A multiplex real-time PCR assay for identification porcine circovirus 2, atypical porcine pestivirus, and classical swine fever virus.

PCV2 infects porcine placental trophoblast cells and remodels the lncRNA-mRNA regulatory network.

Porcine circovirus infections, primarily caused by porcine circovirus type 2 (PCV2), are one of the most significant problems in global pig production. Vaccination is a key tool for control and prevention of porcine circovirus infections. Recombinant analogs of capsid protein (rCap) of porcine circovirus type 2 (PCV2) are key components of modern diagnostic test systems and subunit vaccines. The stability of its antigenic activity is a critical parameter determining the efficiency and reproducibility of the functional product. The aim of this study was to develop and test an enzyme-linked immunosorbent assay system for interoperational monitoring of the specific activity of rCap protein. To obtain recombinant analogues of the PCV-2 capsid protein, the previously constructed recombinant E. coli BL21(DE3)pLysS-ORF2 producer strain (for prokaryotic analogue) and the stably expressing Chinese hamster ovary cell line CHO-K1/PCV-ORF2 (for eukaryotic analogue) were used; protein purification was performed according to previously developed protocols. Chromatographically purified IgG isolated from a pool of sera from PCV-2-seropositive pigs was used for sensitization of the solid phase (polystyrene plates); the specific peroxidase conjugate was obtained by the periodate method. The study resulted in the production of a stable peroxidase conjugate with a labeling density of 1.5:1. The developed test system enabled semiquantitative analysis of recombinant raw materials, demonstrating good reproducibility (coefficient of variation<7%). The test system was found to be universal and capable of assessing the activity of rCap variants obtained in both prokaryotic and eukaryotic expression systems, accurately recognizing their antigenic determinants. Thus, the implementation of the semiquantitative ELISA method in the production cycle will standardize the process of in-process quality control of biomass obtained using recombinant DNA technologies and guarantee stable antigenic characteristics of vaccines and diagnostic products.

Background: Porcine circovirus type 2 (PCV2) remains one of the most important pathogens that infects swine, causing considerable economic losses worldwide. PCV2 vaccines are commercially available, and the development of experimental vaccines that could confer better protection against emerging genotypes is underway. The expression of virus-like particles (VLPs) carrying different PCV2 capsid (Cap) peptides in E. coli was recently reported. These chimeric particles were adjuvated with an oil-in-water emulsion with polymer and induced different titers of serum IgG in BALB/c mice after a single subcutaneous injection. The aim of this study was to assess the immune response and protective efficacy elicited by VLPs carrying the PCV2b Cap carboxy-terminal peptide in the target species. Methods: Domestic pigs (Sus scrofa domesticus) were immunized intramuscularly with 25 μg of adjuvated chimeric VLPs on days 0 and 14 and challenged on day 28 with a PCV2b Mexican isolate. PCV2 peptide-specific IgG seroconversion, serum cytokines, viral load in nasal swabs and organs, and histopathological score were determined. Results: IgG levels peaked 28 days post-immunization. Interleukin-12 and -18 and interferon-gamma increased 21 days after immunization. In addition, genomic material of PCV2 was detected in nasal swabs from one specimen on day 7, two specimens on day 14, and two specimens on day 21 following viral challenge. Finally, histological lesions were not less severe in immunized specimens compared to non-vaccinated/challenged specimens. Conclusions: These results suggest that immunization with chimeric VLPs could contribute to controlling viral shedding in pig herds where a PCV2b genotype is most prevalent.

Porcine circovirus type 2 (PCV2), a widely distributed immunosuppressive virus, causes substantial economic losses in the global swine industry. C1QTNF6 has emerged as a novel immunoregulatory factor attracting increasing research interest due to its dual roles in both pro-inflammatory and antiviral immune responses. However, the role of C1QTNF6 in regulating PCV2 replication remains poorly characterized. Here, we analyzed C1QTNF6 expression patterns and identified its highly specific expression in placental tissues in both humans and pigs. We then overexpressed C1QTNF6 and conducted RNA sequencing analysis. Remarkably, 68 upregulated genes were identified, and most of them were interferon-stimulated genes (ISGs), including MX2 and ISG15. Functional enrichment analysis revealed that these genes were primarily associated with defense response to viruses and antiviral innate immune response. Subsequently, experimental data show that PCV2 infection significantly suppressed inflammatory responses and markedly downregulated the expression of C1qtnf6, MX2, and IFIT2. Moreover, experimental data indicated that C1QTNF6 inhibits PCV2 replication by targeting ISGs, while restoring MX2 expression. To verify whether C1QTNF6-MX2 antiviral axis mediates this antiviral effect, we constructed an MX2 overexpression plasmid and demonstrated that MX2 overexpression indeed significantly suppressed PCV2 replication. Together, these results provide important insights into PCV2-host interactions and the development of novel antiviral strategies.

