Investigating pigeon circovirus infection in a pigeon farm: molecular detection, phylogenetic analysis and complete genome analysis.

BACKGROUND: Pigeon circovirus infections in pigeons (Columba livia domestica) have been reported worldwide. Pigeons should be PiCV-free when utilized as qualified experimental animals. However, pigeons can be freely purchased as experimental animals without any clear guidelines to follow. Herein, we investigated the status quo of PiCV infections on a pigeon farm in Beijing, China, which provides pigeons for experimental use. RESULTS: PiCV infection was verified in at least three types of tissues in all forty pigeons tested. A total of 29 full-length genomes were obtained and deposited in GenBank. The whole genome sequence comparison among the 29 identified PiCV strains revealed nucleotide homologies of 85.8-100%, and these sequences exhibited nucleotide homologies of 82.7-98.9% as compared with those of the reference sequences. The cap gene displayed genetic diversity, with a wide range of amino acid homologies ranging from 64.5% to 100%. Phylogenetic analysis of the 29 full-genome sequences revealed that the PiCV strains in this study could be further divided into four clades: A (17.2%), B (10.4%), C (37.9%) and D (34.5%). Thirteen recombination events were also detected in 18 out of the 29 PiCV genomes obtained in this study. Phylogenetic research using the rep and cap genes verified the recombination events, which occurred between clades A/F, A/B, C/D, and B/D among the 18 PiCV strains studied. CONCLUSIONS: In conclusion, PiCV infection, which is highly genetically varied, is extremely widespread on pigeon farms in Beijing. These findings indicate that if pigeons are to be used as experimental animals, it is necessary to evaluate the impact of PiCV infection on the results.

Implementation of point-of-care platforms for rapid detection of porcine circovirus type 2.

BACKGROUND: Porcine circovirus type 2 (PCV2) infection is ubiquitous around the world. Diagnosis of the porcine circovirus-associated disease requires clinic-pathological elements together with the quantification of viral loads. Furthermore, given pig farms in regions lacking access to sufficient laboratory equipment, developing diagnostic devices with high accuracy, accessibility, and affordability is a necessity. OBJECTIVES: This study aims to investigate two newly developed diagnostic tools that may satisfy these criteria. METHODS: We collected 250 specimens, including 170 PCV2-positive and 80 PCV2-negative samples. The standard diagnosis and cycle threshold (Ct) values were determined by quantitative polymerase chain reaction (qPCR). Then, two point-of-care (POC) diagnostic platforms, convective polymerase chain reaction (cPCR, qualitative assay: positive or negative results are shown) and EZtargex (quantitative assay: Ct values are shown), were examined and analyzed. RESULTS: The sensitivity and specificity of cPCR were 88.23% and 100%, respectively; the sensitivity and specificity of EZtargex were 87.65% and 100%, respectively. These assays also showed excellent concordance compared with the qPCR assay (κ = 0.828 for cPCR and κ = 0.820 for EZtargex). The statistical analysis showed a great diagnostic power of the EZtargex assay to discriminate between samples with different levels of positivity. CONCLUSIONS: The two point-of-care diagnostic platforms are accurate, rapid, convenient and require little training for PCV2 diagnosis. These POC platforms can discriminate viral loads to predict the clinical status of the animals. The current study provided evidence that these diagnostics were applicable with high sensitivity and specificity in the diagnosis of PCV2 infection in the field.

Construction of recombinant pseudorabies virus expressing PCV2 Cap, PCV3 Cap, and IL-4: investigation of their biological characteristics and immunogenicity.

