In Vitro and In Vivo Evaluation of Virus-Induced Innate Immunity in Mouse.

The innate immune system is the first line of host defense against infection by pathogenic microorganisms, among which macrophages are important innate immune cells. Macrophages are widely distributed throughout the body and recognize and eliminate viruses through pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs). In the present chapter, we provide detailed protocols for vesicular stomatitis virus (VSV) amplification, VSV titer detection, isolation of mouse primary peritoneal macrophages, in vitro and in vivo VSV infection, detection of interferon-beta (IFN-ß) expression, and lung injury. These protocols provide efficient and typical methods to evaluate virus-induced innate immunity in vitro and in vivo.

IFN-ß Overexpressing Adipose-Derived Mesenchymal Stem Cells Mitigate Alcohol-Induced Liver Damage and Gut Permeability.

Alcoholic liver disease (ALD) is a form of hepatic inflammation. ALD is mediated by gut leakiness. This study evaluates the anti-inflammatory effects of ASCs overexpressing interferon-beta (ASC-IFN-ß) on binge alcohol-induced liver injury and intestinal permeability. In vitro, ASCs were transfected with a non-viral vector carrying the human IFN-ß gene, which promoted hepatocyte growth factor (HGF) secretion in the cells. To assess the potential effects of ASC-IFN-ß, C57BL/6 mice were treated with three oral doses of binge alcohol and were administered intraperitoneal injections of ASC-IFN-ß. Mice treated with binge alcohol and administered ASC-IFN-ß showed reduced liver injury and inflammation compared to those administered a control ASC. Analysis of intestinal tissue from ethanol-treated mice administered ASC-IFN-ß also indicated decreased inflammation. Additionally, fecal albumin, blood endotoxin, and bacterial colony levels were reduced, indicating less gut leakiness in the binge alcohol-exposed mice. Treatment with HGF, but not IFN-ß or TRAIL, mitigated the ethanol-induced down-regulation of cell death and permeability in Caco-2 cells. These results demonstrate that ASCs transfected with a non-viral vector to induce IFN-ß overexpression have protective effects against binge alcohol-mediated liver injury and gut leakiness via HGF.

Increased IFN-ß Indicates Better Survival in Hepatocellular Carcinoma Treated with Radiotherapy.

Pre-clinical data suggest that type I interferon (IFN) responsiveness is essential for the antitumor effects of radiotherapy (RT). However, its clinical value remains unclear. This study aimed to explore this from a clinical perspective. In cohort 1, data from 152 hepatocellular carcinoma (HCC) patients who received RT were analyzed. Blood samples were taken 1 day before and 2 weeks after RT. RT was found to increase serum levels of IFN-ß (a subtype of IFN-I) in HCC patients (3.42 ± 1.57 to 5.51 ± 2.11 pg/mL, p < 0.01), particularly in those with favorable responses. Higher post-RT serum IFN-ß levels (≥ 4.77 pg/mL) were associated with better progression-free survival (HR = 0.58, p < 0.01). Cohort 2 included 46 HCC patients, including 23 who underwent preoperative RT and 23 matched control HCC who received surgical resection without RT. Formalin-fixed paraffin-embedded samples were obtained. Neoadjuvant RT significantly increased IFN-ß expression in tumor tissues compared to direct surgery (8.13% ± 5.19% to 15.10% ± 5.89%, p < 0.01). Higher post-RT IFN-ß (> median) indicated better disease-free survival (p = 0.049). Additionally, increased CD11c+MHCII+CD141+ antigen presenting cell subsets and CD103+CD39+CD8+ tumor-infiltrating lymphocytes were found in the higher IFN-ß group (p = 0.02, p = 0.03), which may contribute to the favorable prognosis in higher IFN-ß group. Collectively, these findings suggest that IFN-ß response activated by radiation may serve as a prognostic biomarker for HCC patients undergoing RT.

Integrating network pharmacology with pharmacological research to elucidate the mechanism of modified Gegen Qinlian Decoction in treating porcine epidemic diarrhea.

