Differentiated thyroid cancer with osteo-granulomatousinflammation: A case report.

OBJECTIVE: Cryptococcus, a genus of fungi, primarily includes Cryptococcus neoformans and Cryptococcus gattii, both known to cause human infections. Skeletal infections are rare, and there have been no reported cases of bone cryptococcal infection in conjunction with differentiated thyroid carcinoma. SUBJECT AND METHODS: A 56-year-old female presented with a one-month history of "cough and throat irritation." Chest CT revealed scattered small nodules in both lungs,suggestive of metastasis.There was minimal inflammation in both lungs, and scattered lymph nodes were observed in the mediastinum and upper pulmonary hilum. RESULTS: The patient was diagnosed with differentiated thyroid carcinoma complicated by cryptococcal infection. Antifungal treatment with itraconazole 200mg/day was initiated, and after 3 months, clinical symptoms disappeared, with a reduction in lung nodules observed in follow-up chest CT. CONCLUSION: When diagnosing distant metastasis in differentiated thyroid carcinoma, a comprehensive analysis combining imaging studies and serum thyroid globulin plays a complementary role, as illustrated in this case of differentiated thyroid carcinoma concurrent with cryptococcal infection.

RBM14 inhibits the replication of porcine epidemic diarrhea virus by recruiting p62 to degrade nucleocapsid protein through the activation of autophagy and interferon pathway.

Porcine epidemic diarrhea virus (PEDV) results in PED, which is an infectious intestinal disease with the representative features of diarrhea, vomiting, and dehydration. PEDV infects neonatal piglets, causing high mortality rates. Therefore, elucidating the interaction between the virus and host in preventing and controlling PEDV infection is of immense significance. We found a new antiviral function of the host protein, RNA-binding motif protein 14 (RBM14), which can inhibit PEDV replication via the activation of autophagy and interferon (IFN) signal pathways. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV nucleocapsid (N) protein through the RBM14-p62-autophagosome pathway. Furthermore, RBM14 can also improve the antiviral ability of the hosts through interacting with mitochondrial antiviral signaling protein to induce IFN expression. These results highlight the novel mechanism underlying RBM14-induced viral restriction. This mechanism leads to the degradation of viral N protein via the autophagy pathway and upregulates IFN for inhibiting PEDV replication; thus, offering new ways for preventing and controlling PED.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) is a vital reason for diarrhea in neonatal piglets, which causes high morbidity and mortality rates. There is currently no effective vaccine or drug to treat and prevent infection with the PEDV. During virus infection, the host inhibits virus replication through various antiviral factors, and at the same time, the virus antagonizes the host's antiviral reaction through its own encoded protein, thus completing the process of virus replication. Our study has revealed that the expression of RNA-binding motif protein 14 (RBM14) was downregulated in PEDV infection. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV N protein via the RBM14-p62-autophagosome pathway and interacted with mitochondrial antiviral signaling protein and TRAF3 to activate the interferon signal pathway, resulting in the inhibition of PEDV replication.

An Expeditious Neutralization Assay for Porcine Reproductive and Respiratory Syndrome Virus Based on a Recombinant Virus Expressing Green Fluorescent Protein.

Due to the extensive genetic and antigenic variation in Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), as well as its rapid mutability and evolution, PRRS prevention and control can be challenging. An expeditious and sensitive neutralization assay for PRRSV is presented to monitor neutralizing antibodies (NAbs) in serum during vaccine research. Here, a PRRSV expressing eGFP was successfully rescued with reverse genetics based on the infectious clone HuN4-F112-eGFP which we constructed. The fluorescent protein expressions of the reporter viruses remained stable for at least five passages. Based on this reporter virus, the neutralization assay can be easily used to evaluate the level of NAbs by counting cells with green fluorescence. Compared with the classical CPE assay, the newly developed assay increases sensitivity by one- to four-fold at the early antibody response stage, thus saving 2 days of assay waiting time. By using this assay to unveil the dynamics of neutralizing antibodies against PRRSV, priming immunity through either a single virulent challenge or only vaccination could produce limited NAbs, but re-infection with PRRSV would induce a faster and stronger NAb response. Overall, the novel HuN4-F112-eGFP-based neutralization assay holds the potential to provide a highly efficient platform for evaluating the next generation of PRRS vaccines.

Advances in Helicobacter pylori vaccine research: From candidate antigens to adjuvants-A review.