The present study examined the occurrence of the 3 major genotypes of porcine circovirus 2 (PCV2a, PCV2b, PCV2d) in tissue samples from fetuses origination from litters suspicious for stillbirth, mummification, embryonic death, and infertility syndrome (SMEDI) affected litters from Germany.A total of 53 litters suspicious for SMEDI of 27 farms with a total of 469 fetuses sent in for diagnostic purposes between 2021-2023 from veterinarians in the field were available for the present examination. Veterinarians were asked to submit all dead piglets of SMEDI-litters to allow random sampling. Four fetuses per litter were randomly selected for the study. In total, 209 fetuses were examined by qPCR for PCV2 DNA and in case of a positive result, further genotyped by PCV2 genotype-specific qPCR or sequencing of the open reading frame 2. Farm specific data was collected on a voluntary base and included in the analyses.In total 40.7% (11/27) of farms, 37.7% of litters (20/53) and 21.1% (44/209) of fetuses were positive for PCV2 DNA. Genotyping by qPCR was successful in 37 PCV2 positive tissue samples. For 4 additional samples, sequencing of PCV2 DNA was performed to support the genotype assignment. After all, 29.6% (8/27) of farms, 30.2% (16/53) of litters and 19.1% (40/209) of fetuses were positive for PCV2d. One farm (3.7%) had a PCV2a+PCV2d positive fetus (0.5%) in one litter (1.9%).PCV2d could be identified as the main PCV2 genotype in SMEDI-associated fetuses. This finding is in line with the overall observed genotype shift toward PCV2d as the predominating PCV2 genotype in the domestic pig population not only in Germany but also in most all relevant pig producing countries worldwide. Moreover, these findings also indicate that diaplacental transmission may play a major role in the spread of PCV2 to downstream pig populations and thus, also for the observed genotype shift. The high Cq-values in tissue samples indicated that PCV2 was not the etiological pathogen in most cases.The occurrence of SMEDI in a piglet producing herd needs diagnostic attention and may also include PCV2 diagnostics including genotyping in PCV2 associated cases. This approach could enable adjustment of the vaccination protocol on farm level and early detection of newly introduced PCV2 genotype in a pig herd.

Porcine circovirus types 2 (PCV2) and 3 (PCV3) are major pathogens affecting swine health and productivity, yet important gaps remain in understanding their evolution and circulation in Europe, particularly within wild boar populations that may serve as reservoirs. This study examined the genetic diversity and evolutionary dynamics of PCV2 and PCV3 in Portugal, drawing on viral genomes obtained from domestic pigs and wild boars to explore transmission patterns, spillover events and the contribution of recombination to viral emergence. We identified two PCV2 genotypes (PCV2a and PCV2d) and two PCV3 genotypes (PCV3-2a and PCV3-3g) circulating in Portuguese swine. Phylogeographic reconstruction revealed multiple introductions of both PCV2 and PCV3 from China into Europe, followed by regional diversification and subsequent spread within European wild boar populations. Evidence of bidirectional viral exchange between domestic pigs and wild boars was also observed. Recombination played a notable role in PCV2 evolution, with consistent signals detected among PCV2a sequences and indications that the PCV2h genotype likely originated from a recombinant event involving a Portuguese PCV2a strain and a Chinese PCV2d strain. By contrast, no recombination was detected in PCV3, suggesting that its evolution is primarily mutation-driven. Overall, these findings highlight the complex evolutionary history of swine circoviruses in Europe and underscore the importance of continuous genomic surveillance in both domestic and wild hosts. The study reinforces the value of a One Health approach for monitoring and controlling emerging circoviruses with implications for animal health and livestock production.