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to neonatal piglets, particularly due to the limited efficacy of existing vaccines and the scarcity of efficacious therapeutic drugs. Gegen Qinlian Decoction (GQD) has been employed for over two millennia in treating infectious diarrhea. Nonetheless, further scrutiny is required to improve the drug's efficacy and elucidate its underlying mechanisms of action. In this study, a modified GQD (MGQD) was developed and demonstrated its capacity to inhibit the replication of PEDV. Animal trials indicated that MGQD effectively alleviated pathological damage in immune tissues and modulated T-lymphocyte subsets. The integration of network analysis with UHPLC-MS/MS facilitated the identification of active ingredients within MGQD and elucidated the molecular mechanisms underlying its therapeutic effects against PEDV infections. In vitro studies revealed that MGQD significantly impeded PEDV proliferation in IPEC-J2 cells, promoting cellular growth via virucidal activity, inhibition of viral attachment, and disruption of viral biosynthesis. Furthermore, MGQD treatment led to increased expression levels of IFN-α, IFN-ß, and IFN-λ3, while concurrently decreasing the expression of TNF-α, thereby enhancing resistance to PEDV infection in IPEC-J2 cells. In conclusion, our findings suggest that MGQD holds promise as a novel antiviral agent for the treatment of PEDV infections.

Association of chronotype and timing of interferon injection with severity of IFNß-induced flu-like syndrome in Multiple sclerosis (MS) patients.

BACKGROUND: Although Interferon-beta (IFNß) has long been approved as a disease-modifying therapy (DMT) for Multiple sclerosis (MS), flu-like syndrome (FLS) persists as a common adverse effect of interferon therapy. Given the importance of circadian rhythm in regulating physiological processes, we aimed to assess the relationship between patient's chronotype and time of interferon injection with FLS score in MS patients receiving IFNß. METHODS: A cross-sectional study was conducted on 118 MS patients who were referred to the clinic of neurology of Zanjan Vali-e-Asr Hospital for interferon injection. The included were invited to complete a morningness-eveningness questionnaire (MEQ) assessing patients' chronotype. The following data were extracted from patients' record: age, gender, duration of interferon treatment, type of interferon taken, time of interferon injection (morning/evening), FLS score, MS subtype, and usage of pain killers. All data found were imported and statistically analyzed in SPSS ver.26. RESULTS: According to the patients' record, 114 (96.6%) patients had experienced post-interferon injection FLS with different severities. Statistical analysis revealed no significant relationship between the patient's chronotype and FLS score. Nevertheless, the FLS score was significantly higher in those who had evening injections. CONCLUSIONS: Time of interferon injection was significantly associated with FLS score, with higher FLS score following evening injection. However, no significant relationship was found between the FLS score and the patient's chronotype. It is recommended that further studies assessing circadian rhythm using laboratory tests such as melatonin measurement need to be undertaken to investigate the association of circadian rhythm with post-interferon injection FLS.

SLC25A12 inhibits Japanese encephalitis virus replication by interacting with the NS1 and enhancing the type I interferon response.

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic orthoflavivirus causing human encephalitis and reproductive disorders in pigs. Cell-intrinsic antiviral restriction factors are the first line of defense that prevent a virus from establishing a productive infection, while the molecular mechanism of the virus-host interaction is still not fully understood. Our in vitro experiments demonstrated that the Solute Carrier Family 25 Member 12 (SLC25A12) interacted with the JEV nonstructural protein 1 (NS1) and inhibited JEV replication. Furthermore, we showed that knockdown or knockout of SLC25A12 promoted JEV replication, while overexpression of SLC25A12 repressed viral replication. Finally, we demonstrated that SLC25A12 increased IRF7 mRNA levels, which promoted IFN-ß expression and subsequently induced antiviral effects. Collectively, our study revealed that SLC25A12 interacted with NS1, inhibiting viral RNA synthesis and transcription and enhancing type I interferon induction for antiviral effects.

Molecular Cloning, Tissue Distribution and Antiviral Immune Response of Duck Src.