BACKGROUND: Helicobacter pylori is a Gram-negative, spiral-shaped bacterium that infects approximately 50% of the world's population and has been strongly associated with chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoma, and gastric cancer. The elimination of H. pylori is currently considered one of the most effective strategies for the treatment of gastric-related diseases, so antibiotic therapy is the most commonly used regimen for the treatment of H. pylori infection. Although this therapy has some positive effects, antibiotic resistance has become another clinically prominent problem. Therefore, the development of a safe and efficient vaccine has become an important measure to prevent H. pylori infection. METHODS: PubMed and ClinicalTrials.gov were systematically searched from January 1980 to March 2023 with search terms-H. pylori vaccine, adjuvants, immunization, pathogenesis, and H. pylori eradication in the title and/or abstract of literature. A total of 5182 documents were obtained. Based on the principles of academic reliability, authority, nearly publicated, and excluded the similar documents, finally, 75 documents were selected, organized, and analyzed. RESULTS: Most of the candidate antigens used as H. pylori vaccines in these literatures are whole-cell antigens and virulence antigens such as UreB, VacA, CagA, and HspA, and the main types of vaccines for H. pylori are whole bacteria vaccines, vector vaccines, subunit vaccines, nucleic acid vaccines, epitope vaccines, etc. Some vaccines have shown good immune protection in animal trials; however, few vaccines show good in clinical trials. The only H. pylori vaccine passed phase 3 clinical trial is a recombinant subunit vaccine using Urease subunit B (UreB) as the vaccine antigen, and it shows good prophylactic effects. Meanwhile, the adjuvant system for vaccines against this bacterium has been developed considerably. In addition to the traditional mucosal adjuvants such as cholera toxin (CT) and E. coli heat labile enterotoxin (LT), there are also promising safer and more effective mucosal adjuvants. All these advances made safe and effective H. pylori vaccines come into service as early as possible. CONCLUSIONS: This review briefly summarized the advances of H. pylori vaccines from two aspects, candidates of antigens and adjuvants, to provide references for the development of vaccine against this bacterium. We also present our prospects of exosomal vaccines in H. pylori vaccine research, in the hope of inspiring future researchers.

Application of 18F-FDG PET metabolic parameters in evaluating histopathologic grading of soft tissue sarcoma / 中华核医学与分子影像杂志

Objective:To evaluate the value of 18F-FDG PET metabolic parameters in predicting histopathological grade of soft tissue sarcoma (STS). Methods:From December 2012 to December 2021, 51 patients (26 males, 25 females, age range: 32-84 years) who underwent 18F-FDG PET/CT imaging before treatment and confirmed STS pathologically in the First Affiliated Hospital of Dalian Medical University were retrospectively collected. 18F-FDG PET metabolic parameters SUV max, metabolic tumor volume (MTV), total lesion glycolysis (TLG) and intertumoral FDG uptake heterogeneity (IFH) were measured. Kruskal-Wallis rank sum test was used to analyze the differences in metabolic parameters among different groups and Spearman rank correlation analysis was used to analyze the correlation of each metabolic parameter and histological grade. Logistic regression was used to screen and construct the prediction model for high-grade STS. ROC curve was plotted and Delong test was used to analyze the differences among AUCs. Results:The metabolic parameters SUV max, MTV, TLG and IFH were significantly different among French Federation of Cancer Centers Sarcoma Group (FNCLCC)Ⅰ( n=8), Ⅱ( n=10) and Ⅲ ( n=33) grade groups ( H values: 16.24, 10.52, 19.29 and 16.99, all P<0.05), and each metabolic parameter was positively correlated with histological grade ( rs values: 0.58, 0.45, 0.52, and 0.62, all P<0.05). Multivariate logistic regression analysis showed that SUV max(odds ratio ( OR)=1.27, 95% CI: 1.06-1.51, P=0.009) and IFH ( OR=6.83, 95% CI: 1.44-32.27, P=0.015) were independent risk indicators for high-grade STS. The prediction model constructed by combining SUV max and IFH had better diagnostic efficacy for differentiating high-grade STS with the AUC of 0.93, and the sensitivity of 93.9%(31/33) and the specificity of 16/18, respectively. The AUC of prediction model was significant different from SUV max, MTV, TLG and IFH (AUCs: 0.81, 0.78, 0.86 and 0.85; z values: 2.69, 2.53, 1.94 and 1.97, all P<0.05). Conclusions:The metabolic parameters SUV max, MTV, TLG and IFH are valuable predictors for histological grade of STS. The combination of SUV max and IFH may be a more meaningful method than using each of the above metabolic parameters alone.

