Glaesserella parasuis serotype 4 exploits fibronectin via RlpA for tracheal colonization following porcine circovirus type 2 infection.

Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality, but the role played by PCV2 and bacterial and host factors contributing to this process have not been defined. Bacterial attachment is assumed to occur via specific receptor-ligand interactions between adhesins on the bacterial cell and host proteins adsorbed to the implant surface. Mass spectrometry (MS) analysis of PCV2-infected swine tracheal epithelial cells (STEC) revealed that the expression of Extracellular matrix protein (ECM) Fibronectin (Fn) increased significantly on the infected cells surface. Importantly, efficient G. parasuis serotype 4 (GPS4) adherence to STECs was imparted by interactions with Fn. Furthermore, abrogation of adherence was gained by genetic knockout of Fn, Fn and Integrin ß1 antibody blocking. Fn is frequently exploited as a receptor for bacterial pathogens. To explore the GPS4 adhesin that interacts with Fn, recombinant Fn N-terminal type I and type II domains were incubated with GPS4, and the interacting proteins were pulled down for MS analysis. Here, we show that rare lipoprotein A (RlpA) directly interacts with host Fibronectin mediating GPS4 adhesion. Finally, we found that PCV2-induced Fibronectin expression and adherence of GPS4 were prevented significantly by TGF-ß signaling pathway inhibitor SB431542. Our data suggest the RlpA-Fn interaction to be a potentially promising novel therapeutic target to combat PCV2 and GPS4 coinfection.

Genotypic diversity and immunological implications of porcine circovirus: Inspiration from PCV1 to PCV4.

Porcine circovirus (PCV) is a group of DNA viruses that cause diseases in pigs, with multiple genotypes ranging from PCV1 to PCV4. PCV1 is generally considered non-pathogenic, while PCV2 can cause severe immune system damage, especially associated with porcine multisystemic wasting syndrome (PMWS). PCV2 has a genetic homology of about 68 % but differs from PCV1 in antigenicity and phenotype. PCV3 and PCV4 have lower genetic homology with PCV1 and PCV2, with limited research available on their pathogenicity. During virus infection, the host's innate immune system detects PCVs through pattern recognition receptors (PRRs) like TLRs and NLRs. PCV disrupts immune pathways, including interferon and NF-κB pathways, aiding viral replication and causing immunosuppression. This review systematically compares the characteristics and pathogenicity of different genotypes of PCV and their interactions with the host's immune system, aiming to better understand the mechanisms of PCV infection and provide a theoretical basis for prevention and treatment.

Cytotoxicity effect and transcriptome analysis of PCV3-infected cells revealed potential viral pathogenic mechanisms.

Porcine circovirus type 3 (PCV3) has become an important pathogen in the global swine industry and poses a threat to pig health, but its pathogenic mechanism remains unknown. In this study, we constructed an innovative, linear infectious clone of PCV3 for rescuing the virus, and explored the transcriptome of infected cells to gain insights into its pathogenic mechanisms. Subsequently, an in vivo experiment was conducted to evaluate the pathogenicity of the rescued virus in pig. PCV3 nucleic acid was distributed across various organs, indicating systemic circulation via the bloodstream and viremia. Immunohistochemical staining also revealed a significant presence of PCV3 antigens in the spleen, lungs, and lymph nodes, indicating that PCV3 had tropism for these organs. Transcriptome analysis of infected ST cells revealed differential expression of genes associated with apoptosis, immune responses, and cellular metabolism. Notably, upregulation of genes related to the hypoxia-inducible factor-1 pathway, glycolysis, and the AGE/RAGE pathway suggests activation of inflammatory responses, ultimately leading to onset of disease. These findings have expanded our understanding of PCV3 pathogenesis, and the interplay between PCV3 and host factors.

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

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

Prevalence of pigeon rotavirus infections: animal exhibitions as a risk factor for pigeon flocks.