Background: Porcine circovirus type 2 (PCV2) is a globally prevalent and recurrent pathogen that primarily causes slow growth and immunosuppression in pigs. Porcine circovirus type 3 (PCV3), a recently discovered virus, commonly leads to reproductive disorders in pigs and has been extensively disseminated worldwide. Infection with a single PCV subtype alone does not induce severe porcine circovirus-associated diseases (PCVD), whereas concurrent co-infection with PCV2 and PCV3 exacerbates the clinical manifestations. Pseudorabies (PR), a highly contagious disease in pigs, pose a significant threat to the swine industry in China. Methods: In this study, recombinant strains named rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 was constructed by using a variant strain XJ of pseudorabies virus (PRV) as the parental strain, with the TK/gE/gI genes deleted and simultaneous expression of PCV2 Cap, PCV3 Cap, and IL-4. The two recombinant strains obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster Syrian kidney-21 (BHK-21) cells and is safe to mice. Results: rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 exhibited good safety and immunogenicity in mice, inducing high levels of antibodies, demonstrated 100% protection against the PRV challenge in mice, reduced viral loads and mitigated pathological changes in the heart, lungs, spleen, and lymph nodes during PCV2 challenge. Moreover, the recombinant viruses with the addition of IL-4 as a molecular adjuvant outperformed the non-addition group in most indicators. Conclusion: rPRV-2Cap/3Cap and rPRV-2Cap/3Cap/IL4 hold promise as recombinant vaccines for the simultaneous prevention of PCV2, PCV3, and PRV, while IL-4, as a vaccine molecular adjuvant, effectively enhances the immune response of the vaccine.

Prevalence, genotypes, and infection risk factors of psittacine beak and feather disease virus and budgerigar fledgling disease virus in captive birds in Hong Kong.

Psittacine beak and feather disease virus (PBFDV) and budgerigar fledgling disease virus (BFDV) are significant avian pathogens that threaten both captive and wild birds, particularly parrots, which are common hosts. This study involved sampling and testing of 516 captive birds from households, pet shops, and an animal clinic in Hong Kong for PBFDV and BFDV. The results showed that PBFDV and BFDV were present in 7.17% and 0.58% of the samples, respectively. These rates were lower than those reported in most parts of Asia. Notably, the infection rates of PBFDV in pet shops were significantly higher compared to other sources, while no BFDV-positive samples were found in pet shops. Most of the positive samples came from parrots, but PBFDV was also detected in two non-parrot species, including Swinhoe's white-eyes (Zosterops simplex), which had not been reported previously. The ability of PBFDV to infect both psittacine and passerine birds is concerning, especially in densely populated urban areas such as Hong Kong, where captive flocks come into close contact with wildlife. Phylogenetic analysis of the Cap and Rep genes of PBFDV revealed that the strains found in Hong Kong were closely related to those in Europe and other parts of Asia, including mainland China, Thailand, Taiwan, and Saudi Arabia. These findings indicate the presence of both viruses among captive birds in Hong Kong. We recommend implementing regular surveillance for both viruses and adopting measures to prevent contact between captive and wild birds, thereby reducing the transmission of introduced diseases to native species.

Emergence of novel circoviruses in humans and pigs and their possible importance for xenotransplantation and blood transfusions.

BACKGROUND: As sequencing is becoming more broadly available, virus discovery continues. Small DNA viruses contribute to up to 60% of the overall virus load in pigs. Porcine circoviruses (PCVs) are small DNA viruses with a single-stranded circular genome. They are common in pig breeds and have not been properly addressed for their potential risk in xenotransplantation. Whereas PCV1 is non-pathogenic in pigs, PCV2 has been associated with various disease manifestations. Recently two new circoviruses have been described, PCV3 and PCV4. While PCV4 is currently present mainly in Asia, PCV3 is widely distributed, and has been identified in commercial pigs, wild boars, and pigs generated for xenotransplantation. In one case PCV3 was transmitted by pigs to baboons via heart transplantation. PCV3 pathogenicity in pigs was controversial initially, however, the virus was found to be associated with porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and multisystemic inflammation. Inoculation studies with PCV3 infectious clones confirmed that PCV3 is pathogenic. Most importantly, recently discovered human circoviruses (CV) are closely related to PCV3. METHODS: Literature was evaluated and summarized. A dendrogram of existing circoviruses in pigs, humans, and other animal species was created and assessed at the species level. RESULTS: We found that human circoviruses can be divided into three species, human CV1, CV2, and CV3. Human CV2 and CV3 are closest to PCV3. CONCLUSIONS: Circoviruses are ubiquitous. This communication should create awareness of PCV3 and the newly discovered human circoviruses, which may be a problem for blood transfusions and xenotransplantation in immune suppressed individuals.

