Postweaning Multisystemic Wasting Syndrome Research Articles

Macrophage Polarization Modulated by Porcine Circovirus Type 2 Facilitates Bacterial Coinfection.

Polarization of macrophages to different functional states is important for mounting responses against pathogen infections. Macrophages are the major target cells of porcine circovirus type 2 (PCV2), which is the primary causative agent of porcine circovirus–associated disease (PCVAD) leading to immense economic losses in the global swine industry. Clinically, PCV2 is often found to increase risk of other pathogenic infections yet the underlying mechanisms remain to be elusive. Here we found that PCV2 infection skewed macrophages toward a M1 status through reprogramming expression of a subset of M1-associated genes and M2-associated genes. Mechanistically, induction of M1-associated genes by PCV2 infection is dependent on activation of nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK) signaling pathways whereas suppression of M2-associated genes by PCV2 is via inhibiting expression of jumonji domain containing-3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase that regulates M2 activation of macrophages. Finally, we identified that PCV2 capsid protein (Cap) directly inhibits JMJD3 transcription to restrain expression of interferon regulatory factor (IRF4) that controls M2 macrophage polarization. Consequently, sustained infection of PCV2 facilitates bacterial infection in vitro. In summary, these findings showed that PCV2 infection functionally modulated M1 macrophage polarization via targeting canonical signals and epigenetic histone modification, which contributes to bacterial coinfection and virial pathogenesis.

Porcine Circovirus 2 Manipulates the PERK-ERO1α Axis of the Endoplasmic Reticulum To Favor Its Replication by Derepressing Viral DNA from HMGB1 Sequestration within Nuclei.

Porcine circovirus type 2 (PCV2) causes several disease syndromes in grower pigs. PCV2 infection triggers endoplasmic reticulum (ER) stress, autophagy, and oxidative stress, all of which support PCV2 replication. We have recently reported that nuclear HMGB1 is an anti-PCV2 factor by binding to viral genomic DNA. However, how PCV2 manipulates host cell responses to favor its replication has not been explored. Here, we demonstrate that PCV2 infection increased expression of ERO1α, generation of reactive oxygen species (ROS), and nucleocytoplasmic migration of HMGB1 via protein kinase R-like endoplasmic reticulum kinase (PERK) activation in PK-15 cells. Inhibition of PERK or ERO1α repressed ROS production in PCV2-infected cells and increased HMGB1 retention within nuclei. These findings indicate that PCV2-induced activation of the PERK-ERO1α axis would lead to enhanced generation of ROS sufficient to decrease HMGB1 retention in the nuclei, thus derepressing viral DNA from HMGB1 sequestration. The viral Rep and Cap proteins were able to induce PERK-ERO1α-mediated ROS accumulation. Cysteine residues 107 and 305 of Rep or 108 of Cap played important roles in PCV2-induced PERK activation and distribution of HMGB1. Of the mutant viruses, only the mutant PCV2 with substitution of all three cysteine residues failed to activate PERK with reduced ROS generation and decreased nucleocytoplasmic migration of HMGB1. Collectively, this study offers novel insight into the mechanism of enhanced viral replication in which PCV2 manipulates ER to perturb its redox homeostasis via the PERK-ERO1α axis, and the ER-sourced ROS from oxidative folding is sufficient to reduce HMGB1 retention in the nuclei-hence the release of HMGB1-bound viral DNA for replication. IMPORTANCE Considering the fact that clinical porcine circovirus-associated diseases (PCVAD) mostly results from activation of latent PCV2 infection by confounding factors such as coinfection or environmental stresses, we propose that such confounding factors might impose oxidative stress to the animals, where PCV2 in infected cells might utilize the elevated reactive oxygen species (ROS) to promote HMGB1 migration out of nuclei in favor of its replication. An animal infection model with a particular stressor could be approached with or without antioxidant treatment to examine the relationship among the stressor, ROS level, HMGB1 distribution in target tissues, virus replication, and severity of PCVAD. This will help decide the use of antioxidants in the feeding regime on pig farms that suffer from PCVAD. Further investigation could examine if similar strategies are employed by DNA viruses, such as PCV3 and BFDV and if there is cross talk among endoplasmic reticulum (ER) stress, autophagy/mitophagy, and mitochondrial-sourced ROS in favor of PCV2 replication.

