PfAgo-mediated a rapid and visual detection assay for porcine circovirus type 3 based on recombinase-aided amplification.

Efficient, high-quality production is essential to the swine industry, with carcass inspection by health authorities playing a crucial role in detecting abnormalities that may lead to condemnation, including enteritis. Enteritis is, usually, characterized by changes in color, mucosal integrity, intestinal wall thickness and luminal contents and has a multifactorial etiology. This study assessed and classified visual lesions attributed to enteritis and to seek for associations with known pathogens. A scoring system was applied to classify visual changes, and 30 condemned intestines were categorized as mild (< 10 points), moderate (11-20 points), or severe (> 20 points). Laboratory analyses included histopathology; isolation and antimicrobial susceptibility testing of Escherichia coli and Salmonella spp.; and molecular detection of porcine circovirus type 2 (PCV2), Lawsonia intracellularis, Brachyspira pilosicoli, and Brachyspira hyodysenteriae. Lesions were classified as mild (23%), moderate (57%), and severe (20%). PCV2 and L. intracellularis DNA were detected in 60% and 23% of samples, respectively. E. coli was isolated from 57% of intestines, of which 87% exhibited antimicrobial resistance and 56% were multidrug-resistant. No samples tested positive for Salmonella spp., B. pilosicoli, or B. hyodysenteriae. Lower condemnation scores were associated with the presence of PCV2 DNA, mild lesions, and lymph nodes showing necrosis and follicular depletion. Mild lesions were also associated with a lower frequency of multidrug-resistant E. coli. The high prevalence of antimicrobial resistance is concerning from a One Health perspective. These findings underscore the importance of pathogen monitoring, antimicrobial stewardship, and strong animal welfare practices within the swine production chain.

Porcine circovirus associated disease (PCVAD) is an immunosuppressive disease caused by porcine circovirus (PCV), which has led to substantial economic losses in the swine industry. Porcine circovirus type 2 (PCV2) is the primary genotype responsible for PCVAD and the most prevalent pathogenic agent. PCV2 infection can induce lymphoid depletion, immunosuppression, and multi-systemic lesions, severely compromising pig health and production performance. Since no specific antiviral drug against PCV2 is available, vaccination becomes the primary strategy for disease prevention and control. Currently, commercial vaccines, predominantly inactivated and subunit vaccines, play a crucial role in controlling PCVAD. In recent years, advances in genetic engineering have significantly propelled the development of novel vaccines, including virus-like particle vaccines, live vector vaccines, transgenic plant-based vaccines, nanoparticle vaccines, and nucleic acid vaccines. However, the high mutation rate of PCV2 and the emergence of new genotypes have resulted in reduced efficacy of existing vaccines against newly circulating strains, posing ongoing challenges to disease control. This review systematically summarizes the etiological characteristics of PCV2 and the current landscape of vaccine development and application, with a particular focus on commenting on the progress in novel vaccine research. Finally, we prospect future directions for vaccine development, aiming to provide a scientific basis and technical references for the effective prevention and control of PCVAD.

Porcine circovirus type 3 (PCV3) is an emerging viral pathogen associated with multi-systemic inflammation, porcine dermatitis and nephropathy syndrome, and reproductive failure, leading to substantial economic losses in the global swine industry. Although PCV3 has been widely detected in southwestern China, its pathogenic characteristics remain insufficiently understood. In this study, an infectious clone of the PCV3/SC/Sichuan-2023 strain was constructed using the pBluescript SK(+) backbone and subsequently employed to rescue the recombinant virus rPCV3. The recovered rPCV3 replicated efficiently in vitro and induced overt clinical manifestations and pathological lesions in piglets that closely recapitulated those observed under field conditions. Strong viral antigen signals were detected predominantly in the lungs and lymphoid tissues, confirming distinct tissue tropism. Similarly, rPCV3 infection elicited significant upregulation of pro-inflammatory cytokines, reflecting the immune dysregulation characteristic of natural infection. These results demonstrate that rPCV3 reproduces essential pathogenic features observed during experimental infection and aligns closely with reports from field strains. Furthermore, the established infectious clone provides a robust reverse-genetics platform for virus rescue, stable propagation in PK-15 cells, and comprehensive in vitro and in vivo characterization, facilitating future investigations into PCV3 pathogenesis and the development of effective control strategies in southwestern China.IMPORTANCEPorcine circovirus type 3 (PCV3) is widely prevalent in pig populations; however, its biological characteristics and pathogenic mechanisms remain incompletely understood. In this study, we generated a full-length infectious clone of a field-derived PCV3 strain. We demonstrated that the rescued virus faithfully reproduces critical aspects of natural infection, including robust replication, distinct tissue tropism, and cytokine dysregulation in piglets. These findings provide valuable experimental tools and novel insights into PCV3 pathogenesis, supporting enhanced disease control strategies and future vaccine development.

