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.

Elevation of IL-8 secretion induced by PEDV infection via NF-κB signaling pathway.

Porcine epidemic diarrhea virus (PEDV) is associated with severe enteritis, which contributes to high mortality in piglets. The aim of this study was to describe molecular mechanisms associated with proinflammatory cytokine(s) production during PEDV infection. We showed that infection of porcine intestine epithelial cell clone J2 (IPEC-J2) with PEDV induces a gradual increase in interleukin 8 (IL-8) production at different time points, as well as infection of Vero E6 with PEDV. The secretion of IL-8 in these two cell lines infected with PEDV is related to the activation of NF-κB. Furthermore, the cells expressing PEDV M or E protein can induce the upregulation of IL-8. These findings suggest that the IL-8 production can be the initiator of inflammatory response by the host cells upon PEDV infection.

Porcine deltacoronavirus nucleocapsid protein interacts with the Grb2 through its proline-rich motifs to induce activation of the Raf-MEK-ERK signal pathway and promote virus replication.

Porcine deltacoronavirus (PDCoV), an enteropathogenic coronavirus, causes severe watery diarrhoea, dehydration and high mortality in piglets, which has the potential for cross-species transmission in recent years. Growth factor receptor-bound protein 2 (Grb2) is a bridging protein that can couple cell surface receptors with intracellular signal transduction events. Here, we investigated the reciprocal regulation between Grb2 and PDCoV. It is found that Grb2 regulates PDCoV infection and promotes IFN-ß production through activating Raf/MEK/ERK/STAT3 pathway signalling in PDCoV-infected swine testis cells to suppress viral replication. PDCoV N is capable of interacting with Grb2. The proline-rich motifs in the N- or C-terminal region of PDCoV N were critical for the interaction between PDCoV-N and Grb2. Except for Deltacoronavirus PDCoV N, the Alphacoronavirus PEDV N protein could interact with Grb2 and affect the regulation of PEDV replication, while the N protein of Betacoronavirus PHEV and Gammacoronavirus AIBV could not interact with Grb2. PDCoV N promotes Grb2 degradation by K48- and K63-linked ubiquitin-proteasome pathways. Overexpression of PDCoV N impaired the Grb2-mediated activated effect on the Raf/MEK/ERK/STAT3 signal pathway. Thus, our study reveals a novel mechanism of how host protein Grb2 protein regulates viral replication and how PDCoV N escaped natural immunity by interacting with Grb2.

Network of Interactions between the Mut Domains of the E2 Protein of Atypical Porcine Pestivirus and Host Proteins.

Atypical porcine pestivirus (APPV) can cause congenital tremor type A-II in neonatal piglets, posing a significant threat to swine herd health globally. Our previous study demonstrated that the Mut domains, comprising 112 amino acids at the N-terminus, are the primary functional regions of the E2 protein of APPV. This study identified 14 host cellular proteins that exhibit potential interactions with the Mut domains of the E2 protein using yeast two-hybrid screening. Using bioinformatics analysis, we discovered that the Mut domains of the E2 protein might exert regulatory effects on apoptosis by modulating energy metabolism within the mitochondria. We also conducted co-immunoprecipitation, glutathione S-transferase pull-down, and immunofluorescence assays to confirm the interaction between the Mut domains of the E2 protein and cathepsin H and signal sequence receptor subunit 4 (SSR4). Ultimately, SSR4 enhanced APPV replication in vitro. In summary, our study successfully elucidated the interactions between the Mut domains of the E2 protein and host cell protein, predicted the potential pathways implicated in these interactions, and demonstrated SSR4 involvement in APPV infection. These significant findings contribute valuable knowledge toward a deeper understanding of APPV pathogenesis and the role of the Mut domains of the E2 protein in this intricate process.

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.