Generation of porcine circovirus type 4 virus-like particles and their use to detect serum antibodies.

Porcine circovirus type 4 (PCV4), first identified in 2019 as a newly emerging pathogen, has been found in several provinces of China, as well as in Korea and Thailand. Since PCV4 is not included in immunization programs, epidemiological investigations should be conducted for detection of anti-PCV4 antibodies. Virus-like particles (VLPs) are frequently used for serological analysis of pathogen infections. However, there have been no reports on using PCV4 VLPs for serological investigation of PCV4 infection. In this study, we generated self-assembled PCV4 VLPs using an E. coli expression system, purified them using a two-step process, and used them to develop an indirect ELISA. This ELISA method was found to be highly specific, sensitive, and repeatable, making it suitable for PCV4 antibody detection in serum samples. Finally, the ELISA was used to analyze 422 serum samples collected from across several regions in China, 134 of which tested positive. Thus, the PCV4-VLP-based ELISA can effectively detect antibodies against PCV4 in serum samples, making it a useful tool for PCV4 epidemiology.

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

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

Crystal structure of the ATPase domain of porcine circovirus type 2 Rep protein.

PCV2 belongs to the genus Circovirus in the family Circoviridae, whose genome is replicated by rolling circle replication (RCR). PCV2 Rep is a multifunctional enzyme that performs essential functions at multiple stages of viral replication. Rep is responsible for nicking and ligating single-stranded DNA and unwinding double-stranded DNA (dsDNA). However, the structure and function of the Rep are still poorly understood, which significantly impedes viral replication research. This study successfully resolved the structure of the PCV2 Rep ATPase domain (PRAD) using X-ray crystallography. Homologous structure search revealed that Rep belonged to the superfamily 3 (SF3) helicase, and multiple conserved residues were identified during sequence alignment with SF3 family members. Simultaneously, a hexameric PRAD model was generated for analysing characteristic structures and sites. Mutation of the conserved site and measurement of its activity showed that the hallmark motifs of the SF3 family influenced helicase activity by affecting ATPase activity and ß-hairpin just caused the loss of helicase activity. The structural and functional analyses of the PRAD provide valuable insights for future research on PCV2 replication and antiviral strategies.

The Nuclear Localization Signal of Porcine Circovirus Type 4 Affects the Subcellular Localization of the Virus Capsid and the Production of Virus-like Particles.

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.

[Viral co-infections targeting the porcine respiratory system: Consequences and limits of the experimental systems]. / Les co-infections virales affectant le système respiratoire porcin : conséquences et limites des systèmes d'étude.

Coinfections affecting the porcine respiratory system have often been overlooked, in favor of mono-infections, even though they are significantly more common in the field. In pigs, the term 'porcine respiratory complex' is used to describe coinfections involving both viruses, such as, for example, the swine influenza type A virus (swIAV), the porcine respiratory and reproductive syndrome virus (PRRSV), and the porcine circovirus type 2 (PCV-2), as well as bacteria. Until recently, most studies were primarily focused on clinical aspects and paid little attention to the molecular consequences of coinfections. This narrative review addresses the consequences of coinfections in the porcine respiratory system involving viruses. When possible, interactions that can occur between viruses are briefly presented. Conversely, research involving bacteria, protozoa, and fungi has not been considered at all. Finally, the main limitations complicating the interpretation of results from coinfection/superinfection studies are considered, and prospects in this exciting field of health research are presented.

Divergent clinical outcomes depending on the sequential infection order of porcine circovirus type 2 and Mycoplasma hyopneumoniae.

