Search Results (66)

Porcine Rotavirus (PoRV), a predominant causative agent of neonatal diarrhea in piglets, shares substantial genetic homology with human rotavirus and represents a considerable threat to both public health and the global swine industry in the absence of specific antiviral interventions. The VP6 protein, an internal capsid component, is characterized by exceptional sequence conservation and robust immunogenicity, rendering it an ideal candidate for viral genotyping and vaccine development. In the present study, the recombinant plasmid pET28a(+)-VP6 was engineered to facilitate the high-yield expression and purification of the VP6 antigen. BALB/c mice were immunized to generate monoclonal antibodies (mAbs) through hybridoma technology, and the antigenic specificity of the resulting mAbs was stringently validated. Subsequently, a panel of truncated protein constructs was designed to precisely map linear B-cell epitopes, followed by comparative conservation analysis across diverse PoRV strains. Functional validation demonstrated that all three mAbs exhibited high-affinity binding to VP6, with a peak detection titer of 1:3,000,000 and exclusive specificity toward PoRVA. These antibodies effectively recognized representative genotypes such as G3 and X1, while exhibiting no cross-reactivity with unrelated viral pathogens; however, their reactivity against other PoRV serogroups (e.g., types B and C) remains to be further elucidated. Epitope mapping identified two novel linear B-cell epitopes, ¹²⁸YIKNWNLQNR¹³⁷ and ¹³⁸RQRTGFVFHK¹⁴⁷, both displaying strong sequence conservation among circulating PoRV strains. Collectively, these findings provide a rigorous experimental framework for the functional dissection of VP6 and reinforce its potential as a valuable diagnostic and immunoprophylactic target in PoRV control strategies. Full article

Porcine circovirus type 3 (PCV3) is an emerging pathogen that causes porcine dermatitis, nephropathy syndrome-like symptoms, multisystemic inflammation, and reproductive failure. The PCV3 capsid (Cap) protein interacts with DDX21, which functions mainly through controlling interferon (IFN)-β levels. However, how the interaction between DDX21 and PCV3 Cap regulates viral replication remains unknown. In the present study, upon shRNA-mediated DDX21 depletion in PK-15 cells, we observed impaired PCV3 proliferation via a lentivirus-delivered system, as indicated by reduced replicase (Rep) protein levels and viral titers. Furthermore, DDX21 negatively regulated IFN-β and interferon-stimulated gene (ISG) levels, promoting PCV3 replication. Mechanistically, PCV3 Cap co-localized and interacted with DDX21, and the nuclear localization signal (NLS) of PCV3 Cap and ⁷⁶³GSRSNRFQNK⁷⁷² at the C-terminal domain (CTD) of DDX21 were indispensable to the interaction. Moreover, PCV3 infection prevented the repression of DDX21 to facilitate its pro-viral activity. Taken together, these results show that DDX21 promotes PCV3 replication by binding to the PCV3 Cap protein and prohibiting IFN-β response, which provides important insight on the prevention and control of PCV3 infection. Full article

Viruses in the Circoviridae family can infect mammals and birds. Porcine circovirus type 2 (PCV2) significantly affects the livestock industry by causing porcine circovirus-associated diseases, such as postweaning multisystem wasting syndrome, respiratory disease complex, and dermatitis nephropathy syndrome. Additionally, beak and feather disease virus in parrots, canine circovirus in dogs, and columbid circovirus (pigeon circovirus) in racing pigeons induce immunosuppression, followed by secondary infections in these hosts. Although the PCV2 capsid protein has been demonstrated to inhibit type I interferon (IFN) signaling, the molecular mechanisms of Circoviridae-induced immunosuppression are largely unknown. In this study, we examined whether these functions are conserved across Circoviridae capsid proteins. Our results illustrated that although the nuclear localization of capsid proteins is conserved, their effects on IFN-β signaling vary by species, revealing the diverse roles of Circoviridae capsid proteins in modulating immune responses. Full article

