Beak and feather disease virus (BFDV) persists in tissues of asymptomatic wild Crimson Rosellas.

Infectious diseases can drive populations and species to extinction. Beak and feather disease virus (BFDV) is a circovirus of global conservation concern that can infect all Psittaciformes and some other species. Yet some parrot species, such as Crimson rosellas (Platycercus elegans), can live successfully with high BFDV prevalence (>40%) with no clinical signs reported in infected individuals. We assessed BFDV load in 10-12 tissues per bird, from n = 66 P. elegans, to reveal tissue tropism and BFDV persistence in tissues. Here we show that in 94% of individuals, BFDV was detected in one or more tissues. While BFDV replicated to high levels in subadults, in adults (some confirmed seropositive) the virus persisted in various tissues at much lower levels. Our findings reveal that BFDV is much more common in wild P. elegans than previously thought and suggest that current screening practices (mostly on blood) may substantially underestimate BFDV infection estimates, with implications for biosecurity and conservation programs globally.

Isolation of porcine circovirus 3 using primary porcine bone marrow-derived cells.

Porcine circovirus 3 (PCV3) was first reported in the United States in 2016; this virus is considered to be involved in diverse pathologies, such as multisystem inflammation, porcine dermatitis and nephropathy syndrome, and reproductive disorders. However, successful isolation of PCV3 using cultured cells has been rare. In this study, we aimed to isolate PCV3 using primary porcine bone marrow-derived cells. Mononuclear cells were isolated from the femur bones of clinically healthy pigs. These primary cells were cultured for 6-10 days post-seeding and infected with PCV3-containing tissue homogenates. The cells were cultured for up to 37 days, and the culture medium was changed every 3-4 days. The growth curve of PCV3 in porcine bone marrow cells revealed a decline in growth during the first 10 days post-infection, followed by an increase leading to > 1010 genomic copies/mL of the cell culture supernatant; moreover, the virus was capable of passaging. The indirect fluorescent antibody assay for PCV3 infection revealed the presence of PCV3 capsid protein in the cytoplasm and nuclei of infected cells. Bone marrow cells were passaged for more than 20 generations (over 5 months), and PCV3 persistently infected the cells. PCV3-infected bone marrow cells expressed mesenchymal markers. These results reflect that primary porcine bone marrow-derived mesenchymal cells are permissive to PCV3 and continuously replicate a high copy number of the PCV3 genome. These findings regarding the high replication rate of PCV3 in bone marrow-derived mesenchymal cells could enhance our understanding of PCV3 pathogenicity.

E3 ligase RNF2 inhibits porcine circovirus type 3 replication by targeting its capsid protein for ubiquitination-dependent degradation.

Porcine circovirus type 3 (PCV3) is closely associated with various diseases, such as the porcine dermatitis, nephropathy syndrome, and multisystemic clinicopathological diseases. PCV3-associated diseases are increasingly recognized as severe diseases in the global swine industry. Ring finger protein 2 (RNF2), an E3 ubiquitin ligase exclusively located in the nucleus, contributes to various biological processes. This ligase interacts with the PCV3 Cap. However, its role in PCV3 replication remains unclear. This study confirmed that the nuclear localization signal domain of the Cap and the RNF2 N-terminal RING domain facilitate the interaction between the Cap and RNF2. Furthermore, RNF2 promoted the binding of K48-linked polyubiquitination chains to lysine at positions 139 and 140 (K139 and K140) of the PCV3 Cap, thereby degrading the Cap. RNF2 knockdown and overexpression increased or decreased PCV3 replication, respectively. Moreover, the RING domain-deleted RNF2 mutant eliminated the RNF2-induced degradation of the PCV3 Cap and RNF2-mediated inhibition of viral replication. This indicates that both processes were associated with its E3 ligase activity. Our findings demonstrate that RNF2 can interact with and degrade the PCV3 Cap via its N-terminal RING domain in a ubiquitination-dependent manner, thereby inhibiting PCV3 replication.IMPORTANCEPorcine circovirus type 3 is a recently described pathogen that is prevalent worldwide, causing substantial economic losses to the swine industry. However, the mechanisms through which host proteins regulate its replication remain unclear. Here, we demonstrate that ring finger protein 2 inhibits porcine circovirus type 3 replication by interacting with and degrading the Cap of this pathogen in a ubiquitination-dependent manner, requiring its N-terminal RING domain. Ring finger protein 2-mediated degradation of the Cap relies on its E3 ligase activity and the simultaneous existence of K139 and K140 within the Cap. These findings reveal the mechanism by which this protein interacts with and degrades the Cap to inhibit porcine circovirus type 3 replication. This consequently provides novel insights into porcine circovirus type 3 pathogenesis and facilitates the development of preventative measures against this pathogen.