Cell-surface d-glucuronyl C5-epimerase binds to porcine deltacoronavirus spike protein facilitating viral entry.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteric coronavirus with zoonotic potential. The coronavirus spike (S) glycoprotein, especially the S1 subunit, mediates viral entry by binding to cellular receptors. However, the functional receptor of PDCoV remains poorly understood. In this study, we used the soluble PDCoV S1 protein as bait to capture the S1-binding cellular transmembrane proteins in combined immunoprecipitation and mass spectrometry analyses. A single guide RNA screen identified d-glucuronyl C5-epimerase (GLCE), a heparan sulfate-modifying enzyme, as a proviral host factor for PDCoV infection. GLCE knockout significantly inhibited the attachment and internalization stages of PDCoV infection. We also demonstrated the interaction between GLCE and PDCoV S with coimmunoprecipitation in both an overexpression system and PDCoV-infected cells. GLCE could be localized to the cell membrane, and an anti-GLCE antibody suppressed PDCoV infection. Although GLCE expression alone did not render nonpermissive cells susceptible to PDCoV infection, GLCE promoted the binding of PDCoV S to porcine amino peptidase N (pAPN), acting synergistically with pAPN to enhance PDCoV infection. In conclusion, our results demonstrate that GLCE is a novel cell-surface factor facilitating PDCoV entry and provide new insights into PDCoV infection. IMPORTANCE: The identification of viral receptors is of great significance, potentially extending our understanding of viral infection and pathogenesis. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus with the potential for cross-species transmission. However, the receptors or coreceptors of PDCoV are still poorly understood. The present study confirms that d-glucuronyl C5-epimerase (GLCE) is a positive regulator of PDCoV infection, promoting viral attachment and internalization. The anti-GLCE antibody suppressed PDCoV infection. Mechanically, GLCE interacts with PDCoV S and promotes the binding of PDCoV S to porcine amino peptidase N (pAPN), acting synergistically with pAPN to enhance PDCoV infection. This work identifies GLCE as a novel cell-surface factor facilitating PDCoV entry and paves the way for further insights into the mechanisms of PDCoV infection.

Pseudorabies virus infection increases the permeability of the mammalian respiratory barrier to facilitate Pasteurella multocida infection.

Interaction between viruses and bacteria during the development of infectious diseases is a complex question that requires continuous study. In this study, we explored the interactions between pseudorabies virus (PRV) and Pasteurella multocida (PM), which are recognized as the primary and secondary agents of porcine respiratory disease complex (PRDC), respectively. In vivo tests using mouse models demonstrated that intranasal inoculation with PRV at a sublethal dose induced disruption of murine respiratory barrier and promoted the invasion and damages caused by PM through respiratory infection. Inoculation with PRV also disrupted the barrier function of murine and porcine respiratory epithelial cells, and accelerated the adherence and invasion of PM to the cells. In mechanism, PRV infection resulted in decreased expression of tight junction proteins (ZO-1, occludin) and adherens junction proteins (ß-catenin, E-cadherin) between neighboring respiratory epithelial cells. Additionally, PRV inoculation at an early stage downregulated multiple biological processes contributing to epithelial adhesion and barrier functions while upregulating signals beneficial for respiratory barrier disruption (e.g., the HIF-1α signaling). Furthermore, PRV infection also stimulated the upregulation of cellular receptors (CAM5, ICAM2, ACAN, and DSCAM) that promote bacterial adherence. The data presented in this study provide insights into the understanding of virus-bacteria interactions in PRDC and may also contribute to understanding the mechanisms of secondary infections caused by different respiratory viruses (e.g., influenza virus and SARS-CoV-2) in both medical and veterinary medicine. IMPORTANCE: Co-infections caused by viral and bacterial agents are common in both medical and veterinary medicine, but the related mechanisms are not fully understood. This study investigated the interactions between the zoonotic pathogens PRV and PM during the development of respiratory infections in both cell and mouse models, and reported the possible mechanisms which included: (i) the primary infection of PRV may induce the disruption and/or damage of mammal respiratory barrier, thereby contributing to the invasion of PM; (ii) PRV infection at early stage accelerates the transcription and/or expression of several cellular receptors that are beneficial for bacterial adherence. This study may shed a light on understanding the mechanisms on the secondary infection of PM promoted by different respiratory viruses (e.g., influenza virus and SARS-CoV-2) in both medical and veterinary medicine.

Comparative proteomic analysis of PK-15 cells infected with wild-type strain and its EP0 gene-deleted mutant strain of pseudorabies virus.