Expression of alcohol dehydrogenase 1A and vascular endothelial growth factor-A in hepatocellular carcinoma / 安徽医科大学学报

Objective @#To investigate the expression , synergistic relationship and clinical significance of alcohol dehydrogenase (ADH1A) and vascular endothelial growth factor-A (VEGFA) in hepatocellular carcinoma (HCC) . @*Methods @#The expression and correlation of ADH1A and VEGFA in HCC and adjacent normal tissues were ana lyzed by GEPIA . TCGA and GSEA were used to analyze the pathway of ADH1A in HCC . The clinical and patho logical data of 84 patients with HCC were collected , and 54 patients with paracancer normal tissue samples were se lected as controls to analyze the correlation between ADH1A and VEGFA and clinicopathological parameters of HCC . Immunohistochemistry was used to detect the protein expression of ADH1A and VEGFA in cases and con trols , and the correlation between the expression of ADH1A and VEGFA and the clinical progression and prognosis of patients with HCC was analyzed based on clinical pathological parameters and Kaplan Meier.@*Results @#Bioinfor matics analysis found that ADH1A was low expressed in HCC and VEGFA was highly expressed in HCC , and there was a negative correlation between the two ( P < 0.001) ; immunohistochemical detection results showed that the expression of ADH1A in HCC tissue was lower than that in normal tissue adjacent to cancer (P < 0.01) while the expression rate of VEGFA in HCC tissue was significantly higher than that of normal tissue adjacent to cancer (P < 0.01) ; The recurrence rate of vascular thrombus and HCC patients in HCC group with high expression of ADH1A was lower (P < 0.05) . The proportion of tumor diameter > 5 cm , high TNM stage , microsatellite and G2 G3 dif ferentiation in HCC tissues in VEGFA high expression group was higher (P < 0.05) . Kaplan Meier survival analy sis showed that patients with high ADH1A expression and low VEGFA expression had a higher five year survival rate .@*Conclusion @#Low expression of ADH1A and high expression of VEGFA in tumor tissues of patients with HCC indicate tumor progression and can be used as one of the prognostic evaluation indicators for patients with HCC .

Targeting Ferroptosis to Enhance Radiosensitivity of Glioblastoma / 生物化学与生物物理进展

Glioblastoma (GBM), one of the most common malignant tumors in the central nervous system (CNS), is characterized by diffuse and invasive growth as well as resistance to various combination therapies. GBM is the most prevalent type with the highest degree of malignancy and the worst prognosis. While current clinical treatments include surgical resection, radiotherapy, temozolomide chemotherapy, novel molecular targeted therapy, and immunotherapy, the median survival time of GBM patients is only about one year. Radiotherapy is one of the important treatment modalities for GBM, which relies on ionizing radiation to eradicate tumor cells. Approximately 60% to 70% of patients need to receive radiotherapy as postoperative radiotherapy or neoadjuvant radiotherapy during the treatment process. However, during radiotherapy, the radioresistant effect caused by DNA repair activation and cell apoptosis inhibition impedes the therapeutic effect of malignant glioblastoma.Ferroptosis was first proposed by Dr. Brent R. Stockwell in 2012. It is an iron-dependent mode of cell death induced by excessive lipid peroxidation. Although the application of ferroptosis in tumor therapy is still in the exploratory stage, it provides a completely new idea for tumor therapy as a novel form of cell death. Ferroptosis has played a significant role in the treatment of GBM. Specifically, research has revealed the key processes of ferroptosis occurrence, including intracellular iron accumulation, reactive oxygen species (ROS) generation, lipid peroxidation, and a decrease in the activity of the antioxidant system. Among them, glutathione peroxidase 4(GPX4) in the cytoplasm and mitochondria, ferroptosis suppressor protein 1 (FSP1) on the plasma membrane, and dihydroorotate dehydrogenase (DHODH) in the mitochondria constitute an antioxidant protection system against ferroptosis. In iron metabolism, nuclear receptor coactivator 4 (NCOA4) can mediate ferritin autophagy to regulate intracellular iron balance based on intracellular iron content. Heme oxygenase1 (HMOX1) catalyzes heme degradation to release iron and regulate ferroptosis. Radiation can trigger ferroptosis by generating ROS, inhibiting the signaling axis of the antioxidant system, depleting glutathione, upregulating acyl-CoA synthase long chain family member 4 (ACSL4), and inducing autophagy. Interestingly, some articles has documented that exposure to low doses of radiation (6 Gy for 24 h or 8 Gy for 4-12 h) can induce the expression of SLC7A11 and GPX4 in breast cancer and lung cancer cells, leading to radiation resistance, while radiation-induced ferroptosis occurs after 48 h. In contrast, high doses of ionizing radiation (20 Gy and 50 Gy) increase lipid peroxidation after 24 h. This suggests that radiation-induced oxidative stress is a double-edged sword that can regulate ferroptosis in both directions, and the ultimate fate of cells after radiation exposure——developing resistance and achieving homeostasis or undergoing ferroptosis——depends on the degree and duration of membrane lipid damage caused by the radiation dose. In addition, during the process of radiotherapy, methods such as inducing iron overload, damaging the antioxidant system, and disrupting mitochondrial function are used to target ferroptosis, thereby enhancing the radiosensitivity of glioblastoma. By promoting the occurrence of ferroptosis in tumor cells as a strategy to improve radiotherapy sensitivity, we can enhance the killing effect of ionizing radiation on tumor cells, thus providing more treatment options for patients with glioblastoma. In this paper, we reviewed ferroptosis and its mechanism, analyzed the molecular mechanism of radiation-induced ferroptosis, and discussed the effective strategies to regulate ferroptosis in enhancing the sensitivity of radiotherapy, with a view to providing an important reference value for improving the current status of glioblastoma treatment.

