caused great economic losses to the pig industry worldwide. In the existing commercial PCV2 enzyme-linked immunosorbent assay (ELISA) kits both natural infection with PCV2 and vaccine immunization produce results that are positive for PCV2 Cap antibodies and therefore they cannot diagnose PCV2 infection in immunized pig farms.Aim. To establish a PCV2 non-structural protein antibody detection method that distinguishes between antibodies resulting from natural prior exposure (infection) and those induced by subunit vaccine immunization.Methodology. Based on the non-structural Rep' protein, we established an indirect ELISA (iELISA) using sera from guinea pigs and piglets.Results. The results for iELISA for guinea pig serum showed that animals vaccinated with a whole-virus inactivated PCV2 vaccine had 100 % (10/10) Cap antibody positivity and 100 % (10/10) Rep' antibody positivity. Guinea pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (10/10) Cap antibody positivity, while no (0/10) guinea pigs were Rep' antibody-positive. The combined detection results for the Rep' iELISA and a PCV2 Antibody Test kit (Commercial) showed that pigs vaccinated with a whole-virus inactivated PCV2 vaccine or PCV2 SD/2017 had 100 % (5/5) Cap antibody positivity and 100 % (5/5) Rep' antibody positivity. Pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (5/5) Cap antibody positivity, while no (0/10) pigs were Rep' antibody-positive.Conclusion. This paper describes an effective iELISA method that can distinguish natural infection with PCV2 (Cap and Rep positive) or inoculation with a whole-virus inactivated vaccine (Cap and Rep positive) from subunit vaccine immunization (Cap-positive, Rep-negative). These comparative assays could be very useful in the control of PCV2 in pig herds.