Detection of porcine circovirus type 2 (PCV2) in the Philippines and the complexity of PCV2-associated disease diagnosis.

Porcine circovirus type 2 (PCV2), an important pig viral pathogen, can cause porcine circovirus-associated disease (PCVAD), resulting in economic losses associated with decreased growth and mortalities. The diagnosis of PCVAD is complex requiring clinical, pathological and virological approaches. This study assessed PCV2 infection using histopathology and immunohistochemistry (IHC) on tissue samples and quantitative polymerase chain reaction (qPCR) on serum samples from 47 grower-finisher pigs allocated in three clinical groups in the Philippines. Typical PCV2 histopathological lesions were observed in mediastinal lymph nodes (MLN) of eight of 47 pigs. Lymphoid depletion was seen in all eight pigs and granulomatous inflammation in one of these pigs. Four of these eight pigs were PCV2 positive by both IHC and qPCR. IHC revealed PCV2 antigen in 8 pigs in at least one of the following tissues: MLN (5/8), spleen (3/8), tonsils (4/8) and lungs (5/8). PCV2 antigen was observed in 3/8 MLN with lymphoid depletion and in one MLN with depletion and granulomatous inflammation. The qPCR test showed that 33 sera had a non-detectable level, twelve had < 106 and two had > 106 PCV2 DNA copies/ml serum. One pig with lymphoid depletion had > 106 PCV2 DNA copies/ml serum, and another pig without MLN lesions also had > 106 PCV2 DNA copies/ml serum. These findings suggest that PCVAD is present in the Philippines and confirm the challenges of PCVAD diagnosis as different patterns of results were obtained from the different tests.

Porcine circovirus type 2 (PCV2) infection in Hebei Province from 2016 to 2019: a retrospective study

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in swine, the most common of which are postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). To investigate the prevalence and genetic diversity of PCV2 in Hebei Province, Northern China, from 2016 to 2019, a total of 448 suspected cases of PCV2 infection were studied, and 179 samples were positive for PCV2. A pathological and histopathological examination suggested PCV2 to be cause of the observed lesions. Phylogenetic analysis showed that four genotypes were prevalent in Hebei Province: PCV2a, 2b, 2d, and 2e. Analysis of PCV2 strains using RDP4 and SimPlot showed that there were genetic recombination events among PCV2 strains in Hebei Province. A total of 3284 serum samples were screened by ELISA, and the positive rate of PCV2 antibodies was 73.9% (2428/3284). This study provides a scientific reference for the prevention and treatment of PCV2 in Hebei Province.

AlphaB-crystallin promotes porcine circovirus type 2 replication in a cell proliferation-dependent manner

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) and causes heavy economic losses to the porcine industry worldwide. In this study, PK-15 cells were infected with PCV2 for 48 h, then harvested and subjected to label-free quantitative proteomic mass spectrometry. In total, 1212 proteins were differentially expressed in PCV2-infected cells compared with mock-infected cells, including 796 upregulated and 416 downregulated proteins. Gene ontology analysis showed that these differentially expressed proteins were involved in biological processes, cellular components and molecular functions, and these categories included cellular processes, environmental information processing, genetic information processing, disease, metabolism, and body systems. Enrichment analysis of the KEGG pathway showed that innate immune responses were significantly enriched. AlphaB-crystallin (CRYAB) interacts with desmin and cytoplasmic actin to prevent protein misfolding and aggregation, helping to maintain cytoskeletal integrity and promoting cell proliferation. In this study, CRYAB was found to effect the replication of PCV2, as verified by qRT-PCR, TCID50 determination and western blot analysis. Overexpression of CRYAB significantly upregulated PCV2 capsid protein and increased viral titers in both PK-15 cells and culture supernatants, whereas the opposite results were obtained in CRYAB knockdown cells. Furthermore, we revealed that the promotion of PCV2 replication by CRYAB was dependent on cell proliferation. To our knowledge, this is the first report of the effect of CRYAB on PCV2 replication and our findings contribute to a greater understanding of the mechanism of PCV2 replication and pathogenesis, as well as the host’s response to PCV2 infection.

Porcine Bocavirus: A 10-Year History since Its Discovery.