IMPORTANCE: As one of the main etiologic agents of infectious diseases in pigs, pseudorabies virus (PRV) infections have caused enormous economic losses worldwide. EP0, one of the PRV early proteins (EP) plays a vital role in PRV infections, but the mechanisms are unclear. OBJECTIVE: This study examined the function of EP0 to provide a direction for its in-depth analysis. METHODS: In this study, the EP0-deleted PRV mutant was obtained, and Tandem Mass Tag-based proteomic analysis was used to screen the differentially expressed proteins (DEPs) quantitatively in EP0-deleted PRV- or wild-type PRV-infected porcine kidney 15 cells. RESULTS: This study identified 7,391 DEPs, including 120 and 21 up-regulated and down-regulated DEPs, respectively. Western blot analysis confirmed the changes in the expression of the selected proteins, such as speckled protein 100. Comprehensive analysis revealed 141 DEPs involved in various biological processes and molecular functions, such as transcription regulator activity, biological regulation, and localization. CONCLUSIONS AND RELEVANCE: These results holistically outlined the functions of EP0 during a PRV infection and might provide a direction for more detailed function studies of EP0 and the stimulation of lytic PRV infections.

The downregulation of genes encoding muscle proteins have a potential role in the development of scrotal hernia in pigs.

BACKGROUND: Testicular descent is a physiological process regulated by many factors. Eventually, disturbances in the embryological/fetal development path facilitate the occurrence of scrotal hernia, a congenital malformation characterized by the presence of intestinal portions within the scrotal sac due to the abnormal expansion of the inguinal ring. In pigs, some genes have been related to this anomaly, but the genetic mechanisms involved remain unclear. This study aimed to investigate the expression profile of a set of genes potentially involved with the manifestation of scrotal hernia in the inguinal ring tissue. METHODS AND RESULTS: Tissue samples from the inguinal ring/canal of normal and scrotal hernia-affected male pigs with approximately 30 days of age were used. Relative expression analysis was performed using qPCR to confirm the expression profile of 17 candidate genes previously identified in an RNA-Seq study. Among them, the Myosin heavy chain 1 (MYH1), Desmin (DES), and Troponin 1 (TNNI1) genes were differentially expressed between groups and had reduced levels of expression in the affected animals. These genes encode proteins involved in the formation of muscle tissue, which seems to be important for increasing the resistance of the inguinal ring to the abdominal pressure, which is essential to avoid the occurrence of scrotal hernia. CONCLUSIONS: The downregulation of muscular candidate genes in the inguinal tissue clarifies the genetic mechanisms involved with this anomaly in its primary site, providing useful information for developing strategies to control this malformation in pigs and other mammals.

Porcine deltacoronavirus nucleocapsid protein antagonizes JAK-STAT signaling pathway by targeting STAT1 through KPNA2 degradation.

Porcine deltacoronavirus (PDCoV) is an enteric pathogenic coronavirus that causes acute and severe watery diarrhea in piglets and has the ability of cross-species transmission, posing a great threat to swine production and public health. The interferon (IFN)-mediated signal transduction represents an important component of virus-host interactions and plays an essential role in regulating viral infection. Previous studies have suggested that multifunctional viral proteins encoded by coronaviruses antagonize the production of IFN via various means. However, the function of these viral proteins in regulating IFN-mediated signaling pathways is largely unknown. In this study, we demonstrated that PDCoV and its encoded nucleocapsid (N) protein antagonize type I IFN-mediated JAK-STAT signaling pathway. We identified that PDCoV infection stimulated but delayed the production of IFN-stimulated genes (ISGs). In addition, PDCoV inhibited JAK-STAT signal transduction by targeting the nuclear translocation of STAT1 and ISGF3 formation. Further evidence showed that PDCoV N is the essential protein involved in the inhibition of type I IFN signaling by targeting STAT1 nuclear translocation via its C-terminal domain. Mechanistically, PDCoV N targets STAT1 by interacting with it and subsequently inhibiting its nuclear translocation. Furthermore, PDCoV N inhibits STAT1 nuclear translocation by specifically targeting KPNA2 degradation through the lysosomal pathway, thereby inhibiting the activation of downstream sensors in the JAK-STAT signaling pathway. Taken together, our results reveal a novel mechanism by which PDCoV N interferes with the host antiviral response.IMPORTANCEPorcine deltacoronavirus (PDCoV) is a novel enteropathogenic coronavirus that receives increased attention and seriously threatens the pig industry and public health. Understanding the underlying mechanism of PDCoV evading the host defense during infection is essential for developing targeted drugs and effective vaccines against PDCoV. This study demonstrated that PDCoV and its encoded nucleocapsid (N) protein antagonize type I interferon signaling by targeting STAT1, which is a crucial signal sensor in the JAK-STAT signaling pathway. Further experiments suggested that PDCoV N-mediated inhibition of the STAT1 nuclear translocation involves the degradation of KPNA2, and the lysosome plays a role in KPNA2 degradation. This study provides new insights into the regulation of PDCoV N in the JAK-STAT signaling pathway and reveals a novel mechanism by which PDCoV evades the host antiviral response. The novel findings may guide us to discover new therapeutic targets and develop live attenuated vaccines for PDCoV infection.