IntroductionPorcine enteric coronaviruses (PECs) are a group of viruses that cause severe diarrhea in piglets, significantly impacting the pig industry and resulting in huge economic losses. Important PECs include porcine epidemic diarrhea virus (PEDV), porcine enteric alphacoronavirus (PEAV), porcine deltacoronavirus (PDCoV), and transmissible gastroenteritis virus (TGEV). These pathogens cause highly similar clinical symptoms and pathological changes in piglets. Therefore, it is necessary to develop a simultaneous detection method for the precise prevention and control of porcine diarrheal diseases.MethodsTo establish a rapid, simple, and accurate detection method for differential diagnosis on these four pathogens, this study designed specific primers and probes based on the conserved regions of the M gene of PEDV, PEAV, and PDCoV, and the N gene of TGEV. By optimizing the reaction conditions, a quadruple fluorescence real-time quantitative RT-PCR (RT-qPCR) method for simultaneous detection of the four porcine diarrhea viruses was developed.ResultsThis method demonstrated high specificity, with no cross-reactivity with other common porcine pathogens such as porcine reproductive and respiratory syndrome virus, porcine circovirus, classical swine fever virus, porcine rotavirus, and pseudorabies virus. The sensitivity was excellent, with the limit of detection of 100 copies/μL in multiplex real-time RT-qPCR assays and all correlation coefficients (R2) exceeding 0.99. Repeatability was also strong, with the coefficient of variation of the intra- and inter-assay repeatability tests ranging from 0.3% to 1.0%.When applied to 231 clinical samples from Fujian province, the multiplex RT-qPCR method identified PEDV as the predominant pathogen, and often in co-infections. These results were 100% consistent with those from the commercial RT-qPCR kits, demonstrating the high accuracy of the developed method.DiscussionIn summary, this study established a specific, sensitive, and accurate multiplex RT-qPCR assay for the simultaneous detection of PEDV, PEAV, TGEV, and PDCoV, providing a valuable tool for the monitoring and differential diagnosis of these four PECs.

Porcine circovirus type 2 (PCV2) is a major swine pathogen causing significant economic losses in the swine industry worldwide. Continual monitoring of genetic and antigenic diversity is essential for the early detection of emerging variants. This study investigates the evolutionary dynamics and genetic variation of PCV2 circulating in Thailand and across Asia from 2007 to 2024, using integrated phylogenetic and phylodynamic analyses. Analysis of 2739 PCV2 genomes, after excluding 0.99% recombinant strains, revealed four main groups circulating in Asia, with PCV2d as the predominant genotype. PCV2d has diversified into several distinct clades, including a recently identified variant with the 133HDAM136 amino acid motif, which likely originated from earlier variants, the 133ANAL136 and 133ATAL136 motifs. Recombination analysis detected intragenotypic recombination events within PCV2d strains circulating in Thailand, emphasizing the role of genetic recombination in driving the evolutionary changes of the virus. Phylodynamic analysis demonstrated significant fluctuations in the virus population size, correlating with changes in genotype dominance over time. Selective pressure analysis identified positively selected sites in the capsid protein (codons 63, 131, 134, 169, and 190), indicating ongoing adaptation under host immune pressures. Structural modeling and epitope analyses revealed mutations affecting antigenic sites and immune recognition, suggesting concerns for vaccine efficacy. This integrated approach enhances our understanding of PCV2 evolution, informing strategies for effective vaccine development and disease control.

Porcine circovirus type 2 (PCV2) represents a principal infectious agent causing considerable economic detriment to swine production. N-acetyltransferase 10 (NAT10), which catalyzes N4-acetylcytidine (ac4C) deposition, has been implicated in regulating immune responses, RNA stability, and viral replication. However, its role in PCV2 infection remains unclear. In this study, we established a PCV2-infected PK15 cell model and observed a marked downregulation of NAT10 expression following infection. Functional assays demonstrated that NAT10 knockdown significantly suppressed PCV2 replication in PK15 cells. Comparative transcriptomic analysis revealed that NAT10 silencing altered the expression of 81 genes, predominantly involved in immune-related signaling pathways. Notably, integrative omics analysis identified NR1H4 as a potential downstream target of NAT10. Collectively, these findings elucidate the regulatory mechanism of NAT10 in PCV2 replication and provide new insights for identifying NAT10 as a potential biomarker and therapeutic target for PCV2 infection in pigs.

A novel Gaussia luciferase immunoprecipitation assay for the detection of Getah virus antibodies in pigs.

A novel antiviral peptide Laby A1/A3 precursor-SUMO against PCV2 replication with broad-spectrum antiviral potential.

Zinc finger antiviral protein (ZAP) inhibits PCV2 replication by targeting ORF1, ORF2 and ORF3 mRNA.