Porcine bocavirus (PBoV) is a single-stranded DNA virus, belongs to the genus Bocaparvovirus of family Parvoviridae. It was discovered along with porcine circovirus 2 (PCV 2) and torque tenovirus (TTV) in the lymph nodes of pigs suffering from postweaning multisystemic wasting syndrome (PMWS) in Sweden in 2009. PBoV has been reported throughout the world, mostly in weaning piglets, and has a broad range of tissue tropism. Since PBoV is prevalent in healthy as well as clinically infected pigs and is mostly associated with coinfection with other viruses, the pathogenic nature of PBoV is still unclear. Currently, there are no cell lines available for the study of PBoV, and animal model experiments have not been described. This review summarizes the current state of knowledge about PBoV, including the epidemiology, evolution analysis, detection methods, pathogenesis and public health concerns.

Truncated Diphtheria Toxin DT390 Enhances the Humoral Immunogenicity of Porcine Circovirus Type 2 Capsid Antigen in Mice.

Porcine circovirus type 2 (PCV2) is the causative agent of PCV-associated disease, which harms the swine industry worldwide. Open reading frame 2 of PCV2 encodes the principal immunogenic capsid (Cap) protein, which induces neutralizing antibodies and protective immunity. Cap has been developed as a subunit vaccine against PCV2 infection, although its use is hindered by low immunogenicity. Here, we hypothesized that the truncated diphtheria toxin DT390 might enhance the immunogenicity of Cap. To verify this hypothesis, we fused Cap with DT390, which was expressed using the unique diphtheria toxin-resistant Pichia pastoris expression system. We assessed the immunogenicity of DT390-Cap using BALB/c mice. DT390-Cap induced significantly higher Cap-specific and neutralizing antibodies than Cap alone with or without the ISA201 adjuvant. DT390-Cap with ISA201 adjuvant induced production of more Cap-specific antibodies and neutralizing antibodies than Ingelvac CircoFLEX (positive control). DT390-Cap induced slightly higher Th2-associated interleukin-4 production than Cap alone but did not affect Th1-associated interferon-γ production. The protection study demonstrated that DT390-Cap induced more effective protective immunity than Cap alone, when challenged with PCV2. The viral loads in the lungs, liver, and thymus in mice immunized using DT390-Cap were significantly lower than in those immunized with the corresponding Cap with or without the ISA201 adjuvant. Taken together, the engineered DT390 effectively enhanced the immunogenicity and protective immunity of Cap in mice. Thus, DT390-Cap is a promising novel vaccine candidate against PCV2 infection.

Porcine circovirus type 2 infection activates NF-κB pathway and cellular inflammatory responses through circPDCD4/miR-21/PDCD4 axis in porcine kidney 15 cell

Porcine circovirus type 2 (PCV2) causes postweaning multisystemic wasting syndrome (PMWS) as well as other PCV-associated diseases (PCVADs), which seriously affect the development of the swine industry. The association between aberrant expression of microRNA-21 (miR-21) and the pathogenesis of inflammatory diseases is already known. Also, our previous study showed elevated ssc-miR-21-5p (miR-21) levels in PCV2-infected porcine kidney 15 (PK-15) cells. However, the functional expression of miR-21 in PCV2 infection is unknown. Here, we found that the miR-21 levels were substantially upregulated in PCV2-infected cells, while the expression of PDCD4 and a PDCD4-derived circRNA (circPDCD4) were downregulated compared with the noninfected cells. The results of RNA immunoprecipitation and dual-luciferase reporter (DLR) assay revealed that circPDCD4 acted as a miR-21 sponge and PDCD4 was a miR-21 target. Functionally, we found that miR-21 overexpression induced the NF-κB pathway along with inflammation in cells exposed to PCV2. Further, the overexpression of PDCD4 or circPDCD4 downregulated miR-21 levels and subsequently, attenuated the miR-21-induced activation of inflammation and the NF-κB pathway. Thus, PCV2 activated the NF-κB pathway and cellular inflammatory responses through regulating circPDCD4, miR-21, and PDCD4 in PK-15 cells.