Subcellular localization of viral proteins after porcine epidemic diarrhea virus infection and their roles in the viral life cycle.

Porcine epidemic diarrhea virus (PEDV) is one of the most devastating diseases affecting the pig industry globally. Due to the emergence of novel strains, no effective vaccines are available for prevention and control. Investigating the pathogenic mechanisms of PEDV may provide insights for creating clinical interventions. This study constructed and expressed eukaryotic expression vectors containing PEDV proteins (except NSP11) with a 3' HA tag in Vero cells. The subcellular localization of PEDV proteins was examined using endogenous protein antibodies to investigate their involvement in the viral life cycle, including endocytosis, intracellular trafficking, genome replication, energy metabolism, budding, and release. We systematically analyzed the potential roles of all PEDV viral proteins in the virus life cycle. We found that the endosome sorting complex required for transport (ESCRT) machinery may be involved in the replication and budding processes of PEDV. Our study provides insight into the molecular mechanisms underlying PEDV infection. IMPORTANCE: The global swine industry has suffered immense losses due to the spread of PEDV. Currently, there are no effective vaccines available for clinical protection. Exploring the pathogenic mechanisms of PEDV may provide valuable insights for clinical interventions. This study investigated the involvement of viral proteins in various stages of the PEDV lifecycle in the state of viral infection and identified several previously unreported interactions between viral and host proteins. These findings contribute to a better understanding of the pathogenic mechanisms underlying PEDV infection and may serve as a basis for further research and development of therapeutic strategies.

NLRX1 Mediates the Disruption of Intestinal Mucosal Function Caused by Porcine Astrovirus Infection via the Extracellular Regulated Protein Kinases/Myosin Light-Chain Kinase (ERK/MLCK) Pathway.

Porcine astrovirus (PAstV) has a potential zoonotic risk, with a high proportion of co-infection occurring with porcine epidemic diarrhea virus (PEDV) and other diarrheal pathogens. Despite its high prevalence, the cellular mechanism of PAstV pathogenesis is ill-defined. Previous proteomics analyses have revealed that the differentially expressed protein NOD-like receptor X1 (NLRX1) located in the mitochondria participates in several important antiviral signaling pathways in PAstV-4 infection, which are closely related to mitophagy. In this study, we confirmed that PAstV-4 infection significantly up-regulated NLRX1 and mitophagy in Caco-2 cells, while the silencing of NLRX1 or the treatment of mitophagy inhibitor 3-MA inhibited PAstV-4 replication. Additionally, PAstV-4 infection triggered the activation of the extracellular regulated protein kinases/ myosin light-chain kinase (ERK/MLCK) pathway, followed by the down-regulation of tight-junction proteins (occludin and ZO-1) as well as MUC-2 expression. The silencing of NLRX1 or the treatment of 3-MA inhibited myosin light-chain (MLC) phosphorylation and up-regulated occludin and ZO-1 proteins. Treatment of the ERK inhibitor PD98059 also inhibited MLC phosphorylation, while MLCK inhibitor ML-7 mitigated the down-regulation of mucosa-related protein expression induced by PAstV-4 infection. Yet, adding PD98059 or ML-7 did not affect NLRX1 expression. In summary, this study preliminarily explains that NLRX1 plays an important role in the disruption of intestinal mucosal function triggered by PAstV-4 infection via the ERK/MLC pathway. It will be helpful for further antiviral drug target screening and disease therapy.