Post-weaning diarrhoea (PWD) is a disease syndrome that negatively impacts pig health, welfare and productivity. PWD typically occurs within two weeks of weaning and coincides with significant physiological changes, including villus atrophy and increased crypt depth in the gastrointestinal (GI) tract. The GI microbiome of healthy pigs is a complex ecosystem of commensal microorganisms. Disruption of the natural integrity of the GI tract has been associated with increased colonization by both viral and bacterial pathogens. In this study, metagenomic sequencing was used to assess the presence, load, and diversity of viruses in the GI tracts of PWD-affected pigs and age-matched healthy (AMH) cohorts on commercial pig farms in England. In addition, the viromes of archived faecal samples from post-weaned pigs between four and six weeks of age, collected from diagnosis-not-reached (DNR) and diagnosis-reached (DR) enteric cases were investigated through sequencing. Viruses belonging to at least ten virus families were identified in both PWD and AMH pigs including astrovirus, enterovirus, kobuvirus, smacovirus, picobirnavirus, sapovirus, parvovirus, posavirus, teschovirus, sapelovirus, rotavirus, torovirus, anellovirus and adenovirus. Co-infection with four viruses, astrovirus, enterovirus, kobuvirus and smacovirus was detected in all samples from PWD and AMH pigs. No sequence reads matching porcine coronaviruses, porcine reproductive and respiratory disease virus, porcine circoviruses, swine influenza virus, atypical porcine pestivirus or porcine teschovirus-1 were detected in either PWD or AMH faecal samples. Metagenomic analysis also identified several viruses with a higher virus load in PWD cases (astro, entero, sapelo, sapo, posa, adeno and toro-viruses), but the differences from those in AMH cases were not statistically significant. No viruses were detected in samples from archived DNR and DR cases that were not found in the PWD and AMH pigs. This study revealed the complexity of the virus element in the enteric microbiome in the post-weaned pigs. The role of the viruses detected and their interplay with the host and other bacterial or viral flora in inducing PWD, however, remains unclear and warrants further studies.

Receptor-interacting protein kinase-3 (RIPK3), a critical regulator of necroptosis and inflammation, has been implicated in modulating viral infections by either promoting host defense or facilitating viral replication. In this study, it was found that porcine circovirus type 2 (PCV2) infection selectively induced RIPK3 phosphorylation in PK-15 cells without activating its canonical downstream effector MLKL. This indicated that the virus exploits RIPK3 in a necroptosis-independent manner. Inhibition of RIPK3 phosphorylation using GSK872 or RIPK3 knockdown significantly reduced viral replication, as evidenced by viral DNA levels in PK-15 cells, with a concomitant reduction in Rep protein expression. Through functional screening of viral proteins, we found that only ORF3 triggered RIPK3 phosphorylation, while capsid (Cap) and replication-associated (Rep) proteins did not. Both PCV2 infection and ORF3 could induce autophagy. RIPK3 knockdown suppressed PCV2-induced autophagy, and subsequently knockdown of the autophagy-related protein ATG7 resulted in the reduction of PCV2 replication. These findings indicated that PCV2 employed its ORF3 protein to hijack RIPK3 phosphorylation-dependent autophagy, thereby creating a promoted viral replication environment. ‌In conclusion‌, this study demonstrated that PCV2 manipulated host cell machinery through its ORF3 protein, which hijacked RIPK3 phosphorylation to activate autophagy-a mechanism distinct from RIPK3's classical necroptosis function. This ORF3-RIPK3 phosphorylation-autophagy axis represented a novel therapeutic target for PCV2 control.