INVESTIGATION OF EFFECTS OF PORCINE CIRCOVIRUS TYPE 2 (PCV2) VACCINATION ON CELLULAR IMMUNITY IN PIGS IN A TAIWAN PIG FARM

Porcine circovirus type 2 (PCV2) is the major cause of serious porcine circovirus-associated diseases (PCVADs) causing significant economic losses in the pig industry. The PCV2 vaccines have proved effective by lowering serum and tissue viral loads. However, little is known about its effects on the cellular functions of viral target cells in pigs. For this, blood samples from 38 vaccinated and 34 non-vaccinated, 12–14 week-old, clinically healthy and PCV2-infected pigs in a commercial farm were sampled and anticoagulated. The viral load in plasma and the cellular proliferation and mRNA expression levels of IL-1 [Formula: see text], IL-2, IL-4, IL-8, IL-10 and IFN-[Formula: see text] of peripheral blood mononuclear cells (PBMCs) with or without concanavalin A (Con A) and PCV2 stimulation in vitro were then investigated by real-time polymerase chain reaction and flow cytometry. Compared with the non-vaccinated pigs, the average viral loads in the plasma and cultivated PBMCs were reduced in the PCV2-vaccinated pigs. Significant differences in the aspects of increased cell proliferation, decreased average viral loads in PBMCs three days after Con A stimulation, increased mRNA expression levels of IL-2 and IFN-[Formula: see text] after re-exposure to PCV2, and decreased levels of IL-1 [Formula: see text], IL-4 and IL-8 after Con A or PCV2 stimulation were also noted in the PCV2-vaccinated pigs. The results suggested that the PCV2-vaccinated pigs were likely to develop better cell-mediated immunity than the non-vaccinated pigs.

Efficient influence of ssDNA virus PCV2 replication by CRISPR/Cas9 targeting of the viral genome

Porcine circovirus type 2 (PCV2), a ubiquitous pathogen that primary cause of postweaning multisystemic wasting syndrome (PMWS), had caused significant morbidity and mortality in swine populations with huge economic losses in the worldwide swine industry. Currently, looking for effective antiviral drugs for PCV2 infection remains an important works. In our study, CRISPR/Cas9 system was used to further detected the key sites of PCV2 replication. We designed 8 single guide RNAs (sgRNA) by targeting essential genes across the genome of PCV2. Western-blot(WB), Cell counting kit-8 for high-throughput sgRNA screening were applied to detect PCV2 replication levels. The results showed that Oc8, O13, O134, NQT and NPS sgRNAs can edit the PCV2 genome efficiently and inhibit PCV2 replication in PK-15 cell; H3 sgRNA cannot edit the PCV2 genome successfully; NAT sgRNA can edit the PCV2 genome efficiently to improve the PCV2 replication in PK-15 cell; O26 sgRNA can edit the PCV2 genome successfully but it is not known yet of its effect on PCV2 replication, besides the Cas9 expression had no effect on cell viability. These data suggest that CRISPR/Cas9 system targeting PCV2 essential genes may serve as a novel therapeutic agent against PCV2 infection in the future.

Emergence of porcine circovirus 2g (PCV2g) and evidence for recombination between genotypes 2g, 2b and 2d among field isolates from non-vaccinated pigs in Mizoram, India.

The molecular genetics of fourteen Porcine Circovirus 2 (PCV2) isolates from non-vaccinated pigs that died of porcine circovirus associated disease (PCVAD) between 2012 and 2019 in the Mizoram state of North East India, was studied. The PCVAD in these pigs, that had shown characteristic clinical signs and lesions associated with post-weaning multi-systemic wasting syndrome and reproductive failure was confirmed with detection of PCV2 DNA in the tissue samples. Complete viral genomes of these fourteen field isolates were sequenced following in house developed overlapping PCR. The multiple sequence alignment of viral capsid proteins or the open reading frame 2 (ORF2) sequences showed highly conserved residues known for antibody recognition and genotype specificity, however, variations were noticed in the amino acid residues previously known as important for in vitro replication of PCV2. The phylogenetic analyses based on the complete genome sequences enabled identification of genotype PCV2g (9/14, 64.29%) for the first time in India along with genotypes PCV2d (3/14, 21.43%) and PCV2b (2/14, 14.29%). Further, recombination analyses showed evidence for recombination between the genotypes 2b, 2g and 2d. This is the first report on the prevalence of genotype PCV2g and natural inter-genotypic (2g-2b, 2g-2d and 2d-2g) recombinants in India. The findings indicate a non-vaccine driven, natural genotypic shift and signify the need for routine PCV2 surveillance and genotyping. Our analyses also provide a solid ground for future studies to understand the consequences of multiple PCV2 genotypes within a pig population with respect to vaccination, diagnostics and emergence of new genotypes.