PoRVA G9P[23] and G5P[7] infections differentially promote PEDV replication by reprogramming glutamine metabolism.

PoRVA and PEDV coinfections are extremely common in clinical practice. Although coinfections of PoRVA and PEDV are known to result in increased mortality, the underlying mechanism remains unknown. Here, we found that PoRVA infection promoted PEDV infection in vivo and in vitro and that PoRVA G9P[23] (RVA-HNNY strain) enhanced PEDV replication more significantly than did PoRVA G5P[7] (RVA-SXXA strain). Metabolomic analysis revealed that RVA-HNNY more efficiently induced an increase in the intracellular glutamine content in porcine small intestinal epithelial cells than did RVA-SXXA, which more markedly promoted ATP production to facilitate PEDV replication, whereas glutamine deprivation abrogated the effect of PoRVA infection on promoting PEDV replication. Further studies showed that PoRVA infection promoted glutamine uptake by upregulating the expression of the glutamine transporter protein SLC1A5. In SLC1A5 knockout cells, PoRVA infection neither elevated intracellular glutamine nor promoted PEDV replication. During PoRVA infection, the activity and protein expression levels of glutamine catabolism-related enzymes (GLS1 and GLUD1) were also significantly increased promoting ATP production through glutamine anaplerosis into the TCA cycle. Consistent with that, siRNAs or inhibitors of GLS1 and GLUD1 significantly inhibited the promotion of PEDV replication by PoRVA. Notably, RVA-HNNY infection more markedly promoted SLC1A5, GLS1 and GLUD1 expression to more significantly increase the uptake and catabolism of glutamine than RVA-SXXA infection. Collectively, our findings illuminate a novel mechanism by which PoRVA infection promotes PEDV infection and reveal that the modulation of glutamine uptake is key for the different efficiencies of PoRVA G9P[23] and PoRVA G5P[7] in promoting PEDV replication.

Proteomics analysis of PK-15 cells infected with porcine parvovirus and the effect of PCBP1 on PPV replication.

Porcine parvovirus (PPV) is one of the most important pathogens that cause reproductive failure in pigs. However, the pathogenesis of PPV infection remains unclear. Proteomics is a powerful tool to understand the interaction between virus and host cells. In the present study, we analyzed the proteomics of PPV-infected PK-15 cells. A total of 32 and 345 proteins were differentially expressed at the early and replication stages, respectively. Subsequent gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed these differentially expressed proteins were significantly enriched in pathways including toll-like receptor signaling pathway, tumor necrosis factor signaling pathway, and viral carcinogenesis. The expression of poly (rC) binding protein 1 (PCBP1) was observed to decrease after PPV infection. Overexpressed or silenced PCBP1 expression inhibited or promoted PPV infection. Our studies established a foundation for further exploration of the multiplication mechanism of PPV. IMPORTANCE: Porcine parvovirus (PPV) is a cause of reproductive failure in the swine industry. Our knowledge of PPV remains limited, and there is no effective treatment for PPV infection. Proteomics of PPV-infected PK-15 cells was conducted to identify differentially expressed proteins at 6 hours post-infection (hpi) and 36 hpi. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that various pathways participate in PPV infection. Poly (rC) binding protein 1 was confirmed to inhibit PPV replication, which provided potential targets for anti-PPV infection. Our findings improve the understanding of PPV infection and pave the way for future research in this area.

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.

Bacillus subtilis alleviates excessive apoptosis of intestinal epithelial cells in intrauterine growth restriction suckling piglets via the members of Bcl-2 and caspase families.