This study compared the different sequential order of infection of porcine circovirus type 2d (PCV2d) and Mycoplasma hyopneumoniae. Thirty-six pigs were allocated randomly across six different groups. Pigs underwent various inoculation sequences: M. hyopneumoniae administered 14 days before PCV2d, simultaneous PCV2d-M. hyopneumoniae, PCV2d given 14 days before M. hyopneumoniae, PCV2d only, M. hyopneumoniae only, or a mock inoculum. Overall, the pigs inoculated with M. hyopneumoniae 14 days prior to PCV2d (Mhyo-PCV2 group) and those inoculated simultaneously with PCV2d and M. hyopneumoniae (PCV2+Mhyo group) displayed notably higher clinical disease severity and experienced a significant decrease of their average daily weight gain than pigs inoculated with PCV2d 14 days prior to M. hyopneumoniae (PCV2-Mhyo group). M. hyopneumoniae infection potentiated PCV2 blood and lymph node viral loads, as well as PCV2-associated lesions, while the infection of PCV2d did not impact the intensity of M. hyopneumoniae infection. Tumor necrosis factor-α (TNF-α) sera levels were significantly increased in the Mhyo-PCV2 and PCV2+Mhyo groups as compared to the PCV2-Mhyo, PCV2, and Mhyo groups. The most important information was that the potentiation effect of M. hyopneumoniae on PCV2d was found only in pigs inoculated with either M. hyopneumoniae followed by PCV2d (Mhyo-PCV2 group) or a simultaneous inoculation of PCV2d and M. hyopneumoniae (PCV2+Mhyo group). The sequential infection order of PCV2d and M. hyopneumoniae resulted in divergent clinical outcomes.

Simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting analysis.

Birds infected with duck circovirus (DuCV) can potentially cause immunosuppression by damaging lymphoid tissues, causing great losses in the duck breeding industry. Duck circovirus can be divided into two genotypes (DuCV-1 and DuCV-2), but simultaneous detection and differentiation of DuCV-1 and DuCV-2 by high-resolution melting (HRM) analysis is still lacking. Here, we designed specific primers according to the sequence characteristics of the newly identified ORF3 gene and then established a PCR-HRM method for the simultaneous detection and differentiation of DuCV-1 and DuCV-2 via high-resolution melting analysis. Our data showed that the established PCR-HRM assay had the advantages of specificity, with the lowest detection limits of 61.9 copies/µL (for DuCV-1) and 60.6 copies/µL (for DuCV-2). The melting curve of the PCR-HRM results indicated that the amplification product was specific, with no cross-reaction with common waterfowl origin pathogens and a low coefficient of variation less than 1.50% in both intra-batch and inter-batch repetitions, indicating the advantages of repeatability. We found that the percentage of DuCV-2-positive ducks was higher than that of DuCV-1-positive ducks, with 8.62% rate of DuCV-1 and DuCV-2 coinfection. In addition, we found DuCV-2-positive in geese firstly. In conclusion, this study provides a candidate PCR-HRM assay for the detection and accurate differentiation of DuCV-1 and DuCV-2 infection, which will help us for further epidemiological surveillance of DuCVs.

Pluck-pools as diagnostic samples for detecting porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in porcine abortion material and stillbirths.

Investigating infectious agents in porcine abortion material and stillborn piglets poses challenges for practitioners and diagnostic laboratories. In this study, pooled samples of individual reference organs (thymus and heart) from a total of 1000 aborted fetuses and stillborn piglets were investigated using quantitative PCR protocols for porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) and porcine circovirus type 2 (PCV2). Simultaneously, a pluck-pool containing equivalent portions of fetal thymus, heart, and lung tissue was collected, frozen at - 20 °C, and re-analyzed when a certain amount of either PRRSV-1 RNA or PCV2 DNA was detected in individual reference organs. Thirteen pluck-pools were assessed for PRRSV-1, all being PCR-positive. For PCV2, 11 of 15 pluck-pools investigated were PCR-positive. In all pluck-pools testing negative, viral loads in individual pools were low. This study indicates that pluck-pools can be valuable diagnostic material and the consolidation of multiple organs through a single RNA/DNA extraction optimizes the utilization of available laboratory resources. Additional research is required to assess the feasibility of follow-up investigations and to accurately define criteria for interpretation of viral loads in a clinical context.

Platycodon grandiflorus polysaccharide inhibits the inflammatory response of 3D4/21 cells infected with PCV2.