Innovative MgCl2 -mediated two-step precipitation-dissolution strategy for efficient purification of Porcine Circovirus Type 2 capsid protein and self-assembly of virus-like particles.

Epidemiological and evolutionary analysis of porcine circovirus type 2 within pig populations in central China during 2021-2024.

Porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3) have emerged as significant pathogens jeopardizing the swine industry in China in recent years, and caused substantial economic losses to pig farms. This study aimed to conduct an epidemiological investigation and genetic evolutionary analysis of PCV2 and PCV3 in three provinces of Northeast China in 2025. A total of 322 clinical samples suspected of PCV infection were collected from 25 cities across the three provinces of Northeast China. All samples were tested for PCV2 and PCV3 by a duplex nested PCR. The genomes of selected PCV2 and PCV3 strains were sequenced for the subsequent homology and phylogenetic analysis. Among the 322 samples, the number and prevalence of PCV2-positive samples were 68 (21.12%), and the number and prevalence of PCV3-positive samples were 122 (37.89%). The highest positive rate for PCV2 was in Jilin Province, and the highest positive rate for PCV3 was in Liaoning Province. The positive rate of PCV2 and PCV3 were both higher in winter than that in other seasons. Homology and phylogenetic analysis performed on 17 sequenced PCV2 genomes revealed the nucleotide identity ranging from 96.20% to 99.90%, and all strains were classified as the genotype PCV2d. The 17 sequenced PCV3 genomes showed the nucleotide identity between 98.60% and 99.90%. Among these PCV3 strains, 5 strains belonged to the PCV3a, and 12 strains belonged to the PCV3c. The results of the amino acid sequence analysis of Cap showed that the 17 PCV2 strains exhibited multiple amino acid mutations within the antibody recognition domain. Among these, 16 strains harbored variations in the immunodominant decoy epitope. Additionally, amino acid mutations were detected in the nuclear localization signal of 5 strains. The results of PCV3 amino acid analysis showed that the residues at the position 24 and 27 were consistent with the phylogenetic analysis. Beyond these, only four amino acid mutations were observed and all of them were not located within the reported antibody recognition domain of the PCV3 Cap. The currently predominant strains circulating in three provinces of Northeast China in 2025 were PCV2d and PCV3c. Seasonal analysis indicated a higher prevalence in winter for both PCV2 and PCV3. PCV2 Cap displayed amino acid variations at multiple key sites, while the amino acid of PCV3 Cap is relatively conserved. This study provided the crucial data for understanding the epidemiology of PCV2 and PCV3 and for the development of PCVAD control strategies in these regions.

Replication of Senecavirus A (SVA) generates intact virions and empty capsids, some of which disassemble into pentamers. To enable precise quality control of SVA intact viral antigen, we established a double-antibody sandwich ELISA to quantify intact SVA virions and virus-like particles (VLPs). The assay employs a conformation-specific nanobody (SV1) as the capture antibody, which binds both virions and VLPs, and a His-tagged nanobody (SVH1) as the detection antibody. Using purified virions as standards, the assay demonstrated a limit of detection (LOD) of 9.7 ng/mL, with a linear range spanning from 9.7 ng/mL to 1.25 μg/mL. Critically, it showed no cross-reactivity with pentamers and other viral particles, including foot-and-mouth disease virus (FMDV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), classical swine fever virus (CSFV), and encephalomyocarditis virus (EMCV). The coefficients of variation (CV) were <10% for both intra-batch and inter-batch analyses. Validation against sucrose density gradient centrifugation (SDGC) confirmed concordant results. Applied to vaccine development, the ELISA quantified a 36% antigen loss in SVA VLPs after emulsification. In summary, this method provides a rapid, specific tool for SVA vaccine standardization.