BACKGROUND: The intestine is a barrier resisting various stress responses. Intrauterine growth restriction (IUGR) can cause damage to the intestinal barrier via destroying the balance of intestinal epithelial cells' proliferation and apoptosis. Bacillus subtilis has been reported to regulate intestinal epithelial cells' proliferation and apoptosis. Thus, the purpose of this study was to determine if B. subtilis could regulate intestinal epithelial cells' proliferation and apoptosis in intrauterine growth restriction suckling piglets. RESULTS: Compared with the normal birth weight group, the IUGR group showed greater mean optical density values of Ki-67-positive cells in the ileal crypt (P < 0.05). IUGR resulted in higher ability of proliferation and apoptosis of intestinal epithelial cells, by upregulation of the messenger RNA (mRNA) or proteins expression of leucine rich repeat containing G protein coupled receptor 5, Caspase-3, Caspase-7, ß-catenin, cyclinD1, B-cell lymphoma-2 associated agonist of cell death, and BCL2 associated X (P < 0.05), and downregulation of the mRNA or protein expression of B-cell lymphoma-2 and B-cell lymphoma-2-like 1 (P < 0.05). However, B. subtilis supplementation decreased the mRNA or proteins expression of leucine rich repeat containing G protein coupled receptor 5, SPARC related modular calcium binding 2, tumor necrosis factor receptor superfamily member 19, cyclinD1, Caspase-7, ß-catenin, B-cell lymphoma-2 associated agonist of cell death, and Caspase-3 (P < 0.05), and increased the mRNA expression of B-cell lymphoma-2 (P < 0.05). CONCLUSION: IUGR led to excessive apoptosis of intestinal epithelial cells, which induced compensatory proliferation. However, B. subtilis treatment prevented intestinal epithelial cells of IUGR suckling piglets from excessive apoptosis. © 2024 Society of Chemical Industry.

NS7a of SADS-CoV promotes viral infection via inducing apoptosis to suppress type III interferon production.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered swine coronavirus with potential cross-species transmission risk. Although SADS-CoV-induced host cell apoptosis and innate immunity antagonization has been revealed, underlying signaling pathways remain obscure. Here, we demonstrated that infection of SADS-CoV induced apoptosis in vivo and in vitro, and that viral protein NS7a is mainly responsible for SADS-CoV-induced apoptosis in host cells. Furthermore, we found that NS7a interacted with apoptosis-inducing factor mitochondria associated 1 (AIFM1) to activate caspase-3 via caspase-6 in SADS-CoV-infected cells, and enhanced SADS-CoV replication. Importantly, NS7a suppressed poly(I:C)-induced expression of type III interferon (IFN-λ) via activating caspase-3 to cleave interferon regulatory factor 3 (IRF3), and caspase-3 inhibitor protects piglets against SADS-CoV infection in vivo. These findings reveal how SADS-CoV induced apoptosis to inhibit innate immunity and provide a valuable clue to the development of effective drugs for the clinical control of SADS-CoV infection.IMPORTANCEOver the last 20 years, multiple animal-originated coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2, have caused millions of deaths, seriously jeopardized human health, and hindered social development, indicating that the study of animal-originated coronaviruses with potential for cross-species transmission is particularly important. Bat-originated swine acute diarrhea syndrome coronavirus (SADS-CoV), discovered in 2017, can not only cause fatal diarrhea in piglets, but also infect multiple human cells, with a potential risk of cross-species transmission, but its pathogenesis is unclear. In this study, we demonstrated that NS7a of SADS-CoV suppresses IFN-λ production via apoptosis-inducing factor mitochondria associated 1 (AIFM1)-caspase-6-caspase-3-interferon regulatory factor 3 (IRF3) pathway, and caspase-3 inhibitor (Z-DEVD-FMK) can effectively inhibit SADS-CoV replication and protect infected piglets. Our findings in this study contribute to a better understanding of SADS-CoV-host interactions as a part of the coronaviruses pathogenesis and using apoptosis-inhibitor as a drug as potential therapeutic approaches for prevention and control of SADS-CoV infection.

Large animal models for Huntington's disease research.