Importance of sequential infection order of porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus to divergent clinical outcomes.

This study was designed to investigate the different sequential order of infection for porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV). Thirty-six pigs were randomly assigned to six different treatment groups. The first (hereafter referred to as PRRSV-PCV2) group was inoculated with PRRSV first followed by PCV2d. The second (hereafter referred to as PCV2+PRRSV) group was co-infected with both viruses at the same timepoint (42 days of age). The third (hereafter referred to as PCV2-PRRSV) group was inoculated with PCV2d first followed by PRRSV. A fourth group was only inoculated with PCV2d at 42 days of age, while a fifth group was only inoculated with PRRSV at the same timepoint. The sixth group served as a negative control group. The most important observation discovered that PRRSV only had a potentiation effect on PCV2 in both PRRS-PCV2 and PCV2+PRRSV groups. Both PRRSV-PCV2 and PCV2+PRRSV groups experienced a significant reduction in growth performance compared with control pigs. In addition, PRRSV-PCV2 and PCV2+PRRSV groups exhibited a greater severity in their clinical signs, and/or had higher PCV2 blood and lymphoid viral loads that resulted in a stronger severity of lymphoid lesions compared with PCV2-PRRSV group. Serum TNF-α levels were significantly higher in both PRRS-PCV2 and PCV2+PRRSV groups compared with those in PCV2-PRRS, PCV2, and PRRSV groups. The results of this study demonstrated that divergent clinical outcomes are dependent on the sequential infection order of PCV2 and PRRSV.

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.

Porcine circovirus type 2 ORF5 induces an inflammatory response by up-regulating miR-21 levels through targeting nuclear ssc-miR-30d.

Porcine circovirus type 2 (PCV2) infection leads to multi-system inflammation in pigs, and this effect can be achieved by upregulating host miR-21. The underlying mechanism of miR-21 regulates PCV2-induced inflammation is already known, however, how PCV2 regulates miR-21 levels and function using both autonomic and host factors remains to be further revealed. Here we present the first evidence that PCV2 ORF5 induces an inflammatory response by up-regulating miR-21 level through targeting nuclear miR-30d. In this study, we found that overexpression of ORF5 significantly increased miR-21 level and promoted the expression of inflammatory cytokines and activation of the NF-κB pathway, while ORF5 mutation had the opposite effect. Moreover, the differential expression of miR-21 could significantly change the pro-inflammatory effect of ORF5, indicating that ORF5 promotes inflammatory response by up-regulating miR-21. Bioinformatics analysis and clinical detection found that nuclear miR-30d was significantly down-regulated after ORF5 overexpression and PCV2 infection, and targeted pri-miR-21 and PCV2 ORF5. Functionally, we found that miR-30d inhibited the levels of miR-21 and inflammatory cytokines in cells. Mechanistically, we demonstrated that ORF5 inhibits miR-30d expression levels through direct binding but not via the circRNA pathway, and miR-30d inhibits miR-21 levels by targeting pri-miR-21. In summary, the present study revealed the molecular mechanism of ORF5 upregulation of miR-21, further refined the molecular chain of PCV2-induced inflammatory response and elucidated the role of miRNAs in it.

PCV2 Induced Endothelial Derived IL-8 Affects MoDCs Maturation Mainly via NF-κB Signaling Pathway.