As a founding member of the Src family of kinases, Src has been confirmed to participate in the regulation of immune responses, integrin signaling, and motility. Ducks are usually asymptomatic carriers of RNA viruses such as Newcastle disease virus and avian influenza virus, which can be deadly to chickens. The beneficial role of Src in modulating the immune response remains largely unknown in ducks. Here, we characterized the duck Src and found that it contains a 192-base-pair 5' untranslated region, a 1602-base-pair coding region, and a 2541-base-pair 3' untranslated region, encoding 533 amino acid residues. Additionally, duSrc transcripts were significantly activated in duck tissues infected by Newcastle disease virus compared to controls. The duSrc transcripts were notably widespread in all tissues examined, and the expression level was higher in liver, blood, lung, pancreas, and thymus. Moreover, we found the expression levels of IFN-ß, NF-κB, IRF3, and Src were significantly increased in DEFs after infection with 5'ppp dsRNA, but there was no significant difference before and after treatment in DF1 cells. Furthermore, overexpression of duSrc followed by stimulation with 5'ppp dsRNA led to an elevation of IFN-ß levels. The SH3 and PTKc domains of duSrc contributed to promoting the activity of IFN-ß and NF-κB in DEFs stimulated by 5'ppp dsRNA.

Type I Interferon Activates PD-1 Expression through Activation of the STAT1-IRF2 Pathway in Myeloid Cells.

PD-1 (Programmed cell death protein 1) regulates the metabolic reprogramming of myeloid-derived suppressor cells and myeloid cell differentiation, as well as the type I interferon (IFN-I) signaling pathway in myeloid cells in the tumor microenvironment. PD-1, therefore, is a key inhibitory receptor in myeloid cells. However, the regulation of PD-1 expression in myeloid cells is unknown. We report that the expression level of PDCD1, the gene that encodes the PD-1 protein, is positively correlated with the levels of IFNB1 and IFNAR1 in myeloid cells in human colorectal cancer. Treatment of mouse myeloid cell lines with recombinant IFNß protein elevated PD-1 expression in myeloid cells in vitro. Knocking out IFNAR1, the gene that encodes the IFN-I-specific receptor, diminished the inductive effect of IFNß on PD-1 expression in myeloid cells in vitro. Treatment of tumor-bearing mice with a lipid nanoparticle-encapsulated IFNß-encoding plasmid (IFNBCOL01) increased IFNß expression, resulting in elevated PD-1 expression in tumor-infiltrating myeloid cells. At the molecular level, we determined that IFNß activates STAT1 (signal transducer and activator of transcription 1) and IRFs (interferon regulatory factors) in myeloid cells. Analysis of the cd279 promoter identified IRF2-binding consensus sequence elements. ChIP (chromatin immunoprecipitation) analysis determined that the pSTAT1 directly binds to the irf2 promoter and that IRF2 directly binds to the cd279 promoter in myeloid cells in vitro and in vivo. In colon cancer patients, the expression levels of STAT1, IRF2 and PDCD1 are positively correlated in tumor-infiltrating myeloid cells. Our findings determine that IFNß activates PD-1 expression at least in part by an autocrine mechanism via the stimulation of the pSTAT1-IRF2 axis in myeloid cells.

African swine fever virus structural protein p17 inhibits IRF3 activation by recruiting host protein PR65A and inducing apoptotic degradation of STING.

African swine fever virus (ASFV) is notoriously known for evolving strategies to modulate IFN signaling. Despite lots of efforts, the underlying mechanisms have remained incompletely understood. This study concerns the regulatory role of viral inner membrane protein p17. We found that the ASFV p17 shows a preferential interaction with cGAS-STING-IRF3 pathway, but not the RIG-I-MAVS-NF-κB signaling, and can inhibit both poly(I:C)- and poly(A:T)-induced activation of IRF3, leading to attenuation of IFN-ß induction. Mechanistically, p17 interacts with STING and IRF3 and recruits host scaffold protein PR65A, a subunit of cellular phosphatase PP2A, to down-regulate the level of p-IRF3. Also, p17 targets STING for partial degradation via induction of cellular apoptosis that consequently inhibits activation of both p-TBK1 and p-IRF3. Thus, our findings reveal novel regulatory mechanisms for p17 modulation of IFN signaling and shed light on the intricate interplay between ASFV proteins and host immunity.