Coinfections with porcine circovirus types 2, 3, and 4 (PCV2, PCV3, and PCV4) are increasingly being detected in the swine industry. However, there is no commercially available vaccine which prevents coinfection with PCV2, PCV3, and PCV4. The development of a vaccine expressing capsid (Cap) fusion proteins of multiple PCVs represents a promising approach for broadly preventing infection with PCVs. In this study, we developed a PCV subunit vaccine candidate (Cap 2-3-4) by predicting, screening, and fusing antigenic epitopes of Cap proteins of PCV2, PCV3, and PCV4. Immunoprotection assays showed that the prokaryotic expression of Cap 2-3-4 could effectively induce high levels of PCV2, PCV3, and PCV4 Cap-specific antibodies and successfully neutralize both PCV2 and PCV3. Furthermore, Cap 2-3-4 demonstrated a potent ability to activate cellular immunity and thus prevent lung damage in mice. This study provides a new option for the development of broad vaccines against PCVs. Full article

Porcine circovirus 3 (PCV3) is a small non-enveloped circovirus associated with porcine dermatitis and nephropathy syndrome (PDNS). It has occurred worldwide and poses a serious threat to the pig industry. However, there is no commercially available vaccine. PCV3 capsid protein (Cap) is an ideal antigen candidate for serodiagnosis. Here, a novel fully automated chemiluminescence enzyme immunoassay (CLEIA) was developed to detect antibodies (Abs) to Cap in porcine serum. Recombinant PCV3 Cap, self-assembled into virus-like particles (VLPs), was produced using baculovirus and coupled to magnetic particles (Cap-MPs) as carriers. Combined with an alkaline phosphatase (AP)–adamantane (AMPPD) system, Cap-Abs can be rapidly measured on a fully automated chemiluminescence analyzer. Under optimal conditions, a cut-off value of 31,508 was determined, with a diagnostic sensitivity of 96.8% and specificity of 97.3%. No cross-reactivity was observed with PCV1 and PCV2 and other common porcine pathogens, and both intra-assay and inter-assay coefficients were less than 5% and 10%, respectively. Prepared Cap-MPs can be stored at 4 °C for more than 6 months. Importantly, this CLEIA had a good agreement of 95.19% with the commercially available kit, demonstrating excellent analytical sensitivity and significantly reduced operating time and labor. A serological survey was then conducted, and showed that PCV3 continues to spread widely in South China. In conclusion, our CLEIA provides time and labor-saving, and a reliable tool for PCV3 epidemiological surveillance. Full article

Porcine parvoviruses one through eight (PPV1-PPV8) are prevalent in Chinese swine herds. However, the infection status of all these PPVs in slaughtered pigs is still unclarified. In this study, we detected PPV1-PPV8 in 353 tissue samples collected from slaughtered pigs from six regions of China in 2023. At least one species of PPV was detected in 79.32% of the samples (280 out of 353). Six PPV species were detected, except for PPV4 and PPV8, in slaughtered pigs, within which PPV3 (49.86%), PPV2 (42.49%), and PPV7 (42.21%) were predominant, followed by PPV1 (13.31%), PPV6 (13.31%), and PPV5 (8.22%). Noticeably, co-infection was frequently detected, with 67.50% of PPV-positive samples (189 out of 280) co-infecting with two to six PPVs. In addition, one representative genome for each detected PPV was determined. Multiple sequence alignment determined a large number of substitutions in capsid proteins of PPVs. Genome-based phylogenetic analysis confirmed the PCR detection results. Recombination detection identified two potential recombinants (PPV2 GDCZ2023-2088 strain and PPV3 HLJSYS2023-1654 strain) in slaughtered pigs. Overall, this study provides new insights into the prevalence and evolution of PPVs, particularly in slaughtered pigs in China. Full article

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine. Full article

Porcine parvovirus 7 (PPV7) was first discovered in swine in 2016, and PPV7 infection has been detected in aborted pig fetuses and in sows that experienced reproductive failure. The objective of this study was to report the prevalence and genetic characterization of PPV7 in Hunan, China. Seventy of the four hundred and twenty-two (16.6%) serum, semen, and tissue samples collected from pigs were positive for PPV7. One complete PPV7 strain and eighteen complete cap gene sequences were obtained; nucleotide and amino acid identity among the nineteen Cap sequences were 88.1–99.4% and 88.1–100%, respectively. They shared identity with previously discovered sequences ranging from 86.6 to 98.9% and 83.7 to 99.8% at the nucleotide- and amino acid-level, respectively. The phylogenetic tree analysis exhibited that PPV7 strains had two major groups based on the presence or absence of five amino acid (181–185) insertions on the Cap protein. Analysis of the Cap protein demonstrated that PPV7 Cap had significant variability, implying that PPV7 evolved at high substitution rates. Substantial variations of that PPV7 Cap may enable the emergence of newly mutated capsid profiles due to its viral adaptation to host responses. Furthermore, antigenic alteration owing to PPV7 Cap protein amino acid mutations at immune epitopes may enable viruses to escape from the host’s immune system. This study determined the prevalence and genetic characteristics of PPV7 circulating in swine in Hunan, China, and provided the impetus and basis to further investigate the pathogenicity and epidemiology of PPV7. Full article