Porcine circovirus type 2 (PCV2) infection can cause immunosuppressive diseases in pigs. Vascular endothelial cells (VECs), as the target cells for PCV2, play an important role in the immune response and inflammatory regulation. Endothelial IL-8, which is produced by porcine hip artery endothelial cells (PIECs) infected with PCV2, can inhibit the maturation of monocyte-derived dendritic cells (MoDCs). Here, we established a co-culture system of MoDCs and different groups of PIECs to further investigate the PCV2-induced endothelial IL-8 signaling pathway that drives the inhibition of MoDC maturation. The differentially expressed genes related to MoDC maturation were mainly enriched in the NF-κB and JAK2-STAT3 signaling pathways. Both the NF-κB related factor RELA and JAK2-STAT3 signaling pathway related factors (IL2RA, JAK, STAT2, STAT5, IL23A, IL7, etc.) decreased significantly in the IL-8 up-regulated group, and increased significantly in the down-regulated group. The expression of NF-κB p65 in the IL-8 up-regulated group was reduced significantly, and the expression of IκBα was increased significantly. Nuclear translocation of NF-κB p65 was inhibited, while the nuclear translocation of p-STAT3 was increased in MoDCs in the PCV2-induced endothelial IL-8 group. The results of treatment with NF-κB signaling pathway inhibitors showed that the maturation of MoDCs was inhibited and the expression of IL-12 and GM-CSF at mRNA level were lower. Inhibition of the JAK2-STAT3 signaling pathway had no significant effect on maturation, and the expression of IL-12 and GM-CSF at mRNA level produced no significant change. In summary, the NF-κB signaling pathway is the main signaling pathway of MoDC maturation, and is inhibited by the PCV2-induced up-regulation of endothelial-derived IL-8.

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.

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.

Porcine circovirus type 2 and Glaesserella parasuis serotype 4 co-infection activates Snail1 to disrupt the intercellular junctions and facilitate bacteria translocation across the tracheal epithelium.

Clinically, 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. However, the mechanism of PCV2 and G. parasuis serotype 4 (GPS4) co-infection is still not fully understood. In this study, swine tracheal epithelial cells (STEC) were used as a barrier model, and our results showed that PCV2 infection increased the adhesion of GPS4 to STEC, while decreasing the levels of ZO-1, Occludin and increasing tracheal epithelial permeability, and ultimately facilitated GPS4 translocation. Snail1 is a transcriptional repressor, and has been known to induce epithelial-to-mesenchymal transition (EMT) during development or in cancer metastasis. Importantly, we found that Snail1, as a transcriptional repressor, was crucial in destroying the tracheal epithelial barrier induced by PCV2, GPS4, PCV2 and GPS4 coinfection. For the first time, we found that PCV2, GPS4, PCV2 and GPS4 coinfection cross-activates TGF-ß and p38/MAPK signaling pathways to upregulate the expression of Snail1, down-regulate the levels of ZO-1 and Occludin, and thus disrupt the integrity of tracheal epithelial barrier then promoting GPS4 translocation. Finally, PCV2 and GPS4 co-infection also can activate TGF-ß and p38/MAPK signaling pathways in vivo and upregulate Snail1, ultimately down-regulating the expression of ZO-1 and Occludin. Our study elucidates how PCV2 infection promotes GPS4 to breach the tracheal epithelial barrier and aggravate clinical manifestations.

TGF-ß from the Porcine Intestinal Cell Line IPEC-J2 Induced by Porcine Circovirus 2 Increases the Frequency of Treg Cells via the Activation of ERK (in CD4+ T Cells) and NF-κB (in IPEC-J2).

Porcine circovirus 2 (PCV2) causes immunosuppression. Piglets infected with PCV2 can develop enteritis. Given that the gut is the largest immune organ, however, the response of the gut's immune system to PCV2 is still unclear. Here, IPEC-J2 cells with different treatments were co-cultured with PBMC or CD4+ T cells (Transwell). Flow cytometry and Western blotting revealed that PCV2-infected IPEC-J2 increased the frequency of CD4+ T cells among piglets' peripheral blood mononuclear cells (PBMCs) and caused CD4+ T cells to undergo a transformation into Foxp3+ regulatory T cells (Treg cells) via activating CD4+ T ERK. Cytokines production and an inhibitor assay showed that the induction of Tregs by PCV2-infected IPEC-J2 was dependent on TGF-ß induced by PCV2 in IPEC-J2, which was associated with the activation of NF-κB. Taken together, PCV2-infected IPEC-J2 activated NF-κB to stimulate the synthesis of TGF-ß, which enhanced the differentiation of CD4+ T cells into Treg cells through the activation of ERK in CD4+ T cells. This information sheds light on PCV2's function in the intestinal immune system and suggests a potential immunosuppressive mechanism for PCV2 infection.