Silk fibroin methacryloyl hydrogel loaded with Scriptaid regulates polarization of microglia cells / 中国组织工程研究

BACKGROUND:Microglia cells play a major role in maintaining the balance as well as the development and function reconstruction of the central nervous system.As a histone deacetylase inhibitor,Scriptaid can inhibit neuroinflammation and enhance neuroprotection. OBJECTIVE:To investigate the effect of silk fibroin methacryloyl hydrogel loaded with Scriptaid on the behaviors of microglia cells. METHODS:Microglia(BV2 cells)were cultured in medium containing different concentrations of Scriptaid(0,0.1,0.5,1,2,5,10 μmol/L).The optimal concentration of Scriptaid was screened by the CCK-8 assay and live/dead cell staining.Silk fibroin methacryloyl hydrogels loaded with or without Scriptaid were prepared using photocuring.The micromorphology,swelling properties,mechanical properties,slow release properties,and hydrophilicity of the hydrogels were characterized.Microglia(BV2 cells)were inoculated in the subventricular region of 24-well Transwell and cultured in five groups.In the control group,the cell culture medium was added to the lower chamber.In the lipopolysaccharide group,Scriptaid group,hydrogel group,and drug-loaded hydrogel group,cell culture media containing lipopolysaccharide were added into the lower chamber.After 24 hours of lipopolysaccharide intervention,in the Scriptaid group,hydrogel group and drug-loaded hydrogel group,Scriptaid,silk fibroin methacryloyl hydrogel,and silk fibroin methacryloyl hydrogel loaded with Scriptaid were added to the upper chamber,respectively.The culture medium was replaced with ordinary culture medium and continued to culture for 24 hours.The cell viability was detected by CCK-8 assay,and the cell phenotype was detected by immunofluorescence staining of induced nitric oxide synthase and arginase 1. RESULTS AND CONCLUSION:(1)Compared with the group without Scriptaid,the viability and number of BV2 cells were decreased after Scriptaid added.When Scriptaid 2 μmol/L or above was added,the cell viability was lower than the standardized cell viability(70%),and the number of BV2 cells was significantly reduced.Therefore,1 μmol/L Scriptaid was selected to be loaded into the hydrogel.(2)Characterization experiments showed that the addition of Scriptaid did not affect the microscopic morphology,swelling rate of water absorption,compression modulus and hydrophilicity of silk fibroin methacryloyl hydrogel,and silk fibroin methacryloyl hydrogel had slow release performance.(3)The result of CCK-8 assay showed that compared with the control group,silk fibroin methacryloyl hydrogel significantly increased the viability of BV2 cells(P<0.001).(4)Immunofluorescence staining showed that compared with the control group,the expression of inducible nitric oxide synthase was increased in the lipopolysaccharide group(P<0.01);the expression of inducible nitric oxide synthase was decreased(P<0.01)and the expression of arginase 1 was increased(P<0.001)in the drug-loaded hydrogel group;the expression of arginase 1 was increased in the Scriptaid group(P<0.01).(5)The results indicate that Scriptaid-loaded silk fibroin methacryloyl hydrogel is able to promote polarization of microglia to the M2 type after lipopolysaccharide induction.