Porcine circovirus type 3 (PCV3) infection can cause symptoms similar to those of porcine circovirus type 2 (PCV2) infection, and coinfections with both PCV2 and PCV3 are observed in the swine industry. Consequently, developing chimeric vaccines is essential to prevent and control porcine circovirus infections. In this study, we used both E. coli and mammalian expression systems to express PCV3 Cap (Cap3) and a chimeric gene containing the PCV2-neutralizing epitope within the PCV3 Cap (Cap3-Cap2E), which were assembled into virus-like particle (VLP) vaccines. We found that Cap3 lacking nuclear localization signal (NLS) could not form VLPs, while Cap3 with a His-tag successfully assembled into VLPs. Additionally, the chimeric of PCV2-neutralizing epitopes did not interfere with the assembly process of VLPs. Various immunization approaches revealed that pCap3-Cap2E VLP vaccines were capable of activating high PCV3 Cap-specific antibody levels and effectively neutralizing both PCV3 and PCV2. Furthermore, pCap3-Cap2E VLPs demonstrated a potent ability to activate cellular immunity, protecting against PCV3 infection and preventing lung damage in mice. In conclusion, this study successfully developed a PCV3 Cap VLP vaccine incorporating chimeric PCV2-neutralizing epitope genes, providing new perspectives for PCV3 vaccine development. Full article

Human health is dependent on food safety and, therefore, on the health of farm animals. One of the most significant threats in regard to swine diseases is African swine fever (ASF). Infections caused by porcine circoviruses (PCVs) represent another important swine disease. Due to the ubiquitous nature of PCV2, it is not surprising that this virus has been detected in ASFV-affected pigs. However, recent data indicate that coinfection of PCV3 and ASFV also occurs. It is still unclear whether PCV infection plays a role in ASFV infection, and that subject requires further analysis. The aim of this study was to assess whether PCV3 and PCV4 are present in the wild boar population in Poland (real-time PCR). The analysis was performed on wild boar samples collected for routine ASF surveillance in Poland, between 2018 and 2021. By extension, the obtained data were compared in regard to ASFV presence in these samples, thus investigating the odds of ASFV infection on the grounds of the PCV carrier state in free-ranging Suidae in Poland. In addition, sequencing of PCV3 and phylogenetic analysis were performed, based on a full genome and a capsid gene. In the current study, we demonstrated the high prevalence of PCV3 in the wild boar population in Poland; meanwhile, PCV4 was not detected. The odds of ASFV infection on the grounds of the PCV3 carrier state in free-ranging Suidae in Poland was more than twice as high. Ten full genome sequences of PCV3 were obtained, all of them belonging to clade 3a. The similarity between them was in the range of 98.78–99.80%. Full article

Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus-associated disease (PCVAD) that profoundly impacts the swine industry worldwide. While most of the commercial PCV vaccines are developed based on PCV genotype 2a (PCV2a), PCV genotype 2b (PCV2b) has become predominant since 2003. In this study, we developed and evaluated DNA-based bivalent vaccines covering both PCV2a and PCV2b. We generated a new immunogen, PCV2b-2a, by combining consensus sequences of the PCV2a and PCV2b capsid proteins (Cap2a and Cap2b) in a form of fusion protein. We also examined whether modifications of the PCV2b-2a fusion protein with a signal sequence (SS) and granulocyte macrophage-colony stimulating factor (GM-CSF) fusing with interleukine-4 (IL-4) (GI) could further improve the vaccine immunogenicity. An immunogenicity study of BALB/cAJcl mice revealed that the DNA vector pVAX1 co-expressing PCV2b-2a and GI (pVAX1.PCV2b-2a-GI) was most potent at inducing both antibody and cellular immune responses against Cap2a and Cap2b. Interestingly, the vaccines skewed the immune response towards Th1 phenotype (IgG2a > IgG1). By performing ELISA and ELISpot with predicted epitope peptides, the three most immunogenic B cell epitopes and five putative T cell epitopes were identified on Cap2a and Cap2b. Importantly, our DNA vaccines elicited broad immune responses recognizing both genotype-specific and PCV2-conserved epitopes. Sera from mice immunized with the DNAs expressing PCV2b-2a and PCV2b-2a-GI significantly inhibited PCV2a cell entry at serum dilution 1:8. All these results suggest a great potential of our PCV2b-2a-based vaccines, which can be further developed for use in other vaccine platforms to achieve both vaccine efficacy and economical production cost. Full article