A total of 289 cloacal swabs from pigeons from 29 different breeders in Germany were collected. In addition, samples from pigeons exhibited at shows were collected. The detailed health status of the pigeon flocks was recorded. Samples were analysed for the presence of the recently discovered pigeon rotavirus and pigeon circovirus. Pigeon rotavirus was found in 10.3% and pigeon circoviruses was found in 65.5% of sampled pigeon lofts. The study revealed a strong relationship between the attendance of shows and the occurrence of different clinical signs. The higher prevalence of pigeon rotavirus in exhibited animals indicates that exhibitions are a risk factor for the transmission of this pathogen.

Identification and whole-genome characterization of a novel Porcine Circovirus 3 subtype b strain from swine populations in Vietnam.

Porcine circovirus 3 (PCV3) is a novel circovirus detected in pigs suffering from porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and multisystemic infection. In this study, we identified PCV3 infection in aborted fetuses and reported the full-length genome sequence of a PCV3 strain identified from southern Vietnam. The complete genome of this PCV3 strain is 2000 nucleotides in length. We found that it shares 98.5-99.25% sequence identity with other reference sequences and that it clusters with the PCV3b subtype. Several specific mutated sites were found to be unique to this Vietnamese PCV3b strain, including I14M in the Rep protein and K139R, I150F, and P169T in the Cap protein. The sequence data that have been made publically available as part of this study will help investigators to better understand the molecular characteristics, genetic diversity, and evolutionary history of PCV3. Careful and in-depth investigations into the epidemiology, pathogenicity, and the evolution of this novel virus is a matter of urgent economic and agricultural interest in Vietnam.

Development of a dual-labeled, hydrolysis probe-based, real-time quantitative PCR assay for detection of both genotypes of duck circovirus-1 (DuCV-1) and DuCV-2.

In this study, we developed a real-time quantitative polymerase chain reaction (qPCR) assay based on a dual-labeled hydrolysis probe to simultaneously detect both duck circovirus (DuCV) 1 and DuCV-2. The reproducibility, sensitivity and specificity of the primer set and probe were evaluated using other duck pathogens. The detection limit was 20 copies per µL. The intra-assay coefficients of variation (CVs) were ≤ 0.73% and the inter-assay CVs were ≤ 1.89%. No cross-reaction occurred with other duck pathogens. In addition, the qPCR assay was successfully applied to the simultaneous detection of DuCV-1 and DuCV-2 in clinical field samples. Therefore, this assay will be useful for laboratory diagnosis and epidemiological field studies of DuCV.

Synaptogyrin-2 influences replication of Porcine circovirus 2.

Porcine circovirus 2 (PCV2) is a circular single-stranded DNA virus responsible for a group of diseases collectively known as PCV2 Associated Diseases (PCVAD). Variation in the incidence and severity of PCVAD exists between pigs suggesting a host genetic component involved in pathogenesis. A large-scale genome-wide association study of experimentally infected pigs (n = 974), provided evidence of a host genetic role in PCV2 viremia, immune response and growth during challenge. Host genotype explained 64% of the phenotypic variation for overall viral load, with two major Quantitative Trait Loci (QTL) identified on chromosome 7 (SSC7) near the swine leukocyte antigen complex class II locus and on the proximal end of chromosome 12 (SSC12). The SNP having the strongest association, ALGA0110477 (SSC12), explained 9.3% of the genetic and 6.2% of the phenotypic variance for viral load. Dissection of the SSC12 QTL based on gene annotation, genomic and RNA-sequencing, suggested that a missense mutation in the SYNGR2 (SYNGR2 p.Arg63Cys) gene is potentially responsible for the variation in viremia. This polymorphism, located within a protein domain conserved across mammals, results in an amino acid variant SYNGR2 p.63Cys only observed in swine. PCV2 titer in PK15 cells decreased when the expression of SYNGR2 was silenced by specific-siRNA, indicating a role of SYNGR2 in viral replication. Additionally, a PK15 edited clone generated by CRISPR-Cas9, carrying a partial deletion of the second exon that harbors a key domain and the SYNGR2 p.Arg63Cys, was associated with a lower viral titer compared to wildtype PK15 cells (>24 hpi) and supernatant (>48hpi)(P < 0.05). Identification of a non-conservative substitution in this key domain of SYNGR2 suggests that the SYNGR2 p.Arg63Cys variant may underlie the observed genetic effect on viral load.