Comparison of intraoperative balanced salt solution injection or postoperative air filling for superior bullous retinal detachment / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To observe the outcome of intravitreal balanced salt solution(BSS)injection to increase intraocular pressure(IOP)after extrascleral subretinal fluid drainage, then scleral buckling(SB)to treat superior bullous retinal detachment(SBRD), and compare it with the effect of conventional surgery(without any intravitreal filling)and postoperative air filling.METHODS: Retrospective case-control study. A total of 72 patients(73 eyes)who underwent SB for SBRD from January 2018 to December 2022 in ophthalmology department of Xijing Hospital were included. The extrascleral subretinal fluid drainage was performed in all eyes. According to whether intravitreal injection was performed and different injections, patients were divided into three groups: with 24 cases(24 eyes)in the conventional group(no intravitreal injection), 23 cases(23 eyes)in the air group(sterile air was injected after surgery), and 25 cases(26 eyes)in the BSS group(BSS was injected during extrascleral subretinal fluid drainage). All patients were followed up until subretinal fluid was absorbed completely. The average surgery time, postoperative IOP, retinal reattachment rate, subretinal fluid absorption, visual acuity(LogMAR)and major complications were compared.RESULTS: All surgeries were completed successfully. The average surgery time of the conventional group, air group and BSS group were 63.17±13.22, 61.65±15.55 and 57.30±11.70 min, respectively. There had no significant difference among these groups(F=0.825, P=0.443). On the first post-operative day, the average IOP of the conventional group, air group and BSS group were 13.69±2.69, 16.40±2.86 and 18.35±2.88 mmHg, respectively. The average IOP of the air group and the BSS group were significant higher than that of the conventional group(F=17.18, P&#x003C;0.001). Primary reattachment rates were 88%, 96%, and 100%, respectively. The postoperative BCVA was 0.71±0.42, 0.59±0.44, and 0.91±0.50, respectively, which were significantly higher than those before operation(all P&#x003C;0.05), but there was no significant difference among groups(F=3.046, P&#x003E;0.05). The main complications included subretinal hemorrhage in 1 eye from the conventional group and 1 eye from the air group, and a new retinal tear in 1 eye from the air group, resulting in localized retinal detachment.CONCLUSIONS: For SBRD patients with hypotony during SB surgery, intravitreal injection of BSS to properly increase the IOP and then complete the surgery can improve the reattachment rate and reduce postoperative complications. This method is safe and effective for selected SBRD patients.

Identification of Linear Epitopes in the C-Terminal Region of ASFV p72 Protein.

African swine fever, which is induced by the African swine fever virus (ASFV), poses a significant threat to the global pig industry due to its high lethality in domestic pigs and wild boars. Despite the severity of the disease, there is a lack of effective vaccines and drugs against the ASFV. The p72 protein, constituting 31 to 33% of the total virus particle mass, serves as the primary capsid protein of ASFV. It is a crucial antigen for the development of ASF subunit vaccines and serological diagnostic methods. In this investigation, 27 monoclonal antibodies (mAbs) were generated through mouse immunization with the truncated C-terminal p72 protein expressed by Escherichia coli. Among these, six mAbs exhibited binding to the p72 trimer, with their respective recognized epitopes identified as 542VTAHGINLIDKF553, 568GNAIKTP574, and 584FALKPREEY592. All three epitopes were situated within the interval sequences of functional units of the C-terminal jelly-roll barrel of p72. Notably, two epitopes, 568GNAIKTP574 and 584FALKPREEY592, were internal to the p72 trimer, while the epitope 542VTAHGINLIDKF553 was exposed on the surface of the trimer and consistently conserved across all ASFV genotypes. These findings enhance our comprehension of the antigenic function and structure of the p72 protein, facilitating the utilization of p72 in the development of diagnostic techniques for ASFV.