The study was conducted during September and October, 2024 at a local farm in Bareilly, Uttar Pradesh, India to investigate the etiologies and pathology of concurrent viral infections in piglets. Post mortem examinations of three crossbred piglet carcasses (38-day-old male, 42-day-old female and 50-day-old female) were conducted having history of reduced feed intake, severe dullness, progressive lethargy, diarrhoea and multifocal cutaneous circumscribed scabs followed by death. Necropsy revealed pericarditis with serous fluid accumulation, pulmonary consolidation with edema, hepatic congestion and necrosis, intestinal lymph nodes congestion, renal tubular degeneration and characteristic cutaneous pock lesions with umbilicated crusts. Histopathology showed interstitial pneumonia, hepatic sinusoidal congestion, sloughing of intestinal villus epithelium, lymphoid depletion in lymph nodes and characteristic swine pox lesions like vacuolar degeneration of keratinocytes in the epidermis with eosinophilic intracytoplasmic inclusion bodies. All tissue samples from the three affected piglets were found to be positive for porcine circovirus 2 (PCV2) and swinepox virus (SWPV) by PCR amplification of ORF2 and P42 genes, respectively. Organ-wise viral distribution analysis revealed PCV2 predominantly in lymphoid tissues, heart and lungs, while SWPV was predominantly detected in skin and lymphoid tissues. Based on pathomorphological and molecular investigations, the cause of death of the piglets was diagnosed as concurrent infections of PCV2 and SWPV in piglets maintained under poor biosecurity conditions. PCV2-induced immunosuppression likely exacerbated swinepox severity through enhanced viral replication and systemic dissemination.

Abstract Porcine Circovirus 3 (PCV3) has been detected in pigs worldwide, including Thailand. It is associated with a range of clinical problems, including reproductive disorders, but its pathogenic role remains unclear. To date, PCV3 detection in Thailand has been reported only in pigs with respiratory signs. An outbreak of stillbirths and mummified fetuses occurred at a farrow-to-nursery farm in 2023. Clinical assessment, farm management review, and molecular testing for African swine fever virus (ASFV), porcine parvovirus (PPV1, PPV2, PPV3, PPV4, PPV5, PPV6, and PPV7), porcine reproductive and respiratory syndrome virus (PRRSV-1 and PRRSV-2), PCV2, PCV3, and PCV4 were performed. PCV3 DNA was detected in all the samples, whereas other major reproductive pathogens were not detected. A positive PCV3 sample was subjected to full-genome sequencing and phylogenetic analysis. The complete genome (2,000 nt) of this strain shares 99.35%-99.70% identity with other PCV3 strains in the GenBank database and is classified into the genotypes PCV3a1 or PCV3b, similar to previously reported strains causing reproductive disorders in other countries. No significant environmental or nutritional stressors were identified. The findings of this study provide the first molecular evidence in Thailand, suggesting that PCV3 may be the cause of reproductive failure and possible vertical transmission. PCV3 should be considered in the differential diagnosis of reproductive disorders in swine production.

Our previous work revealed that the anti-diarrhea effects of alkaline mineral complex (AMC) water improve metabolism and protect the gut during weaning stress. However, whether AMC water can inhibit viral replication and treat viral diarrhea is unknown. The aim of this study was to explore the ability of AMC water to improve nutrient metabolism and protect against infection. In this study, porcine epidemic diarrhea virus (PEDV) or porcine deltacoronavirus (PDCoV) were used as RNA model viruses, and pseudorabies virus (PRV) or porcine circovirus (PCV) were used as DNA model viruses. Compared with those in the infected group, the virus content in the piglets fed AMC water was reduced, and the intestinal mucosal barrier was repaired. Transcriptome and metabolome results revealed that AMC water regulated lipid metabolism through GPAT2, DGKA, OAT3, FXR, LIPC and SULT2A1. Further studies showed that glycerol, cholesterol, and bilirubin levels increased after viral infection, and that AMC water inhibited cholesterol content and promoted bile acid synthesis. In a cellular model, AMC water reduced lipid droplet density by activating the glycerolipid and bile secretion pathways of the GPAT2/SULT2A1 axis. In addition, knockdown of DGKA and overexpression of SULT2A1 significantly affected the expression of the GPAT2/SULT2A1 axis, and the expression of viral proteins colocalized with lipid droplets was significantly decreased. Our findings suggest that AMC water promotes cholesterol metabolism by activating the GPAT2/SULT2A1 axis, inhibiting viral infection in piglets. This study provides theoretical support for the use of nutritional regulation to inhibit viral infection and provides a new method for antiviral therapy.