Prevalence of Porcine Circovirus Type 2 (PCV-2) in Smallholder Pig Farms in Thung-Yai, Nakhon Si Thammarat, Thailand

Porcine circovirus type 2 (PCV2), which is a member of the Circovirus genus in the family of Circoviridae, is a small non-enveloped, closed-circular ssDNA. The PCV2-associated disease is one of the most important infectious agents on pig productivity worldwide, including China, India, Malaysia, and Thailand. It caused 2 major syndromes; postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). In addition, PCV2 may cause complexity with a pathogenic agent into porcine circovirus associated diseases (PCVADs). This study aimed to determine the prevalence of PCV2 in smallholder farms in Thung Yai district, Nakhon Si Thammarat, Thailand. A cross-sectional study was performed; 100 blood samples were collected from 13 smallholder pig farms. The samples were classified into 4 groups based on the pig’s age; gilts, 6 sows, nursery-to-starter, and from growing to finishing pigs. Blood samples were carried out for DNA +extraction and nested-PCR. The epidemiological study showed 9 % positive by genetic detection. The result suggested that growing-to-finishing pigs had significant PCV2 infection, followed by nursery-to-starter pigs and sow groups. In addition, multiple farms showed a high positive and significant correlation (Cr ≈ 0.245). These results reveal a low prevalence of PCV2 in endemic regions in southern Thailand, which may help in the local control evaluation and eradication programs. Furthermore, the phylogenic study of local strain should be investigated for the occurrence of PCV2 genetic evolution in Thailand and neighboring countries.

Characterization of two newly emerged torque teno sus virus isolates from a large-scale pig farm in China, in 2018

Torque teno sus virus (TTSuV) infection is common in China's pig herd. Although of uncertain pathogenicity, TTSuVs have been reported as a worsening factor of other porcine diseases, including porcine circovirus associated disease (PCVAD), porcine respiratory diseases complex (PRDC) or porcine dermatitis and nephropathy syndrome (PDNS). To better understand the genetic diversity in TTSuVs, the complete genomes of two newly emerged isolates, referred to as HeN1-A9 and HeN1-A11, collected from pig samples at a large-scale pig farm in China, were analyzed. Phylogenetic relationships of TTSuV sequences separated TTSuV1 and TTSuVk2a groups and divided TTSuV1 into two major subtypes, including TTSuV1a and TTSuV1b; HeN1-A9 and HeN1-A11 strains classified into the TTSuV1a subtype. Recombination analysis demonstrated HeN1-A9 and HeN1-A11 were generated via recombination in the overlapping ORF1/ORF3 region of TTSuV1a genome, which we report for the first time. Furthermore, we found that HeN1-A9 could be replicated in cultured MARC-145 cells for 18 passages. Our findings may be useful for elucidating the characteristics and epidemic status of TTSuVs in China.

Epidemiological investigation of porcine circovirus type 2 and its coinfection rate in Shandong province in China from 2015 to 2018

BackgroundPorcine circovirus type 2 (PCV2) is one of the crucial swine viral pathogens, caused porcine circovirus associated diseases (PCVAD). Shandong province is one of the most important pork producing areas and bears a considerable economic loss due to PCVAD. However, there is limited information on epidemiology and coinfection rate of PCV2 with other critical swine diseases in this area, such as porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Pseudorabies virus (PRV), and porcine epidemic diarrhea virus (PEDV).ResultsOverall, 89.59% serum samples and 36.98% tissue samples were positive for PCV2 specified ELISA and PCR positive for PCV2, respectively. The coinfection rates of PCV2 with PRRSV, PRV, CSFV, and PEDV were 26.73%, 18.37%, 13.06%, and 3.47%, respectively. Moreover, genetic characteristic of PCV2 were analyzed based on the cap genes showing that PCV2d is the dominant sub-genotype circulating in the province.ConclusionsOur findings reveal that PCV2d, as the dominant strain, is prevailing in pig farms in Shandong province at high levels. There was a high frequency of coinfection of PCV2 and PRRSV.

PK15 cell line stably overexpressing IL2 enhances PCV2 replication.