CCR5 Δ32 and CTLA-4 +49 A/G Gene Polymorphisms and Interferon-ß Treatment Response in Croatian and Slovenian Multiple Sclerosis Patients.

The aim of the present study was to investigate the impact of CCR5 Δ32 and CTLA-4 polymorphisms on the response to IFN-ß treatment in our cohort of MS patients from Croatia and Slovenia. Genomic DNA was obtained from 295 MS patients (230 female; 65 male) classified as responders (n = 173) and non-responders (n = 122) based on clinical criteria for treatment efficacy. Genotyping was performed via PCR/PCR-RFLP. No significant differences in the genotype/allele frequencies of CCR5Δ32 and CTLA-4 +49 A/G were detected between male responders and non-responders. A significantly higher prevalence (p = 0.039) of the CTLA-4 +49 AA genotype was found in female responders (42.1%) compared to non-responders (28.9%). Using multiple forward regression analysis, the CTLA-4 +49 AA genotype significantly predicted a positive response to IFN-ß therapy in females (p = 0.011) and contributed to 4.5% of response variability. Furthermore, the combined presence of the CCR5Δ32 wtwt/CTLA-4 +49 AA genotype significantly predicted a positive response to treatment in females (p = 0.025). The age at disease onset, pretreatment relapse rate, and baseline EDSS score were not reliable predictors of treatment response in MS patients. Our results indicate that the presence of the CCR5Δ32 polymorphism was not associated with the response to IFN-ß treatment, whereas the CTLA-4 +49 polymorphism showed a positive correlation with an optimal response in female patients.

Successful treatment of invasive mycobacterium infection with interferon beta in a patient with Interferon-Gamma Receptor 1 deficiency.

Mendelian susceptibility to mycobacterial disease (MSMD) is caused by approximately 21 genetic defects, including a mutation in Interferon-Gamma Receptor 1 (IFNGR1). IFNGR1 deficiency leads to a loss of cellular responsiveness to type II Interferon (IFN-γ), which plays a significant role in controlling intracellular bacteria. This study explored the response of IFN-ß therapy in a patient with partial IFNGR1 deficiency to treat invasive mycobacterial infection. The biological therapy was used successfully as an adjuvant to anti-mycobacterial medications to treat a 17-year-old girl with partial IFNGR1 deficiency who presented with a recurrent mycobacterial infection that extended to her central nervous system, which resulted in clinical and radiological improvement. This report suggests that activation of type I IFN through Signal Transducers and Activators of Transcription1 (STAT1) could bypass the early IFN-γ signaling defects and activate IFN-γ production. For that reason, IFN-ß might be used as a beneficial adjuvant therapy for managing extensive central nervous system mycobacterial infection, especially in patients with IFNGR1 deficiency.

The inhibitory effect of swine TAB1 on the replication of pseudorabies virus.

TAK1-binding protein 1 (TAB1) assembles with TAK1 through its C-terminal domain, leading to the self-phosphorylation and activation of TAK1, which plays an important role in the activation of NF-κB and MAPK signaling pathway. Pseudorabies virus (PRV) is the pathogen of Pseudorabies (PR), which belongs to the Alphaherpesvirus subfamily and causes serious economic losses to the global pig industry. However, the impact of swine TAB1 (sTAB1) on PRV infection has not been reported. In this study, evidence from virus DNA copies, virus titer and western blotting confirmed that sTAB1 could inhibit PRV replication and knockout of sTAB1 by CRISPR-Cas9 gene editing system could promote PRV replication. Further mechanistic studies by real-time PCR and luciferase reporter gene assay demonstrated that sTAB1 could enhance the production of inflammatory factors and chemokines, IFN-ß transcription level and IFN-ß promoter activity after PRV infection. In summary, we clarify the underlying mechanism of sTAB1 in inhibiting PRV replication for the first time, which provides a new idea for preventing PRV infection and lays a foundation for PRV vaccine development.