Porcine Circovirus 2 Activates the PERK-Reactive Oxygen Species Axis To Induce p53 Phosphorylation with Subsequent Cell Cycle Arrest at S Phase in Favor of Its Replication.

Porcine circovirus type 2 (PCV2), the causative agent of porcine circovirus-associated diseases (PCVAD), is known to induce oxidative stress, activate p53 with induction of cell cycle arrest, and trigger the PERK (protein kinase R-like endoplasmic reticulum kinase) branch of the endoplasmic reticulum (ER) stress pathway. All these cellular responses could enhance PCV2 replication. However, it remains unknown whether PERK activation by PCV2 is involved in p53 signaling with subsequent changes of cell cycle. Here, we demonstrate that PCV2 infection induced cell cycle arrest at S phase to favor its replication via the PERK-reactive oxygen species (ROS)-p53 nexus. PCV2 infection promoted phosphorylation of p53 (p-p53) at Ser15 in porcine alveolar macrophages. Inhibition of PERK by RNA silencing downregulated total p53 (t-p53) and p-p53. Treatment with the MDM2 inhibitor nutlin-3 led to partial recovery of t-p53 in perk-silenced and PCV2-infected cells. perk silencing markedly downregulated ROS production. Scavenging of ROS with N-acetylcysteine (NAC) of PCV2-infected cells downregulated t-p53 and p-p53. Increased accumulation of p-p53 in the nuclei during PCV2 infection could be downregulated by silencing of perk or NAC treatment. Further studies showed that perk silencing or NAC treatment alleviated S phase accumulation and downregulated cyclins E1 and A2 in PCV2-infected cells. These findings indicate that the PCV2-activated PERK-ROS axis promotes p-p53 and contributes to cell cycle accumulation at S phase when more cellular enzymes are available to favor viral DNA synthesis. Overall, our study provides a novel insight into the mechanism how PCV2 manipulates the host PERK-ROS-p53 signaling nexus to benefit its own replication via cell cycle arrest. IMPORTANCE Coinfections or noninfectious triggers have long been considered to potentiate PCV2 infection, leading to manifestation of PCVAD. The triggering mechanisms remain largely unknown. Recent studies have revealed that PERK-mediated ER stress, oxidative stress, and cell cycle arrest during PCV2 infection are conducive to viral replication. However, how PCV2 employs such host cell responses requires further research. Here, we provide a novel mechanism of PCV2-induced ER stress and enhanced viral replication: the PCV2-activated PERK-ROS-p53 nexus increases S phase cell population, a cell cycle period of DNA synthesis favorable for PCV2 replication. The fact that PCV2 deploys the simple ROS molecules to activate p53 to benefit its replication provides novel insights into the triggering factors, that is, certain stimuli or management measures that induce ER stress with subsequent generation of ROS would exacerbate PCVAD. Use of antioxidants is justified on farms where PCVAD is severe.