Porcine Circovirus 2 Uses a Multitude of Weak Binding Sites To Interact with Heparan Sulfate, and the Interactions Do Not Follow the Symmetry of the Capsid.

Porcine circovirus 2 (PCV2) is the smallest pathogenic virus capable of autonomous replication within its host. Infections result in immunosuppression and subsequent death of the host and are initiated via the attachment of the PCV2 icosahedral capsid to heparan sulfate (HS) and chondroitin sulfate B (CSB) glycosaminoglycans on the cell surface. However, the underlying mechanism of structural recognition remains to be explored. Using heparin, a routinely used analog of heparan sulfate, we demonstrate that increasing lengths of heparin exhibit a greater affinity toward PCV2. Our competition assays indicate that dextran sulfate (8 kDa) has a higher affinity for PCV2 than heparin (12 kDa), chondroitin sulfate B (41 kDa), hyaluronic acid (1.6 MDa), and dextran (6 kDa). This suggests that polymers high in sulfate content are capable of competing with the PCV2-heparan sulfate interaction and, thus, have the potential to inhibit PCV2 infection. Finally, we visualized the interaction between heparin and the PCV2 capsid using cryo-electron microscopy single-particle analysis, symmetry expansion, and focused classification. The image reconstructions provide the first example of an asymmetric distribution of heparin on the surface of an icosahedral virus capsid. We demonstrate that each of the 60 capsid subunits that generate the T=1 capsid can bind heparin via one of five binding sites. However, not all of the binding sites were occupied by heparin, and only one-third to two-thirds of the binding sites were occupied. The binding sites are defined by arginine, lysine, and polar amino acids. Mutating the arginine, lysine, and polar amino acids to alanine diminished the binding capacity of PCV2 to heparin.IMPORTANCE It has been demonstrated that porcine circovirus 2 (PCV2) attaches to cells via heparan sulfate (HS) and chondroitin sulfate B (CSB) glycosaminoglycans; however, the underlying structural mechanism describing the HS/CSB recognition by PCV2 remains to be explored. We used cryo-electron microscopy with single-particle analysis, symmetry expansion, and focused classification to visualize the interaction between the PCV2 capsid and heparin, an analog of heparan sulfate, to better than 3.6-Å resolution. We observed that the interaction between PCV2 and heparin does not adhere to the icosahedral symmetry of the capsid. To the best of our knowledge, this is the first example where the interaction between heparin and an icosahedral capsid does not follow the symmetry elements of the capsid. Our findings also suggest that anionic polymers, such as dextran sulfate, may act to inhibit PCV2 infection.

Porcine Circovirus Type 2 Induces ORF3-Independent Mitochondrial Apoptosis via PERK Activation and Elevation of Cytosolic Calcium.

