Single-cell profiling of African swine fever virus disease in the pig spleen reveals viral and host dynamics.

African swine fever, one of the major viral diseases of swine, poses an imminent threat to the global pig industry. The high-efficient replication of the causative agent African swine fever virus (ASFV) in various organs in pigs greatly contributes to the disease. However, how ASFV manipulates the cell population to drive high-efficient replication of the virus in vivo remains unclear. Here, we found that the spleen reveals the most severe pathological manifestation with the highest viral loads among various organs in pigs during ASFV infection. By using single-cell-RNA-sequencing technology and multiple methods, we determined that macrophages and monocytes are the major cell types infected by ASFV in the spleen, showing high viral-load heterogeneity. A rare subpopulation of immature monocytes represents the major population infected at late infection stage. ASFV causes massive death of macrophages, but shifts its infection into these monocytes which significantly arise after the infection. The apoptosis, interferon response, and antigen-presentation capacity are inhibited in these monocytes which benefits prolonged infection of ASFV in vivo. Until now, the role of immature monocytes as an important target by ASFV has been overlooked due to that they do not express classical monocyte marker CD14. The present study indicates that the shift of viral infection from macrophages to the immature monocytes is critical for maintaining prolonged ASFV infection in vivo. This study sheds light on ASFV tropism, replication, and infection dynamics, and elicited immune response, which may instruct future research on antiviral strategies.

Impaired interferon response in senecavirus A infection and identification of 3Cpro as an antagonist.

Senecavirus A (SVA), a picornavirus, causes vesicular diseases and epidemic transient neonatal losses in swine, resulting in a multifaceted economic impact on the swine industry. SVA counteracts host antiviral response through multiple strategies facilitatng viral infection and transmission. However, the mechanism of how SVA modulates interferon (IFN) response remains elusive. Here, we demonstrate that SVA 3C protease (3Cpro) blocks the transduction of Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway to antagonize type I IFN response. Mechanistically, 3Cpro selectively cleaves and degrades STAT1 and STAT2 while does not target JAK1, JAK2, and IRF9, through its protease activity. Notably, SVA 3Cpro cleaves human and porcine STAT1 on a Leucine (L)-Aspartic acid (D) motif, specifically L693/D694. In the case of STAT2, two cleavage sites were identified: glutamine (Q) 707 was identified in both human and porcine, while the second cleavage pattern differed, with residues 754-757 (Valine-Leucine-Glutamine-Serine motifs) in human STAT2 and Q758 in porcine STAT2. These cleavage patterns by SVA 3Cpro partially differ from previously reported classical motifs recognized by other picornaviral 3Cpro, highlighting the distinct characteristics of SVA 3Cpro. Together, these results reveal a mechanism by which SVA 3Cpro antagonizes IFN-induced antiviral response but also expands our knowledge about the substrate recognition patterns for picornaviral 3Cpro.IMPORTANCESenecavirus A (SVA), the only member in the Senecavirus genus within the Picornaviridae family, causes vesicular diseases in pigs that are clinically indistinguishable from foot-and-mouth disease (FMD), a highly contagious viral disease listed by the World Organization for Animal Health (WOAH). Interferon (IFN)-mediated antiviral response plays a pivotal role in restricting and controlling viral infection. Picornaviruses evolved numerous strategies to antagonize host antiviral response. However, how SVA modulates the JAK-STAT signaling pathway, influencing the type I IFN response, remains elusive. Here, we identify that 3Cpro, a protease of SVA, functions as an antagonist for the IFN response. 3Cpro utilizes its protease activity to cleave STAT1 and STAT2, thereby diminishing the host IFN response to promote SVA infection. Our findings underscore the significance of 3Cpro as a key virulence factor in the antagonism of the type I signaling pathway during SVA infection.

From homeostasis to defense: Exploring the role of selective autophagy in innate immunity and viral infections.

The process of autophagy, a conservative evolutionary mechanism, is responsible for the removal of surplus and undesirable cytoplasmic components, thereby ensuring cellular homeostasis. Autophagy exhibits a remarkable level of selectivity by employing a multitude of cargo receptors that possess the ability to bind both ubiquitinated cargoes and autophagosomes. In the context of viral infections, selective autophagy plays a crucial role in regulating the innate immune system. Notably, numerous viruses have developed strategies to counteract, evade, or exploit the antiviral effects of selective autophagy. This review encompasses the latest research progress of selective autophagy in regulating innate immunity and virus infectious.

PROTAC EZH2 degrader-1 overcomes the resistance of podophyllotoxin derivatives in refractory small cell lung cancer with leptomeningeal metastasis.