Expression of ISG60 is induced by TLR3 signaling in BEAS­2B bronchial epithelial cells: Possible involvement in CXCL10 expression.

Viral infections in the respiratory tract are common, and, in recent years, severe acute respiratory syndrome coronavirus 2 outbreaks have highlighted the effect of viral infections on antiviral innate immune and inflammatory reactions. Specific treatments for numerous viral respiratory infections have not yet been established and they are mainly treated symptomatically. Therefore, understanding the details of the innate immune system underlying the airway epithelium is crucial for the development of new therapies. The present study aimed to investigate the function and expression of interferon (IFN)­stimulated gene (ISG)60 in non­cancerous bronchial epithelial BEAS­2B cells exposed to a Toll­like receptor 3 agonist. BEAS­2B cells were treated with a synthetic TLR3 ligand, polyinosinic­polycytidylic acid (poly IC). The mRNA and protein expression levels of ISG60 were analyzed using reverse transcription­quantitative PCR and western blotting, respectively. The levels of C­X­C motif chemokine ligand 10 (CXCL10) were examined using an enzyme­linked immunosorbent assay, and the effects of knockdown of IFN­ß, ISG60 and ISG56 were examined using specific small interfering RNAs. Notably, ISG60 expression was increased in proportion to poly IC concentration, and recombinant human IFN­ß also induced ISG60 expression. By contrast, knockdown of IFN­ß and ISG56 decreased ISG60 expression, and ISG60 knockdown reduced CXCL10 and ISG56 expression. These findings suggested that ISG60 is partly implicated in CXCL10 expression and that ISG60 may serve a role in the innate immune response of bronchial epithelial cells. The present study highlights ISG60 as a potential target for new therapeutic strategies against viral infections in the airway.

TLR21 is involved in the NF-κB and IFN-ß pathways in largemouth bass (Micropterus salmoides) and interacts with TRIF but not with the Myd88 adaptor.

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-ß (IFN-ß) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-ß (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

UBR7 E3 Ligase Suppresses Interferon-ß Mediated Immune Signaling by Targeting Sp110 in Hepatitis B Virus-Induced Hepatocellular Carcinoma.

A newly discovered E3 ubiquitin ligase, UBR7, plays a crucial role in histone H2BK120 monoubiquitination. Here, we report a novel function of UBR7 in promoting hepatitis B virus (HBV) pathogenesis, which further leads to HBV-induced hepatocellular carcinoma (HCC). Transcriptomics analysis from HCC patients revealed the deregulation of UBR7 in cancer. Remarkably, targeting UBR7, particularly its catalytic function, led to a significant decrease in viral copy numbers. We also identified the speckled family protein Sp110 as an important substrate of UBR7. Notably, Sp110 has been previously shown to be a resident of promyelocytic leukemia nuclear bodies (PML-NBs), where it remains SUMOylated, and during HBV infection, it undergoes deSUMOylation and exits the PML body. We observed that UBR7 ubiquitinates Sp110 at critical residues within its SAND domain. Sp110 ubiquitination downregulates genes in the type I interferon response pathway. Comparative analysis of RNA-Seq from the UBR7/Sp110 knockdown data set confirmed that the IFN-ß signaling pathway gets deregulated in HCC cells in the presence of HBV. Single-cell RNA-Seq analysis of patient samples further confirmed the inverse correlation between the expression of Sp110/UBR7 and the inflammation score. Notably, silencing of UBR7 induces IRF7 phosphorylation, thereby augmenting interferon (IFN)-ß and the downstream interferon-stimulated genes (ISGs). Further, wild-type but not the ubiquitination-defective mutant of Sp110 could be recruited to the type I interferon response pathway genes. Our study establishes a new function of UBR7 in non-histone protein ubiquitination, promoting viral persistence, and has important implications for the development of therapeutic strategies targeting HBV-induced HCC.

Gasdermin E benefits CD8+T cell mediated anti-immunity through mitochondrial damage to activate cGAS-STING-interferonß axis in colorectal cancer.