Porcine circovirus 4 (PCV4) is a newly identified virus belonging to PCV of the Circoviridae family, the Circovirus genus. We previously found that PCV4 is pathogenic in vitro, while the virus’s replication in cells is still unknown. In this study, we evaluated the N-terminal of the PCV4 capsid (Cap) and identified an NLS at amino acid residues 4–37 of the N-terminus of the PCV4 Cap, ⁴RSRYSRRRRNRRNQRRRGLWPRASRRRYRWRRKN³⁷. The NLS was further divided into two fragments (NLS-A and NLS-B) based on the predicted structure, including two α-helixes, which were located at ⁴RSRYSRRRRNRRNQRR¹⁹ and ²⁴PRASRRRYRWRRK³⁶, respectively. Further studies showed that the NLS, especially the first α-helixes formed by the NLS-A fragment, determined the nuclear localization of the Cap protein, and the amino acid ⁴RSRY⁷ in the NLS of the PCV4 Cap was the critical motif affecting the VLP packaging. These results will provide a theoretical basis for elucidating the infection mechanism of PCV4 and developing subunit vaccines based on VLPs. Full article

Viruses frequently contain overlapping genes, which encode functionally unrelated proteins from the same DNA or RNA region but in different reading frames. Yet, overlapping genes are often overlooked during genome annotation, in particular in DNA viruses. Here we looked for the presence of overlapping genes likely to encode a functional protein in human parvovirus B19 (genus Erythroparvovirus), using an experimentally validated software, Synplot2. Synplot2 detected an open reading frame, X, conserved in all erythroparvoviruses, which overlaps the VP1 capsid gene and is under highly significant selection pressure. In a related virus, human parvovirus 4 (genus Tetraparvovirus), Synplot2 also detected an open reading frame under highly significant selection pressure, ARF1, which overlaps the VP1 gene and is conserved in all tetraparvoviruses. These findings provide compelling evidence that the X and ARF1 proteins must be expressed and functional. X and ARF1 have the exact same location (they overlap the region of the VP1 gene encoding the phospholipase A2 domain), are both in the same frame (+1) with respect to the VP1 frame, and encode proteins with similar predicted properties, including a central transmembrane region. Further studies will be needed to determine whether they have a common origin and similar function. X and ARF1 are probably translated either from a polycistronic mRNA by a non-canonical mechanism, or from an unmapped monocistronic mRNA. Finally, we also discovered proteins predicted to be expressed from a frame overlapping VP1 in other species related to parvovirus B19: porcine parvovirus 2 (Z protein) and bovine parvovirus 3 (X-like protein). Full article

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. Full article

Adeno-associated virus (AAV) vectors are among the most widely used delivery vehicles for in vivo gene therapy as they mediate robust and sustained transgene expression with limited toxicity. However, a significant impediment to the broad clinical success of AAV-based therapies is the widespread presence of pre-existing humoral immunity to AAVs in the human population. This immunity arises from the circulation of non-pathogenic endemic human AAV serotypes. One possible solution is to use non-human AAV capsids to pseudotype transgene-containing AAV vector genomes of interest. Due to the low probability of human exposure to animal AAVs, pre-existing immunity to animal-derived AAV capsids should be low. Here, we characterize two novel AAV capsid sequences: one derived from porcine colon tissue and the other from a caprine adenovirus stock. Both AAV capsids proved to be effective transducers of HeLa and HEK293T cells in vitro. In vivo, both capsids were able to transduce the murine nose, lung, and liver after either intranasal or intraperitoneal administration. In addition, we demonstrate that the porcine AAV capsid likely arose from multiple recombination events involving human- and animal-derived AAV sequences. We hypothesize that recurrent recombination events with similar and distantly related AAV sequences represent an effective mechanism for enhancing the fitness of wildtype AAV populations. Full article