Our previous studies demonstrated that porcine circovirus type 2 (PCV2) triggers an unfolded protein response (UPR) in porcine kidney PK-15 cells by activating the protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) pathway of endoplasmic reticulum (ER) stress, which in turn facilitates viral replication (Y. Zhou et al., Viruses 8:e56, 2016, https://doi.org/10.3390/v8020056; Y. Zhou et al., J Zhejiang Univ Sci B 18:316-323, 2017, https://doi.org/10.1631/jzus.B1600208). PCV2 is found to cause oxidative stress and upregulation of cytoplasmic Ca2+ levels. The virus is reported to employ its open reading frame 3 (ORF3) to induce apoptosis. We wondered whether and how PCV2-induced UPR would lead to apoptosis independent of ORF3. Using an ORF3-deficient PCV2 mutant (ΔORF3), apoptotic responses in infected PK-15 and porcine alveolar macrophage (PAM) cells were still apparent, although lower than in the parental PCV2 strain. We hypothesized that apoptosis induced by ΔORF3 might result from the UPR. We found that ΔORF3-induced apoptosis was significantly reduced when the infected cells were treated with the selective PERK blocker GSK2606414 (GSK) or the general ER stress attenuator 4-phenylbutyrate (4-PBA). Such treatments also ameliorated elevation of cytoplasmic Ca2+ and reactive oxygen species (ROS) levels in PK-15 and PAM cells, two predisposing factors for apoptosis via disruption of the ER-mitochondrion units. Treatment of ΔORF3-infected cells with GSK and 4-PBA also decreased the mitochondrial Ca2+ load and increased the mitochondrial membrane potential (MMP). With transient expression of the structural protein capsid (Cap) in combination with PERK silencing, we found that Cap induced MMP collapse and mitochondrial apoptosis could result from the UPR and elevation of Ca2+ and ROS levels, which were inhibitable by downregulation of PERK. We propose that PCV2-driven ER stress is Cap dependent and could lead to mitochondrial apoptotic responses independent of ORF3 via perturbation of intracellular Ca2+ homeostasis and accumulation of ROS.IMPORTANCE PCV2 encodes protein ORF3, a putative protein with proapoptotic activity. Our early studies showed that PCV2 infection triggers ER stress via selective activation of the PERK pathway, a branch of the ER stress pathways, in permissive cells for enhanced replication and infection increased cytosolic Ca2+ and ROS levels. Here we clearly show that PCV2 infection or Cap expression induces ORF3-independent apoptosis via increased cytosolic and mitochondrial Ca2+ levels and cellular ROS levels as a result of activation of the PERK pathway.

Full-genome sequences of porcine circovirus 3 (PCV3) and high prevalence in mummified fetuses from commercial farms in Brazil.

The clinical implications of recently discovered porcine circovirus 3 (PCV3) infections are still unknown. The potential role of this emerging virus in reproductive loss in swine has been described. Herein, we report a high prevalence of PCV3 in mummified fetuses from sows maintained in modern farms in Rio Grande do Sul, Santa Catarina, Paraná, Goiás, and Mato Grosso do Sul states, Brazil. For this analysis, 276 mummified fetuses from 11 commercial swine farms were included. The presence of PCV3 DNA was confirmed using PCR, and the complete sequence of five different viral strains was obtained. Sequences of PCV3 genomes available on GenBank were then used for phylogenetic tree construction. Of the 276 mummified fetuses examined, 270 (nearly 97%) were positive for PCV3. In 93.1% of the fetuses, co-infections with at least one of the following agents were identified: porcine parvovirus (PPV), porcine circovirus 2 (PCV2) and Leptospira spp. Twelve fetuses were positive for PCV3 alone. The amino acid sequence of the capsid gene for the five viral strains shared 98-100% homology among them. Analysis of the DNA sequence indicates that the viruses identified in this study belong to the PCV3a1 subgroup. In summary, PCV3 DNA was detected in mummified fetuses at a surprisingly high rate. The role of PCV3 in porcine circovirus-associated disease (PCVAD) is still uncertain. However, considering that PCV3 has been detected in a variety of conditions, even in healthy animals, the present results confirm the need to investigate PCV3 as a causative agent of fetal mummification in swine.

Identification and genetic characterization of porcine circovirus 3 on pig farms in Serbia.

BACKGROUD: The presence of PCV3 genome has been detected in pigs affected by different clinical and pathological conditions as well as in healthy animals. Its presence has been reported in many countries of North and South America, Asia and Europe. However, there is no evidence of the presence and genetic characteristics of PCV3 in many European countries and especially the countries of the Balkan Peninsula. PURPOSE: The major objective of this study was to investigate the presence and obtain further genetic characterization of PCV-3 in the pig populations in Serbia. METHODS: To demonstrate the presence of PCV-3 DNA a conventional PCR assay was performed. The samples where no PCR product was observed (n=32), were further tested with a real-time PCR assay. The six PCR samples that were strongly positive for PCV-3 were subjected to amplification and sequencing of their entire cap genes and complete viral genome. RESULTS: We report on the first identification, genetic diversity and potential association in pathogenesis of some systemic and respiratory swine diseases of PCV-3 in Serbia. CONCLUSION: The results imply that PCV-3 circulates widely in the pig population and has a high similarity with previously reported isolates. Detected PCV-3 can be associated with some swine systemic and respiratory diseases but these associations are strongly influenced by the clinical or pathological condition of the animals. Our findings demonstrate that there are certain PCV-3 loads in pigs suffering from active PRRSV infection, Glässer's disease, APP pleuropneumonia, pneumonic pasteurellosis and PRDC; however, the significance of this viral load, as well as the mechanism by which PCV-3 may act as a secondary agent in aggravating the severity during co-infections of these pathogens, requires further research.