Huntington's disease (HD) is a hereditary neurodegenerative disorder for which there is currently no effective treatment available. Consequently, the development of appropriate disease models is critical to thoroughly investigate disease progression. The genetic basis of HD involves the abnormal expansion of CAG repeats in the huntingtin ( HTT) gene, leading to the expansion of a polyglutamine repeat in the HTT protein. Mutant HTT carrying the expanded polyglutamine repeat undergoes misfolding and forms aggregates in the brain, which precipitate selective neuronal loss in specific brain regions. Animal models play an important role in elucidating the pathogenesis of neurodegenerative disorders such as HD and in identifying potential therapeutic targets. Due to the marked species differences between rodents and larger animals, substantial efforts have been directed toward establishing large animal models for HD research. These models are pivotal for advancing the discovery of novel therapeutic targets, enhancing effective drug delivery methods, and improving treatment outcomes. We have explored the advantages of utilizing large animal models, particularly pigs, in previous reviews. Since then, however, significant progress has been made in developing more sophisticated animal models that faithfully replicate the typical pathology of HD. In the current review, we provide a comprehensive overview of large animal models of HD, incorporating recent findings regarding the establishment of HD knock-in (KI) pigs and their genetic therapy. We also explore the utilization of large animal models in HD research, with a focus on sheep, non-human primates (NHPs), and pigs. Our objective is to provide valuable insights into the application of these large animal models for the investigation and treatment of neurodegenerative disorders.

Rotavirus infection inhibits SLA-I expression on the cell surface by degrading ß2 M via ERAD-proteasome pathway.

Group A Rotavirus (RVA) is a major cause of diarrhea in infants and piglets. ß2-microglobulin (ß2 M), encoded by the B2M gene, serves as a crucial subunit of the major histocompatibility complex class I (MHC-I) molecules. ß2 M is indispensable for the transport of MHC-I to the cell membrane. MHC-I, also known as swine leukocyte antigen class I (SLA-I) in pigs, presents viral antigens to the cell surface. In this study, RVA infection down-regulated ß2 M expression in both porcine intestinal epithelial cells-J2 (IPEC-J2) and MA-104 cells. RVA infection did not down-regulate the mRNA level of the B2M gene, indicating that the down-regulation of ß2 M occurred on the protein level. Mechanismly, RVA infection triggered ß2 M aggregation in the endoplasmic reticulum (ER) and enhanced the Lys48 (K48)-linked ubiquitination of ß2 M, leading to the degradation of ß2 M through ERAD-proteasome pathway. Furthermore, we found that RVA infection significantly impeded the level of SLA-I on the surface, and the overexpression of ß2 M could recover its expression. In this study, our study demonstrated that RVA infection degrades ß2 M via ERAD-proteasome pathway, consequently hampering SLA-I expression on the cell surface. This study would enhance the understanding of the mechanism of how RVA infection induces immune escape.

Clinical and gonadal transcriptome analysis of 38,XX disorder of sex development pigs†.

Pigs serve as a robust animal model for the study of human diseases, notably in the context of disorders of sex development (DSD). This study aims to investigate the phenotypic characteristics and molecular mechanisms underlying the reproductive and developmental abnormalities of 38,XX ovotestis-DSD (OT-DSD) and 38,XX testis-DSD (T-DSD) in pigs. Clinical and transcriptome sequencing analyses were performed on DSD and normal female pigs. Cytogenetic and SRY analyses confirmed that OT/T-DSD pigs exhibited a 38,XX karyotype and lacked the SRY gene. The DSD pigs had higher levels of follicle-stimulating hormone, luteinizing hormone, and progesterone, but lower testosterone levels when compared with normal male pigs. The reproductive organs of OT/T-DSD pigs exhibit abnormal development, displaying both male and female characteristics, with an absence of germ cells in the seminiferous tubules. Sex determination and development-related differentially expressed genes shared between DSD pigs were identified in the gonads, including WT1, DKK1, CTNNB1, WTN9B, SHOC, PTPN11, NRG1, and NXK3-1. DKK1 is proposed as a candidate gene for investigating the regulatory mechanisms underlying gonadal phenotypic differences between OT-DSD and T-DSD pigs. Consequently, our findings provide insights into the molecular pathogenesis of DSD pigs and present an animal model for studying into DSD in humans.