Porcine circovirus type 2 (PCV2) infection cause multi-systemic inflammation in pigs. Platycodon grandiflorus polysaccharide (PGPSt) has been reported to have the effects of immune regulation and disease resistance. Nevertheless, the role and mechanism of PGPSt in the inflammatory response of 3D4/21 cells induced by PCV2 infection remain unclear. The present study aims to investigate effects of PGPSt on inflammatory response and its possible underlying mechanisms in vitro models. Cells were treated with PCV2 for 36 h to construct a cell inflammation model. The 3D4/21 cell lines were pretreated with or without PGPSt, and the changes of inflammation-related markers and the signaling pathway were detected by CCK-8, ELISA, qPCR and Western blot. The results showed that PGPSt was non-toxic to cells and protected PCV2-infected cells from inflammatory damage. PGPSt could significantly inhibit the high acetylation of histone H3 (AcH3) and histone H4 (AcH4), down-regulate HAT and up-regulate HDAC activity, and reduce the expression of pro-inflammatory enzymes iNOS and COX-2 proteins levels. Then the levels of IL-1ß, IL-6 and TNF-α were significantly inhibited, and the level of IL-10 was promoted. We also observed that PGPSt inhibited the phosphorylation of p65, p38 and Erk1/2, which subsequently inhibited nuclear translocation of NF-κB p65 to express pro-inflammatory factors. In conclusion, PGPSt can reduce the inflammatory response by regulating histone acetylation, reducing the release of inflammatory factors, reducing the expression of pro-inflammatory enzymes, and inhibiting the activation of NF-κB and MAPKs signaling pathways. This suggests that PGPSt had an anti-inflammatory effect on the inflammatory response caused by PCV2 infection, which provided theoretical data support for the research.

First molecular detection and genetic characterization of porcine circovirus 4 in the Gansu Province of China.

Since its initial discovery in the Hunan province of China, genomic DNA of porcine circovirus 4 (PCV4) has been detected in pigs across multiple provinces in China, as well as in South Korea. However, the prevalence of porcine circovirus type 4 in Gansu Province, China, remains unknown. To address this gap, we undertook an extensive study where we gathered 121 clinical samples displaying diverse clinical manifestations from pig farms in Gansu Province between 2022 and 2023. Employing a real-time fluorescence quantification method, we identified the presence of PCV4 genome. Out of the 121 clinical samples analyzed, 13 samples tested positive for PCV4, resulting in a positive rate of 10.74% (13/121). This finding confirms the presence of PCV4 in pig farms within Gansu Province, China. Furthermore, we successfully sequenced and analyzed the complete genomes of two distinct PCV4 strains, comparing them with 60 reference sequences archived in the GenBank database. The results revealed a high nucleotide homology (98.2-98.8%) between the strains obtained in this study and the PCV4 reference strains, indicating a relatively low evolutionary rate of the PCV4 genome. Phylogenetic analysis revealed that two strains in this study belong to PCV4a and PCV4c. As far as we know, this study marks the inaugural report on the molecular identification and genomic attributes of PCV4 in Gansu Province, China, offering valuable insights for devising preventive and control strategies against this emerging virus.

Porcine circovirus type 2 infection promotes the SUMOylation of nucleophosmin-1 to facilitate the viral circular single-stranded DNA replication.

The mechanism of genome DNA replication in circular single-stranded DNA viruses is currently a mystery, except for the fact that it undergoes rolling-circle replication. Herein, we identified SUMOylated porcine nucleophosmin-1 (pNPM1), which is previously reported to be an interacting protein of the viral capsid protein, as a key regulator that promotes the genome DNA replication of porcine single-stranded DNA circovirus. Upon porcine circovirus type 2 (PCV2) infection, SUMO2/3 were recruited and conjugated with the K263 site of pNPM1's C-terminal domain to SUMOylate pNPM1, subsequently, the SUMOylated pNPM1 were translocated in nucleoli to promote the replication of PCV2 genome DNA. The mutation of the K263 site reduced the SUMOylation levels of pNPM1 and the nucleolar localization of pNPM1, resulting in a decrease in the level of PCV2 DNA replication. Meanwhile, the mutation of the K263 site prevented the interaction of pNPM1 with PCV2 DNA, but not the interaction of pNPM1 with PCV2 Cap. Mechanistically, PCV2 infection increased the expression levels of Ubc9, the only E2 enzyme involved in SUMOylation, through the Cap-mediated activation of ERK signaling. The upregulation of Ubc9 promoted the interaction between pNPM1 and TRIM24, a potential E3 ligase for SUMOylation, thereby facilitating the SUMOylation of pNPM1. The inhibition of ERK activation could significantly reduce the SUMOylation levels and the nucleolar localization of pNPM1, as well as the PCV2 DNA replication levels. These results provide new insights into the mechanism of circular single-stranded DNA virus replication and highlight NPM1 as a potential target for inhibiting PCV2 replication.