Porcine circovirus type 2 upregulates endothelial-derived IL-8 production in porcine iliac artery endothelial cells via the RIG-I/MDA-5/MAVS/JNK signaling pathway.

BACKGROUND: Dysfunction of endothelial cells and vascular system is one of the most important pathological changes of porcine circovirus disease (PCVD) caused by porcine circovirus type 2 (PCV2). PCV2-infected endothelial cells can upregulate the production of endothelial-derived IL-8, which can inhibit the maturation of dendritic cells. Endothelial-derived IL-8 has different structural and biological characteristics compared with monocyte-derived IL-8. However, the mechanism of endothelial-derived IL-8 production is still unclear. RESULTS: Key molecules of RIG-I-like signaling pathway RIG-I, MDA-5, MAVS and a key molecule of JNK signaling pathway c-Jun in PCV2-infected porcine iliac artery endothelial cells (PIECs) were upregulated significantly detected with quantitative PCR, Western blot and fluorescence confocal microscopy, while no significant changes were found in NF-κB signaling pathway. Meanwhile, the expression of endothelial-derived IL-8 was downregulated after RIG-I, MDA-5, or MAVS genes in PIECs were knocked down and PIECs were treated by JNK inhibitor. CONCLUSIONS: PCV2 can activate RIG-I/MDA-5/MAVS/JNK signaling pathway to induce the production of endothelial-derived IL-8 in PIECs, which provides an insight into the further study of endothelial dysfunction and vascular system disorder caused by PCV2.

Assessing circovirus gene flow in multiple spill-over events.

The establishment of viral pathogens in new host environments following spillover events probably requires adaptive changes within both the new host and pathogen. After many generations, signals for ancient cross-species transmission may become lost and a strictly host-adapted phylogeny may mimic true co-divergence while the virus may retain an inherent ability to jump host species. The mechanistic basis for such processes remains poorly understood. To study the dynamics of virus-host co-divergence and the arbitrary chances of spillover in various reservoir hosts with equal ecological opportunity, we examined structural constraints of capsid protein in extant populations of Beak and feather disease virus (BFDV) during known spillover events. By assessing reservoir-based genotype stratification, we identified co-divergence defying signatures in the evolution BFDV which highlighted primordial processes of cryptic host adaptation and competing forces of host co-divergence and cross-species transmission. We demonstrate that, despite extensive surface plasticity gathered over a longer span of evolution, structural constraints of the capsid protein allow opportunistic host switching in host-adapted populations. This study provides new insights into how small populations of endangered psittacine species may face multidirectional forces of infection from reservoirs with apparently co-diverging genotypes.

Truncated Rep protein of porcine circovirus 2 (PCV2) caused by a naturally occurring mutation reduced virus replication in PK15 cells.

BACKGROUND: Porcine circovirus 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVADs). The infection of PCV2 is widespread and has serious consequence, thereby causing significant economic losses in the swine industry worldwide. Previously, we found that a strain named YiY-3-2-3 has a naturally occurring point mutation (G710 to A710) in ORF1 region, which leads to a shorten product of the rep gene (945 to 660 base pair). Importantly, the Rep protein is responsible for genome replication of PCV2. To explore the effects of this mutation on the PCV2 replication, in the current study we constructed infectious clone of this IF-YiY-3-2-3, as well as those of its two parental strains of IF-YiY-3-2-1 and IF-YiY-3-2-10. Subsequently, these infectious clones which have 1.1 copy of PCV2 genome of their corresponding strains were transfected into PK15 cells to obtain rescued viruses, respectively. RESULTS: Though all of the three infectious clones could be rescued, the copy number and infectivity of these rescued viruses were significantly different, as analyzed by fluorescence quantitative PCR, Tissue culture infectious dose 50 (TCID50), and indirect immunofluorescence assay (IFA). Notably, whether the PCV2 copy number, viral titer or the infectivity of rescued viruses from infectious clone IF-YiY-3-2-3 was significantly less than those of its parental clones. Meanwhile, the spatial structure of the Rep protein from the IF-YiY-3-2-3 displayed an apparent truncation at the C-terminal. CONCLUSIONS: These findings therefore suggest that the Rep protein with truncated C-terminal would reduce virus replication and infectivity, and there might also exist both favorable and unfavorable mutations in the ORF1 of PCV2 in the process of its evolution.