Synergistic Pathogenicity by Coinfection and Sequential Infection with NADC30-like PRRSV and PCV2 in Post-Weaned Pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCVs) are two major viruses that affect pigs. Coinfections between PRRSV and PCV2 are frequently reported in most outbreaks, with clinical presentations involving dyspnea, fever, reduced feed intake, weight loss, and death in fattening pigs. The NADC30-like PRRSV and PCV2d are the main circulating virus strains found in China. This study determines the impact of NADC30-like PRRSV and PCV2d mono-infection and coinfection on the immune system, organ pathology, and viral shedding in five-week-old post-weaned pigs. Pigs were randomly divided into six groups: PBS, PRRSV, PCV2, PRRSV-PCV2 coinfection (co), and PRRSV-PCV2 or PCV2-PRRSV sequential infections. Fever, dyspnea, decreased feed intake, weight loss, and pig deaths occurred in groups infected with PRRSV, Co-PRRSV-PCV2, and PRRSV-PCV2. The viral load was higher in Co-PRRSV-PCV2, PRRSV-PCV2, and PCV2-PRRSV than those mono-infected with PRRSV or PCV2. Additionally, cytokines (IFN-γ, TNF-α, IL-4, and IL-10) produced by pigs under Co-PRRSV-PCV2 and PRRSV-PCV2 groups were more intense than the other groups. Necropsy findings showed hemorrhage, emphysema, and pulmonary adhesions in the lungs of pigs infected with PRRSV. Smaller alveoli and widened lung interstitium were found in the Co-PRRSV-PCV2 and PRRSV-PCV2 groups. In conclusion, PRRSV and PCV2 coinfection and sequential infection significantly increased viral pathogenicity and cytokine responses, resulting in severe clinical signs, lung pathology, and death.

Comprehensive Analysis of Codon Usage Patterns in Chinese Porcine Circoviruses Based on Their Major Protein-Coding Sequences.

Porcine circoviruses (PCVs) are distributed in swine herds worldwide and represent a threat to the health of domestic pigs and the profits of the swine industry. Currently, four PCV species, including PCV-1, PCV-2, PCV-3 and PCV-4, have been identified in China. Considering the ubiquitous characteristic of PCVs, the new emerged PCV-4 and the large scale of swine breeding in China, an overall analysis on codon usage bias for Chinese PCV sequences was performed by using the major proteins coding sequences (ORF1 and ORF2) to better understand the relationship of these viruses with their host. The data from genome nucleotide frequency composition and relative synonymous codon usage (RSCU) analysis revealed an overrepresentation of AT pair and the existence of a certain codon usage bias in all PCVs. However, the values of an effective number of codons (ENC) revealed that the bias was of low magnitude. Principal component analysis, ENC-plot, parity rule two analysis and correlation analysis suggested that natural selection and mutation pressure were both involved in the shaping of the codon usage patterns of PCVs. However, a neutrality plot revealed a stronger effect of natural selection than mutation pressure on codon usage patterns. Good host adaptation was also shown by the codon adaptation index analysis for all these viruses. Interestingly, obtained data suggest that PCV-4 might be more adapted to its host compared to other PCVs. The present study obtained insights into the codon usage pattern of PCVs based on ORF1 and ORF2, which further helps the understanding the molecular evolution of these swine viruses.

Harnessing the Genetic Plasticity of Porcine Circovirus Type 2 to Target Suicidal Replication.

Porcine circovirus type 2 (PCV2), the causative agent of a wasting disease in weanling piglets, has periodically evolved into several new subtypes since its discovery, indicating that the efficacy of current vaccines can be improved. Although a DNA virus, the mutation rates of PCV2 resemble RNA viruses. The hypothesis that recoding of selected serine and leucine codons in the PCV2b capsid gene could result in stop codons due to mutations occurring during viral replication and thus result in rapid attenuation was tested. Vaccination of weanling pigs with the suicidal vaccine constructs elicited strong virus-neutralizing antibody responses. Vaccination prevented lesions, body-weight loss, and viral replication on challenge with a heterologous PCV2d strain. The suicidal PCV2 vaccine construct was not detectable in the sera of vaccinated pigs at 14 days post-vaccination, indicating that the attenuated vaccine was very safe. Exposure of the modified virus to immune selection pressure with sub-neutralizing levels of antibodies resulted in 5 of the 22 target codons mutating to a stop signal. Thus, the described approach for the rapid attenuation of PCV2 was both effective and safe. It can be readily adapted to newly emerging viruses with high mutation rates to meet the current need for improved platforms for rapid-response vaccines.

The Isolated in Utero Environment Is Conducive to the Emergence of RNA and DNA Virus Variants.