Mycoplasma hyopneumoniae (Mhyo) and porcine circovirus type 2 (PCV2) infections cause enormous economic losses in the swine production sector. Vaccination remains the most effective strategy against the complex clinical entities caused by these pathogens. While effective vaccines against PCV2 are available, immunisation against Mhyo continues to be challenging. In parallel, intradermal (ID) vaccination can optimise time and costs while providing faster and user-safe administration. This study compares the safety and efficacy of commercial porcine vaccines against Mhyo and PCV2, including intramuscular (IM) and ID ones: (1) ID recombinant Mhyo-PCV2, (2) ID recombinant PCV2 + ID inactivated Mhyo, (3) IM bivalent inactivated Mhyo and recombinant PCV2, (4) IM bivalent inactivated Mhyo and inactivated chimeric PCV1-PCV2 and (5) IM inactivated Mhyo + IM inactivated PCV2. Safety, evaluated through clinical signs, adverse effects, local reactions, and average daily weight gain, has been confirmed for all vaccines. Following challenge with both pathogens, all products reduced PCV2 viremia and faecal shedding, proving efficacy against PCV2 infection. In contrast, only the ID recombinant Mhyo-PCV2 and the IM inactivated Mhyo + IM inactivated PCV2 vaccines, significantly reduced lung lesions related to Mhyo infection, indicating a better protection compared to the other vaccines.

The distribution and genetic diversity of economically significant pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), and porcine parvovirus 1 (PPV1), across extensive Russian territory within wild boars that serve as reservoirs remain poorly characterized. This study aimed to conduct a molecular epidemiological survey of these viruses in wild boar populations. The samples of 476 wild boars, collected across Russia between 2021 and 2025, were tested by PCR for the detection of viral genomes. While PRRSV was not detected, we found high detection rates for PCV2 (34.9%), PCV3 (35.5%), and PPV1 (25.4%). For PCV2, the co-circulation of two genotypes, PCV2b (5/53) and PCV2d (48/53), was observed. All 29 PCV3 sequences belonged to the PCV3a genotype. For PPV1, the presence of the PPV1a, PPV1b, and PPV1d genotype, as well as unclassified isolates, was shown. Co-infection of different viruses was detected: PCV2/PCV3 (16.0%), PCV2/PPV1 (6.9%), PCV2/PCV3/PPV1 (6.9%), and PCV3/PPV1 (4.4%). This is the first comprehensive study that demonstrates the wide dissemination and genetic diversity of PCV2, PCV3, and PPV1 within the wild boar population in Russia and highlights their role as a potential reservoir in viral evolution and spread.

Porcine circovirus (PCV) is a major viral pathogen associated with multiple systemic diseases in pigs, inflicting significant economic losses to the global swine industry. To investigate the epidemiology and genetic evolution of porcine circovirus in Liaoning Province, China, PCV was detected by qPCR in 1224 clinical samples. Subsequent genetic evolution analysis of the Cap gene was conducted, and an indirect ELISA and monoclonal antibody were established. The results demonstrated PCV2 and PCV3 positivity rates of 14.13% and 21.90%, respectively, with a coinfection rate of 4.08%. All six sequences were identified as belonging to the PCV3b subtype. A representative PCV3 strain was expressed in a prokaryotic expression system and used to immunize 6-week-old female BALB/c mice, resulting in serum antibody titers reaching 1:512,000. The positive hybridoma cell line 3E6 was selected and identified as expressing IgM heavy chains and κ light chains. The prepared monoclonal antibody 3E6 exhibited specific reactivity with the Cap protein. Collectively, this study elucidates the recent epidemiological status and evolutionary characteristics of PCV in pig populations in Liaoning Province, thereby providing an important theoretical basis and reference data for disease prevention, control, diagnosis, and vaccine development.