Porcine circovirus type 2 (PCV2) is the primary agent responsible for porcine circovirus-associated diseases (PCVADs), which is acknowledged as one of the most economically important diseases for the swine industry worldwide. Currently, the development of PCV2 vaccine against PCVADs and for other applications require large amounts of viralparticles. The low propagation rate of PCV2 in vitro limits vaccine production. Previous studies showed that a cell line transfected with the porcine interleukin (IL)-2 gene gave higher PCV2 yield in vitro. However, transient transfection may become less effective and unstable after serial generations. In this work, we constructed a PK15 cell line with stable expression of porcine IL2 by lentivirus transfection. The results demonstrated that the transgenic cell line stably expressed IL2 protein significantly enhanced PCV2 replication. Thus, the transgenic PK15 cell line could be a promising cell line for vaccine production.

Expression and evaluation of porcine circovirus type 2 capsid protein mediated by recombinant adeno-associated virus 8.

BackgroundPorcine circovirus type 2 (PCV2) is an important infectious pathogen implicated in porcine circovirus-associated diseases (PCVAD), which has caused significant economic losses in the pig industry worldwide.ObjectivesA suitable viral vector-mediated gene transfer platform for the expression of the capsid protein (Cap) is an attractive strategy.MethodsIn the present study, a recombinant adeno-associated virus 8 (rAAV8) vector was constructed to encode Cap (Cap-rAAV) in vitro and in vivo after gene transfer.ResultsThe obtained results showed that Cap could be expressed in HEK293T cells and BABL/c mice. The results of lymphocytes proliferative, as well as immunoglobulin G (IgG) 2a and interferon-γ showed strong cellular immune responses induced by Cap-rAAV. The enzyme-linked immunosorbent assay titers obtained and the IgG1 and interleukin-4 levels showed that humoral immune responses were also induced by Cap-rAAV. Altogether, these results demonstrated that the rAAV8 vaccine Cap-rAAV can induce strong cellular and humoral immune responses, indicating a potential rAAV8 vaccine against PCV2.ConclusionsThe injection of rAAV8 encoding PCV2 Cap genes into muscle tissue can ensure long-term, continuous, and systemic expression.

Applicability of Raman spectroscopy on porcine parvovirus and porcine circovirus type 2 detection

Porcine parvovirus (PPV) is one of the major infectious causes of reproductive failure of swine. This disease is characterized by embryonic and fetal infection and death, responsible for important economic losses. PPV is also implicated as a trigger in the development of post-weaning multisystemic wasting syndrome (PMWS) caused by Porcine circovirus type 2 (PCV2). Their detection is PCR-based, which is quite sensitive and specific, but laborious, costly and time-demanding. Therefore, this study aimed to assess Raman spectroscopy (RS) as a diagnostic tool for PPV and PCV2 due to its label-free properties and unique ability to search and identify molecular fingerprints. Briefly, swine testis (ST) cells were inoculated with PPV or PCV2 and in vitro cultured (37 °C, 5% CO2) for four days. Fixed cells were then submitted to RS investigation using a 633 nm laser. A total of 225 spectra centered at 1300 cm−1 was obtained for each sample (5 spectra/cell; 15 cells/replicate; 3 replicates) of PPV-, PCV2-infected and uninfected (control) ST cells. Clear statistical discrimination between samples from both virus-infected cells was achieved with a Principal Component – Linear Discriminant Analysis (PCA-LDA) model, reaching sensitivity rates from 95.55% to 97.77%, respectively to PCV2- and PPV-infected cells. These results were then submitted to a Leave-One-Out (LOO) validation algorithm resulting in 99.97% of accuracy. Extensive band assignment was analyzed and compiled for better understanding of PPV and PCV2 virus-cell interaction, demonstrating that specific protein, lipids and DNA/RNA bands are the most important assignments related to discrimination of virus-infected from uninfected cells. In conclusion, these results represent promising bases for RS application on PCV2 and PPV detection for future diagnostic applications.

Co-infection status of novel parvovirus's (PPV2 to 4) with porcine circovirus 2 in porcine respiratory disease complex and porcine circovirus-associated disease from 1997 to 2012.