The host's immune status may affect virus evolution. Little is known about how developing fetal and placental immune milieus affect virus heterogeneity. This knowledge will help us better understand intra-host virus evolution and how new virus variants emerge. The goal of our study was to find out whether the isolated in utero environment-an environment with specialized placental immunity and developing fetal immunity-supports the emergence of RNA and DNA virus variants. We used well-established porcine models for isolated Zika virus (RNA virus) and porcine circovirus 2 (DNA virus) fetal infections. We found that the isolated in utero environment was conducive to the emergence of RNA and DNA virus variants. Next-generation sequencing of nearly whole virus genomes and validated bioinformatics pipelines identified both unique and convergent single nucleotide variations in virus genomes isolated from different fetuses. Zika virus and PCV2 in utero evolution also resulted in single nucleotide variations previously reported in the human and porcine field samples. These findings should encourage further studies on virus evolution in placenta and fetuses, to better understand how virus variants emerge and how in utero viral evolution affects congenital virus transmission and pathogenicity.

Viruses Infecting the European Catfish (Silurus glanis).

In aquaculture, disease management and pathogen control are key for a successful fish farming industry. In past years, European catfish farming has been flourishing. However, devastating fish pathogens including limiting fish viruses are considered a big threat to further expanding of the industry. Even though mainly the ranavirus (Iridoviridea) and circovirus (Circoviridea) infections are considered well- described in European catfish, more other agents including herpes-, rhabdo or papillomaviruses are also observed in the tissues of catfish with or without any symptoms. The etiological role of these viruses has been unclear until now. Hence, there is a requisite for more detailed information about the latter and the development of preventive and therapeutic approaches to complete them. In this review, we summarize recent knowledge about viruses that affect the European catfish and describe their origin, distribution, molecular characterisation, and phylogenetic classification. We also highlight the knowledge gaps, which need more in-depth investigations in the future.

Mutation of the sixth amino acid of the Rep protein has no effect on porcine circovirus 2b but enhances porcine circovirus 2d replication in vitro.

Porcine circovirus type 2 (PCV2) is the etiological agent of post-weaning multisystemic wasting syndrome (PMWS). The original prevalent genotype, PCV2a, has been replaced by genotypes 2b and 2d in the swine population worldwide. The Rep protein is critical for viral replication. Comparison of a large number of Rep protein amino acid (aa) sequences showed that three sites distinguish genotype 2b from genotype 2d. In order to analyze the effect of exchanging the amino acids (asparagine and serine) at position 6 in the Rep proteins of PCV2b and PCV2d, two wild-type and two mutant viruses were rescued. Real-time quantitative PCR and a one-step growth curve were used to determine the viral load to assess the replication ability of the rescued viruses. The results showed that there was no significant difference in in vitro performance between the wild-type PCV2b and the mutated virus, while the mutation of PCV2d enhanced viral replication.

First detection and phylogenetic analysis of porcine circovirus 3 in female donkeys with reproductive disorders.

BACKGROUND: PCV3 is a pathogen associated with porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, reproductive failure, and cardiac and multiorgan inflammation, which was newly identified in 2016 in sows in USA. Recently, PCV3 has also been identified from several non-porcine species like (cattle, dog, wild boar, deer, mice and ticks). However, PCV3 infection in donkey is not well established. Since 2019, 300 blood samples were collected from female donkey, which was characterized by abortion and sterility, in Liaocheng city of China. RESULTS: In the present study, an investigation of PCV3 in donkey blood samples was undertaken employing by real time PCR. Positive rates of PCV3 in donkeys reach to 21.0 %. In addition, one full-length PCV3 genome sequence was obtained, and it had a highest identity with porcine circovirus 3 PCV3/CN/Nanjing2017 strain and is clustered to PCV3a genotype based on ORF2 sequences. CONCLUSIONS: This is the first report of detection of PCV3 from female donkeys presenting reproductive failure in large-scale donkey farms, China. In addition, the PCV3 strain identified in this study shared the closest relationship with those from porcine, suggesting that PCV3 may be transmitted from pigs to donkeys. Totally, PCV3 infection in donkey should be concerned although the association between it and reproductive failure are not better understood.

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.