Establishment and characterization of a telomerase-immortalized porcine bronchial epithelial cell line.

Primary porcine bronchial epithelial cells (PBECs) are an ideal model to study the molecular and pathogenic mechanisms of various porcine respiratory pathogens. However, the short lifespan of primary PBECs greatly limit their application. Here, we isolated and cultured primary PBECs and established immortalized PBECs by transfecting primary PBECs with the pEGFP-hTERT recombinant plasmid containing human telomerase reverse transcriptase (hTERT). Immortalized PBECs (hTERT-PBECs) retained the morphological and functional features of primary PBECs as indicated by cytokeratin 18 expression, telomerase activity assay, proliferation assays, karyotype analysis, and quantitative reverse-transcriptase polymerase chain reaction. Compared to primary PBECs, hTERT-PBECs had higher telomerase activity, extended replicative lifespan, and displayed enhanced proliferative activity. Moreover, this cell line is not transformed in vitro and does not exhibit a malignant phenotype in vivo, suggesting that it can be safely used in further studies. Besides, hTERT-PBECs were susceptible to swine influenza virus of H3N2 subtype and porcine circovirus type 2. In conclusion, the immortalized hTERT-PBECs represent a valuable in vitro model, which can be widely used in the study of porcine respiratory pathogenic infections.

Possible risks posed by single-stranded DNA viruses of pigs associated with xenotransplantation.

Routine large-scale xenotransplantation from pigs to humans is getting closer to clinical reality owing to several state-of-the-art technologies, especially the ability to rapidly engineer genetically defined pigs. However, using pig organs in humans poses risks including unwanted cross-species transfer of viruses and adaption of these pig viruses to the human organ recipient. Recent developments in the field of virology, including the advent of metagenomic techniques to characterize entire viromes, have led to the identification of a plethora of viruses in many niches. Single-stranded DNA (ssDNA) viruses are the largest group prevalent in virome studies in mammals. Specifically, the ssDNA viral genomes are characterized by a high rate of nucleotide substitution, which confers a proclivity to adapt to new hosts and cross-species barriers. Pig-associated ssDNA viruses include torque teno sus viruses (TTSuV) in the Anelloviridae family, porcine parvoviruses (PPV), and porcine bocaviruses (PBoV) both in the family of Parvoviridae, and porcine circoviruses (PCV) in the Circoviridae family, some of which have been confirmed to be pathogenic to pigs. The risks of these viruses for the human recipient during xenotransplantation procedures are relatively unknown. Based on the scant knowledge available on the prevalence, predilection, and pathogenicity of pig-associated ssDNA viruses, careful screening and monitoring are required. In the case of positive identification, risk assessments and strategies to eliminate these viruses in xenotransplantation pig stock may be needed.

Pathogen elimination and prevention within a regulated, Designated Pathogen Free, closed pig herd for long-term breeding and production of xenotransplantation materials.