Molecular basis for the different PCV2 susceptibility of T-lymphoblasts in Landrace and Piétrain pigs.

Clinically, Landrace pigs are more susceptible to porcine circovirus-associated diseases (PCVADs) than Piétrain pigs. We previously found that porcine circovirus type 2 (PCV2) can infect T-lymphoblasts. The present study examined the replication kinetics of six PCV2 strains in the lymphoblasts of Landrace and Piétrain pigs. The results showed that T-lymphoblasts from Landrace pigs are much more susceptible to PCV2 infection than those from Piétrain pigs. In addition, PCV2 replication was strain-dependent. PCV2 binding to T-lymphoblasts was partially mediated by chondroitin sulfate (CS) and dermatan sulfate (DS). Phosphacan, an effective internalization mediator in monocytes that contains several CS chains, was also demonstrated to be involved in PCV2 internalization. Viral binding and internalization were not different between the two breeds, however, the subsequent step, the disassembly was. Although inhibition of serine proteases blocked PCV2 replication in both Landrace and Piétrain pigs, this only occurred at a neutral pH in Piétrain pigs, whereas this occurred also at a low pH in Landrace. This suggested that more proteases can cleave PCV2 in Landrace lymphoblasts than in Piétrain lymphoblasts, explaining the better replication. Through co-localization studies of viral particles with endo-lysosomal markers, and quantitative analysis of organelle sizes during viral internalization, it was observed that PCV2 may exhibit a higher propensity for viral escape from late endosomes in Landrace pigs (smaller) compared to Piétrain pigs. These results provide new understandings of the different PCV2 susceptibility in Landrace and Piétrain pigs.

Efficacy evaluation of a bivalent subunit vaccine against classical swine fever virus and porcine circovirus type 2.

Classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV2) are two of the most devastating and economically significant pathogens affecting pig populations worldwide. Administration of a combination of vaccines against swine pathogens has been demonstrated to be as efficacious as the administration of single vaccines. In this study, we developed and tested a novel bivalent subunit vaccine against CSFV and PCV2. The safety and efficacy of this vaccine were demonstrated in mice and specific pathogen-free (SPF) piglets. In addition to investigating the serological responses after immunization, challenge studies with both viruses were also conducted. The results showed that this CSFV/PCV2 bivalent vaccine elicited a high level of neutralizing antibodies against both viruses and provided protection in challenge studies. In conclusion, the CSFV/PCV2 bivalent vaccine is safe and effective against CSFV or PCV2 challenge.

From obscurity to urgency: a comprehensive analysis of the rising threat of duck circovirus.

Duck circovirus (DuCV) is a small, nonenveloped, single-stranded DNA virus with immunosuppressive effects on ducks that leads to slow growth and elevated mortality following mixed infections. Its infection manifests as feather loss, slow growth, swelling of respiratory tissue, and damage to immune organs in ducks. Although single infections with DuCV do not cause noticeable clinical symptoms, its ability to compromise the immune system and facilitate infections caused by other pathogens poses a serious threat to duck farming. Given the prevalence of this disease and the increasing infection rates in recent years, which have resulted in significant economic losses in duck farming and related sectors, research and control of DuCV infection have become especially important. The aim of this review is to provide a summary of the current understanding of DuCV, serving as a reference for subsequent research and effective control of the virus. We focus mainly on the genetics and molecular biology, epidemiology, clinical symptoms, and pathology of DuCV. Additionally, topics such as the isolation and culture of the virus, vaccines and antiviral therapies, diagnostics, and preventative measures are discussed.

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

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