As global pig health diseases, porcine respiratory disease complex (PRDC) and porcine circovirus-associated disease (PCVAD) generate substantial economic losses despite pigs been vaccinated against the primary causative virus, highlighting the importance of understanding virome interactions and specifically co-factor infections. Established primary endemic pathogens for PRDC include porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSv) and swine influenza virus (SIV), and PCV2 aetiology in interaction with other co-infecting viruses can result in PCVAD. Porcine parvovirus (PPV) 1 is a well-characterized virus with an available vaccine preventing reproductive failure in sows. However, whilst novel PPV 2 to 7 viruses have been identified since 2001, their viral pathogenic potential in clinical and subclinical disease remains to be determined. Therefore, this study has sought to develop a better understanding of their potential role as associated co-infections in PRDC and PCVAD by examining archival samples for the presence of PCV2 and the novel parvoviruses PPV2-4 from clinically diseased pigs across production age stages. Epidemiologically, the novel PPV2 was found to be the most prevalent within the fattener age group with PPV2-4 statistically associated with pig respiratory disease and enteric ulcers. Additionally, statistical modelling by latent class analysis (LCA) on veterinary pathology scored pigs found a clustering co-factor association between PPV2 and PCV2, suggesting the novel PPV may be involved in PRDC and PCVAD. Phylogenetic analysis of novel PPVs revealed the PPV2 capsid evolution to be diverged from the original strains with a low nucleotide homology of 88%-96% between two distinct clades. These findings determine that novel PPV 2-4 viruses are statistically associated as co-infectors in a diseased pig population, and significantly detected PPV2 clustering co-infection frequency with PCV2 in PRDC and PCVAD diseased pigs through LCA analysis.

A novel PCV2 ORF5-interacting host factor YWHAB inhibits virus replication and alleviates PCV2-induced cellular response

Porcine circovirus type 2 (PCV2) infection causes porcine circovirus associated diseases (PCVAD) worldwide. Identification of host factors that interact with viral proteins is a fundamental step to understand the pathogenesis of PCV2. Our previous study reported that ORF5, a newly identified PCV2 viral protein supports PCV2 replication and interacts with multiple host factors. Here, we showed that a host factor YWHAB is an ORF5-interacting protein and plays essential roles during PCV2 infection. By using protein-protein interaction assays, we confirmed that YWHAB directly interacts with PCV2-ORF5 protein. We further showed that YWHAB expression was potently induced upon ORF5 overexpression and PCV2 infection. Remarkably, we found that the YWHAB strongly inhibited PCV2 replication, suggesting its role in defending PCV2 infection. By using the ectopic overexpression and gene knockdown approaches, we revealed that YWHAB inhibits PCV2-induced endoplasmic reticulum stress (ERS), autophagy, reactive oxygen species (ROS) production and apoptosis, suggesting its vital role in alleviating PCV2-induced cellular damage. Together, this study demonstrated that an ORF5-interacting host factor YWHAB affects PCV2 infection and PCV2-induced cellular response, which expands the current understanding of YWHAB biological function and might serves as a new therapeutic target to manage PCV2 infection-associated diseases.

Immune responses induced by a combined vaccination with a recombinant chimera of Mycoplasma hyopneumoniae antigens and capsid virus-like particles of porcine circovirus type 2

BackgroundMycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are two important pathogens causing Mycoplasma pneumonia of swine (MPS) and porcine circovirus diseases and porcine circovirus-associated diseases (PCVDs/PCVADs), respectively, and resulted in considerable economic loss to the swine industry worldwide. Currently, vaccination is one of the main measures to control these two diseases; however, there are few combination vaccines that can prevent these two diseases. To determine the effect of combination immunization, we developed capsid-derived (Cap) virus-like particles (VLPs) of PCV2 and a new recombinant chimera composed of the P97R1, P46, and P42 antigens of Mhp. Then we investigated the immune responses induced by the immunization with this combination vaccine in mice and piglets.ResultsThe high level antibodies against three protein antigens (P97R1, P46, and P42 of Mhp) were produced after immunization, up to or higher than 1:400,000; the antibody levels in Pro group continuously increased throughout the 42 days for all the antigens tested. The lymphocyte proliferative response in PCV2 group was stronger than that in PBS, VP, Mhp CV in mice. The antibody levels for Cap remained stable and reached the peak at 35 DAI. The IFN-γ and IL-4 in sera were significantly enhanced in the Pro group than that in the negative control-VP group on Day 14 and 28 post-the first immunization in piglets.ConclusionsAbove all, the combination immunization could induce humoral and cellular immune responses against all four antigens in mice and piglets. Therefore, our approach is a simple and effective vaccination strategy to protect pigs against MPS and PCVD/PCVAD.