Porcine circoviruses Review article

The yeast Kluyveromyces marxianus (K. marxianus), characterized by its thermotolerance and rapid growth, is emerging as a promising new platform organism for the production of recombinant proteins. In this study, we constructed an expression vector designed for the efficient expression of exogenous proteins in K. marxianus. Initially, qPCR was employed to assess the expression efficiency of endogenous promoters within the yeast. The PDC1 promoter was selected, and its ability to drive the expression of EGFP was validated. The constructed vector exhibited high stability, maintaining approximately 5.2-fold higher copy numbers than the K. marxianus genome after 72 hours of cultivation without hygromycin selection. Notably, the fluorescence signal intensity of K. marxianus harboring the vector was approximately 15.6-fold higher than that of the wild-type strain at 72 h. Subsequently, the cap gene of porcine circovirus type 3 (PCV3) was integrated into the vector, resulting in the production of soluble PCV3 cap protein. Electron microscopy analysis revealed that the PCV3 cap protein self-assembled into virus-like particles (VLPs). This study successfully established the expression vector and characterized its key elements in K. marxianus, which will facilitate further research on the expression of exogenous proteins in this yeast species. Moreover, the soluble expression of the PCV3 cap protein and its formation of VLPs provide a solid foundation for the future development of PCV3 vaccines.

The online version contains supplementary material available at 10.1007/s13337-025-00947-5.

Nuclear transport factor IPO4 modifies localization by PCV2 and facilitates viral replication.

Background/Objectives: Porcine Circovirus Type 2 (PCV2) and Mycoplasma hyopneumoniae are primary pathogens causing respiratory disease in pigs. Recently, a Ready-to-Use bivalent PCV2 and M. hyopneumoniae vaccine has been registered in China. The aim of this randomized, side-by-side trial was to evaluate the efficacy and safety of this vaccine under field conditions in a Chinese commercial pig farm. Methods: In total, 938 piglets were allocated to three groups—A (tested vaccine), B, C—and vaccinated according to different schemes. Efficacy was assessed by Average Daily Gain (ADG), pneumonia lesions at slaughter and PCV2 viremia. Systemic reactions were recorded after vaccination to evaluate safety. Results: ADG was higher in group A compared with other vaccination schemes. The prevalence of pneumonia lesions was significantly lower in group A. PCV2 viremia was overall low in all groups, with no reported differences. No severe or moderate systemic reactions were observed after vaccination. Only four pigs showed mild reactions (A: 2/320, B: 2/309; C: 0/309). Conclusions: Under these conditions, the tested vaccine was proved to be efficacious in increasing ADG and reducing pneumonia at slaughter by protecting against both PCV2 and M. hyopneumoniae field infections. It can also be concluded that the Ready-To-Use bivalent vaccine was safe.

Coinfection of porcine circovirus type 2 (PCV2) with Mycoplasma hyopneumoniae (M. hyo) causes major worldwide economic losses within the swine industry. This study aimed to assess the safety and efficacy of a single dose of a bivalent vaccine containing PCV2d and M. hyo antigen (Cirbloc® M Hyo) under field conditions. Two studies were performed under the GCP (Good Clinical Practice) requirements on farrow-to-finish farms in Hungary and Cyprus. On both farms, the presence of both PCV2 and M. hyo infection was demonstrated. For both studies, safety parameters were observed and measured from inclusion at 21 (±3) days of age until 14 days after vaccination. Efficacy parameters were observed and measured from inclusion until slaughter. Administration of the vaccine was safe in both studies, as no general, immediate, or local reactions were observed. The efficacy of the vaccine was confirmed in both studies as the following parameters were significantly reduced in the vaccinated groups compared to the control groups: viraemia, faecal shedding, viral load in lungs and in all collected lymphoid tissues, M. hyo-specific lung lesions, and average daily body weight gain. These results collectively support the vaccine’s potential as an effective tool for disease control.

ABSTRACTBackgroundA field efficacy trial was conducted on three farms to evaluate a novel combined vaccine containing porcine circovirus types 2a/d (PCV2a/d), Mycoplasma hyopneumoniae, and M. hyorhinis.MethodsThree farms with a history of subclinical PCV2 infection, enzootic pneumonia, and polyserositis were enrolled. Each farm included 40 piglets (18 days old), which were randomly allocated to vaccinated or unvaccinated groups. Pigs in the vaccinated group received a single 2‐mL intramuscular dose of the combined vaccine at 21 days of age, while unvaccinated pigs received phosphate‐buffered saline.ResultsVaccination significantly improved (p < 0.05) body weight and average daily weight gain on all three farms compared with unvaccinated pigs. Vaccinated pigs mounted protective humoral and cellular immune responses against PCV2, M. hyopneumoniae, and M. hyorhinis. Furthermore, vaccination reduced viral and mycoplasmal loads in serum, laryngeal, and nasal samples and decreased the severity of associated lesions.ConclusionsThe combined vaccine demonstrated strong efficacy under field conditions, providing protection against subclinical PCV2 infection, enzootic pneumonia caused by M. hyopneumoniae, and polyserositis caused by M. hyorhinis. These findings support its potential as an effective intervention for improving both health and productivity in swine herds.