BACKGROUND: Pyroptosis belongs to a unique type of programmed cell death among which GSDME is reported to exert anti-tumor immunity. However, the underlying mechanisms of how to boost tumor-infiltrating lymphocytes and whether it could benefit the efficacy of ICIs are still unknown. METHODS: CRC samples were used to analyze its relationship with CD8+T cells. GSDME in mouse CRC cell lines CT26/MC38 was overexpressed. The infiltration of CD8+T cells in grafted tumors was determined by multiplex flow cytometric analysis and immunohistochemistry. Transcriptomic analysis was performed in cell lines to define key signatures related to its overexpression. The mechanism of how mtDNA was released by GSDME-induced mitochondrial damage and activated cGAS-STING pathway was observed. Whether GSDME benefited ICIs and the relationships with the genotypes of CRC patients were investigated. RESULTS: It had favorable prognostic value in CRC and was positively associated with increased number and functionality of CD8+T cells both in human samples and animal models. This was due to mitochondrial damage and activation of cGAS-STING-IFNß pathway for the recruitment of CD8+T cells. Mechanically, GSDME overexpression enhanced N-GSDME level, leading to the mitochondrial damage and mtDNA was released into cytosol. Finally, GSDME benefited with ICIs and exhibited positive relationships with MSI in CRC patients. CONCLUSION: We presented the mechanism of GSDME in anti-tumor immunity through activating cGAS-STING-IFNß axis mediated by mitochondrial damage, leading to more infiltration of CD8+T cells with synergistic efficacy with ICIs.

A novel host restriction factor MRPS6 mediates the inhibition of PDCoV infection in HIEC-6 cells.

Introduction: Porcine deltacoronavirus (PDCoV) is a zoonotic pathogen with a global distribution, capable of infecting both pigs and humans. To mitigate the risk of cross-species transmission and potential outbreaks, it is crucial to characterize novel antiviral genes, particularly those from human hosts. Methods: This research used HIEC-6 to investigate PDCoV infection. HIEC-6 cells were infected with PDCoV. Samples were collected 48 h postinfection for proteomic analysis. Results: We discovered differential expression of MRPS6 gene at 48 h postinfection with PDCoV in HIEC-6 cells. The gene expression initially increased but then decreased. To further explore the role of MRPS6 in PDCoV infection, we conducted experiments involving the overexpression and knockdown of this gene in HIEC-6 and Caco2 cells, respectively. Our findings revealed that overexpression of MRPS6 significantly inhibited PDCoV infection in HIEC-6 cells, while knockdown of MRPS6 in Caco2 cells led to a significant increase of virus titer. Furthermore, we investigated the correlation between PDCoV infection and the expression of MRPS6. Subsequent investigations demonstrated that MRPS6 exerted an augmentative effect on the production of IFN-ß through interferon pathway activation, consequently impeding the progression of PDCoV infection in cellular systems. In conclusion, this study utilized proteomic analysis to investigate the differential protein expression in PDCoV-infected HIEC-6 cells, providing evidence for the first time that the MRPS6 gene plays a restrictive role in PDCoV virus infection. Discussion: Our findings initially provide the validation of MRPS6 as an upstream component of IFN-ß pathway, in the promotion of IRF3, IRF7, STAT1, STAT2 and IFN-ß production of HIEC-6 via dual-activation from interferon pathway.

Hunnivirus structural protein VP2 inhibits beta interferon production by targeting the IRF3 essential modulator.