BACKGROUND: We established a Source Animal (barrier) Facility (SAF) for generating designated pathogen-free (DPF) pigs to serve as donors of viable organs, tissues, or cells for xenotransplantation into clinical patients. This facility was populated with caesarian derived, colostrum deprived (CDCD) piglets, from sows of conventional-specific (or specified) pathogen-free (SPF) health status in six cohorts over a 10-month period. In all cases, CDCD piglets fulfilled DPF status including negativity for porcine circovirus (PCV), a particularly environmentally robust and difficult to inactivate virus which at the time of SAF population was epidemic in the US commercial swine production industry. Two outbreaks of PCV infection were subsequently detected during sentinel testing. The first occurred several weeks after PCV-negative animals were moved under quarantine from the nursery into an animal holding room. The apparent origin of PCV was newly installed stainless steel penning, which was not sufficiently degreased thereby protecting viral particles from disinfection. The second outbreak was apparently transmitted via employee activities in the Caesarian-section suite adjacent to the barrier facility. In both cases, PCV was contained in the animal holding room where it was diagnosed making a complete facility depopulation-repopulation unnecessary. METHOD: Infectious PCV was eliminated during both outbreaks by the following: euthanizing infected animals, disposing of all removable items from the affected animal holding room, extensive cleaning with detergents and degreasing agents, sterilization of equipment and rooms with chlorine dioxide, vaporized hydrogen peroxide, and potassium peroxymonosulfate, and for the second outbreak also glutaraldehyde/quaternary ammonium. Impact on other barrier animals throughout the process was monitored by frequent PCV diagnostic testing. RESULT: After close monitoring for 6 months indicating PCV absence from all rooms and animals, herd animals were removed from quarantine status. CONCLUSION: Ten years after PCV clearance following the second outbreak, due to strict adherence to biosecurity protocols and based on ongoing sentinel diagnostic monitoring (currently monthly), the herd remains DPF including PCV negative.

Reduced antigen presentation capability and modified inflammatory/immunosuppressive cytokine expression of induced monocyte-derived dendritic cells from peripheral blood of piglets infected with porcine circovirus type 2.

The efficiency of immune responses and host defense against pathogens largely depends on the function of dendritic cells (DCs). Porcine circovirus type 2 (PCV2) infection causes viremia and extensive modulation of immune activities in the blood. The objective of the present study was to investigate the effects of PCV2 infection in vivo on the immunological function of DCs induced from peripheral blood monocytes (MoDCs). At different points after infection with PCV2, peripheral blood monocytes from PCV2-infected pigs were used to induce differentiation of DCs in vitro. Flow cytometry and quantitative real-time reverse transcription PCR were conducted to detect mRNA expression of surface markers related to antigen presentation and inflammatory/immunosuppressive cytokines of the induced MoDCs. The ability of induced MoDCs to stimulate T cells was measured using an MTS assay. In the early phase of infection at 3 days post-inoculation (DPI), IL-10, IL-8 and MIP-1ß in MoDCs were upregulated significantly. By the peak of virus proliferation at 7 DPI, antigen presentation molecules SLA-DR (MHC II) and CD80/86 together with cytokines IL-12 and IL-10 had decreased, accompanied by a rapid reduction of IL-8 and MIP-1ß. The T cell stimulation index of induced MoDCs in PCV2 groups after different infection times declined to some extent, with a significant difference at 7 DPI. PCV2 infection in vivo functionally reduced the antigen presentation capability of induced MoDCs from peripheral blood and modified expression of inflammatory/immunosuppressive cytokines that may be related to PCV2-induced immunosuppression.

Co-infection with porcine bocavirus and porcine circovirus 2 affects inflammatory cytokine production and tight junctions of IPEC-J2 cells.

Porcine bocavirus (PBoV) has a high prevalence in both healthy and diseased swine around the world. It was recently reported that PBoV and porcine circovirus type 2 (PCV2)-which contribute to porcine diarrheal disease-have a high rate of co-infection. To clarify the pathogenesis of PBoV, we examined the co-infection rate and effects of these two pathogens in IPEC-J2 porcine intestinal enterocytes. Both single and co-infection had cytopathic effects in IPEC-J2 cells. The apoptosis and proliferation rates of cells infected with both viruses did not differ significantly from those of cells infected with either one alone. PBoV and PCV2 induced the upregulation of inflammatory cytokines and the downregulation of the tight junction proteins occludin and claudin 1 in the early stage of infection, leading to destruction of epithelial barrier integrity and enhanced cytotoxicity. These findings provide insight into the pathogenic mechanisms of PBoV and PCV2 and a basis for developing effective strategies to prevent the spread of gastrointestinal diseases in pigs and other livestock.