Porcine circovirus type 2 (PCV2), the primary etiological agent of porcine circovirus-associated diseases (PCVADs), continues to cause substantial economic losses worldwide. Although commercially available vaccines have been widely deployed, they often fail to induce sufficient cellular immunity, which necessitates the development of improved vaccination strategies. This study reports the design and evaluation of a messenger RNA (mRNA)-based vaccine candidate targeting PCV2. The vaccine encodes the full-length capsid (Cap) protein, the principal target for neutralizing antibodies. For enhanced delivery and immunogenicity, the mRNA was encapsulated in lipid nanoparticles (LNP). Expression of the PCV2 Cap protein was confirmed in HEK-293T cells following transfection with the mRNA-LNP construct. In a mouse model, this vaccine elicited high titers of PCV2-specific IgG and potent virus-neutralizing antibodies, compared to a recombinant Cap subunit vaccine. Furthermore, the vaccine induced a robust Th1-polarized cellular immune response, marked by significant proliferation of antigen-specific CD8+ T cells and CD4+ T cells, enhanced interferon-gamma (IFN-γ) production, and expansion of T follicular helper cells and germinal center B cells in lymph nodes. Co-administration with a CpG ODN adjuvant further enhanced immunogenicity without compromising the vaccine's favorable safety profile. Collectively, these results indicate that the PCV2 mRNA-LNP vaccine represents a promising vaccine candidate worthy of further development against PCV2.IMPORTANCEPorcine circovirus type 2 (PCV2) is a widespread virus that causes severe disease in pigs, leading to significant economic losses in the swine industry worldwide. Existing vaccines often fail to stimulate strong cellular immunity, which is essential for long-lasting protection. In this study, we developed a novel messenger RNA (mRNA)-based vaccine encapsulated in lipid nanoparticles that encodes the PCV2 capsid protein. Our vaccine not only triggers potent antibody responses but also activates key immune cells, enhancing both humoral and cellular immunity. This represents the first mRNA-lipid nanoparticle vaccine against PCV2 and demonstrates the potential of mRNA technology to overcome limitations of traditional veterinary vaccines, offering a promising new tool for disease control in animals.

Myroides odoratimimus is an emerging opportunistic pathogen increasingly implicated in infections across human and animal populations. We previously reported the first outbreak of pneumonia in post-weaning piglets associated with M. odoratimimus, marking a significant shift in its recognized host range. The affected swine herd exhibited co-infection with Porcine Circovirus types 2 and 3 (PCV2 and PCV3), likely contributing to proliferative dermatitis and nephropathy syndrome (PDNS), reproductive abnormalities and immunosuppression. This study presents the genomic characterization of a multidrug-resistant strain (M. odoratimimus pgdne) isolated during the outbreak. Whole-genome sequencing revealed multiple antimicrobial resistance determinants-including the chromosomally encoded blaMUS-1 metallo-β-lactamase-and several virulence-associated factors. Genotype-phenotype correlation demonstrated strong concordance. Phylogenetic analysis (distance method, 1,000 bootstrap replicates) showed close relatedness with human isolates, suggesting zoonotic potential. These findings underscore the pathogenic capacity of M. odoratimimus in immunocompromised animal hosts and highlight its relevance to public health and thereby reinforces the need for integrated One Health-based surveillance and control strategies targeting emerging multidrug-resistant pathogens.

Livestock aggregated samples for monitoring viruses infecting animals and potentially zoonotic viral pathogens

Experimental production of synthetic infectious porcine circovirus 2b particles in swine testicular cells.