Water buffalo Hunnivirus (BufHuV) belongs to the family Picornaviridae and is a newly discovered member of the Hunnivirus A genus. It causes intestinal diseases in cattle, mainly lead to subclinical infections, thereby seriously threatening the health of cattle herds. In addition, it can also bring about various clinical disease syndromes which results in severe economic losses to the cattle industry. To date, there have been no reports worldwide on the study of Hunnivirus virus infecting host cells and causing innate immune responses. In this study, we found that interferon treatment effectively blocked BufHuV replication and infection with the virus weakened the host antiviral responses. Inhibiting the transcription of IFN-ß and ISGs induced by either Sendai virus (SeV) or poly(I:C) in MDBK and HCT-8 cells, were dependent on the IRF3 or NF-κB signaling pathways, and this inhibited the activation of IFN-ß promoter by TBK1 and its upstream molecules, RIGI and MDA5. By constructing and screening five BufHuV proteins, we found that VP2, 2 C, 3 C and 3D inhibited the activation of IFN-ß promoter induced by SeV. Subsequently, we showed that VP2 inhibited the activation of IRF3 induced by SeV or poly (I:C), and it inhibited IRF3 activation by inhibiting its phosphorylation and nuclear translocation. In addition, we confirmed that VP2 inhibited the activation of IFNß induced by signaling molecules, MDA5 and TBKI. In summary, these findings provide new insights into the pathogenesis of Hunnivirus and its mechanisms involved in evading host immune responses.

Allogeneic stem cells engineered to release interferon ß and scFv-PD1 target glioblastoma and alter the tumor microenvironment.

Highly malignant brain tumors, glioblastomas (GBM), are immunosuppressive, thereby limiting current promising immunotherapeutic approaches. In this study, we created interferon receptor 1 knockout allogeneic mesenchymal stem cells (MSC) to secrete dual-function pro-apoptotic and immunomodulatory interferon (IFN) ß (MSCKO-IFNß) using a single lentiviral vector CRISPR/Cas9 system. We show that MSCKO-IFNß induces apoptosis in GBM cells and upregulates the cell surface expression of programmed death ligand-1 in tumor cells. Next, we engineered MSCKO to release a secretable single-chain variable fragment (scFv) to block programmed death (PD)-1 and show the ability of MSCKO-scFv-PD1 to enhance T-cell activation and T-cell-mediated tumor cell killing. To simultaneously express both immune modulators, we engineered MSCKO-IFNß to co-express scFv-PD1 (MSCKO-IFNß-scFv-PD1) and show the expression of both IFNß and scFv-PD1 in vitro leads to T-cell activation and lowers the viability of tumor cells. Furthermore, to mimic the clinical scenario of GBM tumor resection and subsequent treatment, we show that synthetic extracellular matrix (sECM) encapsulated MSCKO-IFNß-scFv-PD1 treatment of resected tumors results in the increase of CD4+ and CD8+ T cells, mature conventional dendritic cells type II and activation of microglia as compared to the control treatment group. Overall, these results reveal the ability of MSCKO-IFNß-scFv-PD1 to shape the tumor microenvironment and enhance therapeutic outcomes in GBM.

Nsp10-interacting host protein SAP18 restricts PEDV replication in Marc-145 cells via enhancing dephosphorylation of RIG-I.

PEDV, a single-stranded RNA virus, causes significant economic losses in the pig industry. Sin3-associated protein 18 (SAP18) is known for its role in transcriptional inhibition and RNA splicing. However, research on SAP18's involvement in PEDV infection is limited. Here, we identified an interaction between SAP18 and PEDV nonstructural protein 10 (Nsp10) using immunoprecipitation-mass spectrometry (IP-MS) and confirmed it through immunoprecipitation and laser confocal microscopy. Additionally, PEDV Nsp10 reduced SAP18 protein levels and induced its cytoplasmic accumulation. Overexpressing SAP18 suppressed PEDV replication, meanwhile its knockdown via short interfering RNA (siRNA) enhanced replication. SAP18 overexpression boosted IRF3 and NF-κB P65 phosphorylation, nuclear translocation, and IFN-ß antiviral response. Furthermore, SAP18 upregulated RIG-I expression and facilitated its dephosphorylation, while SAP18 knockdown had the opposite effect. Finally, SAP18 interacted with phosphatase 1 (PP1) catalytic subunit alpha (PPP1CA), promoting PPP1CA-RIG-I interaction during PEDV infection. These findings highlight SAP18's role in activating the type I interferon pathway and inhibiting viral replication by promoting RIG-I dephosphorylation through its interaction with PPP1CA.