BACKGROUND: Leptomeningeal metastasis (LM) of small cell lung cancer (SCLC) is a highly detrimental occurrence associated with severe neurological disorders, lacking effective treatment currently. Proteolysis-targeting chimeric molecules (PROTACs) may provide new therapeutic avenues for treatment of podophyllotoxin derivatives-resistant SCLC with LM, warranting further exploration. METHODS: The SCLC cell line H128 expressing luciferase were mutated by MNNG to generate H128-Mut cell line. After subcutaneous inoculation of H128-Mut into nude mice, H128-LM and H128-BPM (brain parenchymal metastasis) cell lines were primarily cultured from LM and BPM tissues individually, and employed to in vitro drug testing. The SCLC-LM mouse model was established by inoculating H128-LM into nude mice via carotid artery and subjected to in vivo drug testing. RNA-seq and immunoblotting were conducted to uncover the molecular targets for LM. RESULTS: The SCLC-LM mouse model was successfully established, confirmed by in vivo live imaging and histological examination. The upregulated genes included EZH2, SLC44A4, VEGFA, etc. in both BPM and LM cells, while SLC44A4 was particularly upregulated in LM cells. When combined with PROTAC EZH2 degrader-1, the drug sensitivity of cisplatin, etoposide (VP16), and teniposide (VM26) for H128-LM was significantly increased in vitro. The in vivo drug trials with SCLC-LM mouse model demonstrated that PROTAC EZH2 degrader-1 plus VM26 or cisplatin/ VP16 inhibited H128-LM tumour significantly compared to VM26 or cisplatin/ VP16 alone (P < 0.01). CONCLUSION: The SCLC-LM model effectively simulates the pathophysiological process of SCLC metastasis to the leptomeninges. PROTAC EZH2 degrader-1 overcomes chemoresistance in SCLC, suggesting its potential therapeutic value for SCLC LM.

Application of 3D computed tomography in emphysematous parenchyma patients scheduled for bronchoscopic lung volume reduction.

Bronchoscopic lung volume reduction (BLVR) is a feasible, safe, effective and minimally invasive technique to significantly improve the quality of life of advanced severe chronic obstructive pulmonary disease (COPD). In this study, three-dimensional computed tomography (3D-CT) automatic analysis software combined with pulmonary function test (PFT) was used to retrospectively evaluate the postoperative efficacy of BLVR patients. The purpose is to evaluate the improvement of lung function of local lung tissue after operation, maximize the benefits of patients, and facilitate BLVR in the treatment of patients with advanced COPD. All the reported cases of advanced COPD patients treated with BLVR with one-way valve were collected and analysed from 2017 to 2020. Three-dimensional-CT image analysis software system was used to analyse the distribution of low-density areas <950 Hounsfield units in both lungs pre- and post- BLVR. Meanwhile, all patients performed standard PFT pre- and post-operation for retrospective analysis. We reported six patients that underwent unilateral BLVR with 1 to 3 valves according to the range of emphysema. All patients showed a median increase in forced expiratory volume in 1 second (FEV1) of 34%, compared with baseline values. Hyperinflation was reduced by 16.6% (range, 4.9%-47.2%). The volumetric measurements showed a significant reduction in the treated lobe volume among these patients. Meanwhile, the targeted lobe volume changes were inversely correlated with change in FEV1/FEV1% in patients with heterogeneous emphysematous. We confirm that 3D-CT analysis can quantify the changes of lung volume, ventilation and perfusion, to accurately evaluate the distribution and improvement of emphysema and rely less on the observer.

Foot-and-mouth disease virus VP1 degrades YTHDF2 through autophagy to regulate IRF3 activity for viral replication.

Many viruses, including foot-and-mouth disease virus (FMDV), can promote the degradation of host proteins through macroautophagy/autophagy, thereby promoting viral replication. However, the regulatory mechanism between autophagy and innate immune responses is not fully understood during FMDV infection. Here, we found that the host GTPBP4/NOG1 (GTP binding protein 4) is a negative regulator of innate immune responses. GTPBP4 deficiency promotes the antiviral innate immune response, resulting in the ability of GTPBP4 to promote FMDV replication. Meanwhile, GTPBP4-deficient mice are more resistant to FMDV infection. To antagonize the host's antiviral immunity, FMDV structural protein VP1 promotes the expression of GTPBP4, and the 209th site of VP1 is responsible for this effect. Mechanically, FMDV VP1 promotes autophagy during virus infection and interacts with and degrades YTHDF2 (YTH N6-methyladenosine RNA binding protein F2) in an AKT-MTOR-dependent autophagy pathway, resulting in an increase in GTPBP4 mRNA and protein levels. Increased GTPBP4 inhibits IRF3 binding to the Ifnb/Ifn-ß promoter, suppressing FMDV-induced type I interferon production. In conclusion, our study revealed an underlying mechanism of how VP1 negatively regulates innate immunity through the autophagy pathway, which would contribute to understanding the negative regulation of host innate immune responses and the function of GTPBP4 and YTHDF2 during FMDV infection.Abbreviation: 3-MA:3-methyladenine; ACTB: actin beta; ATG: autophagy related; ChIP:chromatin immunoprecipitation; CQ: chloroquine; DAPI:4',6-diamidino-2-phenylindole; dpi: days post-infection; EV71:enterovirus 71; FMDV: foot-and-mouth disease virus; GTPBP4/NOG1: GTPbinding protein 4; HIF1A: hypoxia inducible factor 1 subunit alpha;hpt:hours post-transfection; IFNB/IFN-ß:interferon beta; IRF3: interferon regulatory factor 3; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MAVS: mitochondriaantiviral signaling protein; MOI: multiplicity of infection; MTOR:mechanistic target of rapamycin kinase; m6A: N(6)-methyladenosine;qPCR:quantitativePCR; SIRT3:sirtuin 3; SQSTM1/p62: sequestosome 1; STING1: stimulator ofinterferon response cGAMP interactor 1; siRNA: small interfering RNA;TBK1: TANK binding kinase 1; TCID50:50% tissue culture infectious doses; ULK1: unc-51 like autophagyactivating kinase 1; UTR: untranslated region; WT: wild type; YTHDF2:YTH N6-methyladenosine RNA binding protein F2.

KIF5B-mediated internalization of FMDV promotes virus infection.

Foot-and-mouth disease (FMD) is a highly contagious and economically important disease, which is caused by the FMD virus (FMDV). Although the cell receptor for FMDV has been identified, the specific mechanism of FMDV internalization after infection remains unknown. In this study, we found that kinesin family member 5B (KIF5B) plays a vital role during FMDV internalization. Moreover, we confirmed the interaction between KIF5B and FMDV structural protein VP1 by co-immunoprecipitation (Co-IP) and co-localization in FMDV-infected cells. In particular, the stalk [amino acids (aa) 413-678] domain of KIF5B was indispensable for KIF5B-VP1 interaction. Moreover, overexpression of KIF5B dramatically enhanced FMDV replication; consistently, knockdown or knockout of KIF5B suppressed FMDV replication. Furthermore, we also demonstrated that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating. KIF5B also promotes the transmission of viral particles to early and late endosomes during the early stages of infection. In conclusion, our results demonstrate that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating and intracellular transport. This study may provide a new therapeutic target for developing FMDV antiviral drugs.

Picornavirus VP3 protein induces autophagy through the TP53-BAD-BAX axis to promote viral replication.

Macroautophagy/autophagy and apoptosis are pivotal interconnected host cell responses to viral infection, including picornaviruses. Here, the VP3 proteins of picornaviruses were determined to trigger autophagy, with the autophagic flux being triggered by the TP53-BAD-BAX axis. Using foot-and-mouth disease virus (FMDV) as a model system, we unraveled a novel mechanism of how picornavirus hijacks autophagy to bolster viral replication and enhance pathogenesis. FMDV infection induced both autophagy and apoptosis in vivo and in vitro. FMDV VP3 protein facilitated the phosphorylation and translocation of TP53 from the nucleus into the mitochondria, resulting in BAD-mediated apoptosis and BECN1-mediated autophagy. The amino acid Gly129 in VP3 is essential for its interaction with TP53, and crucial for induction of autophagy and apoptosis. VP3-induced autophagy and apoptosis are both essential for FMDV replication, while, autophagy plays a more important role in VP3-mediated pathogenesis. Mutation of Gly129 to Ala129 in VP3 abrogated the autophagic regulatory function of VP3, which significantly decreased the viral replication and pathogenesis of FMDV. This suggested that VP3-induced autophagy benefits viral replication and pathogenesis. Importantly, this Gly is conserved and showed a common function in various picornaviruses. This study provides insight for developing broad-spectrum antivirals and genetic engineering attenuated vaccines against picornaviruses.Abbreviations: 3-MA, 3-methyladenine; ATG, autophagy related; BAD, BCL2 associated agonist of cell death; BAK1, BCL2 antagonist/killer 1; BAX, BCL2 associated X, apoptosis regulator; BBC3/PUMA, BCL2 binding component 3; BCL2, BCL2 apoptosis regulator; BID, BH3 interacting domain death agonist; BIP-V5, BAX inhibitor peptide V5; CFLAR/FLIP, CASP8 and FADD like apoptosis regulator; CPE, cytopathic effects; CQ, chloroquine; CV, coxsackievirus; DAPK, death associated protein kinase; DRAM, DNA damage regulated autophagy modulator; EV71, enterovirus 71; FMDV, foot-and-mouth disease virus; HAV, hepatitis A virus; KD, knockdown; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MOI, multiplicity of infection; MTOR, mechanistic target of rapamycin kinase; PML, promyelocytic leukemia; PV, poliovirus; SVA, Seneca Valley virus; TCID50, 50% tissue culture infectious doses; TOR, target of rapamycin. TP53/p53, tumor protein p53; WCL, whole-cell lysate.