The CD2v protein of African swine fever virus inhibits macrophage migration and inflammatory cytokines expression by downregulating EGR1 expression through dampening ERK1/2 activity.

African swine fever virus (ASFV) is a highly contagious and deadly virus that leads to high mortality rates in domestic swine populations. Although the envelope protein CD2v of ASFV has been implicated in immunomodulation, the molecular mechanisms underlying CD2v-mediated immunoregulation remain unclear. In this study, we generated a stable CD2v-expressing porcine macrophage (PAM-CD2v) line and investigated the CD2v-dependent transcriptomic landscape using RNA-seq. GO terms enrichment analysis and gene set enrichment analysis revealed that CD2v predominantly affected the organization and assembly process of the extracellular matrix. Wound healing and Transwell assays showed that CD2v inhibited swine macrophage migration. Further investigation revealed a significant decrease in the expression of transcription factor early growth response 1 (EGR1) through inhibiting the activity of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Notably, EGR1 knockout in swine macrophages restricted cell migration, whereas EGR1 overexpression in PAM-CD2v restored the ability of macrophage migration, suggesting that CD2v inhibits swine macrophage motility by downregulating EGR1 expression. Furthermore, we performed chromatin immunoprecipitation and sequencing for EGR1 and the histone mark H3K27 acetylation (H3K27ac), and we found that EGR1 co-localized with the activated histone modification H3K27ac neighboring the transcriptional start sites. Further analysis indicated that EGR1 and H3K27ac co-occupy the promoter regions of cell locomotion-related genes. Finally, by treating various derivatives of swine macrophages with lipopolysaccharides, we showed that depletion of EGR1 decreased the expression of inflammatory cytokines including TNFα, IL1α, IL1ß, IL6, and IL8, which play essential roles in inflammation and host immune response. Collectively, our results provide new insights into the immunomodulatory mechanism of ASFV CD2v.

PGAM5 degrades PDCoV N protein and activates type I interferon to antagonize viral replication.

IMPORTANCE: As a member of the δ-coronavirus family, porcine deltacoronavirus (PDCoV) is a vital reason for diarrhea in piglets, which can contribute to high morbidity and mortality rates. Initially identified in Hong Kong in 2012, the virus has rapidly spread worldwide. During PDCoV infection, the virus employs evasion mechanisms to evade host surveillance, while the host mounts corresponding responses to impede viral replication. Our research has revealed that PDCoV infection down-regulates the expression of PGAM5 to promote virus replication. In contrast, PGAM5 degrades PDCoV N through autophagy by interacting with the cargo receptor P62 and the E3 ubiquitination ligase STUB1. Additionally, PGAM5 interacts with MyD88 and TRAF3 to activate the IFN signal pathway, resulting in the inhibition of viral replication.

PEDV N protein capture protein translation element PABPC1 and eIF4F to promote viral replication.

Porcine epidemic diarrhea (PED) is an acute, highly infectious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), which seriously endangers the healthy development of the pig industry. PEDV N protein is the most abundant viral structural protein, which can be combined with viral genomic RNA to form ribonucleoprotein complexes, thereby participating in the transcription and replication of the virus. However, how PEDV hijacks the host transcription translation system to promote viral proliferation remains unclear. In this study, we found that there is an interaction between PEDV N, polyadenylate-binding protein cytoplasmic 1 (PABPC1) and eukaryotic initiation factor 4F (eIF4F) proteins through coimmunoprecipitation, GST pulldown and fluorescence microscopy experiments. PABPC1 could bind to the poly(A) tail of the mRNA, and eIF4F could bind to the 5' end cap structure of the mRNA, so the interaction of PABPC1 and eIF4F could facilitate mRNA forming a circular shape to promote translation to the proteins. To further explore the effect of N protein capture protein translation element PABPC1 and eIF4F on PEDV replication, we overexpressed PABPC1, eIF4F (containing eIF4A, eIF4E and eIF4G) separately on Vero cells and LLC-PK1 cells, and we found that the PABPC1 and eIF4F protein could promote PEDV replication. Taken together, our data suggested that PEDV N protein promoted cyclization of viral mRNA carried by N protein through binding with PABPC1 and eIF4F proteins, thus promoting viral transcription and facilitating viral replication.

Expression of ASFV p17 in CHO cells and identification of one novel epitope using a monoclonal antibody.

As a highly pathogenic large DNA virus, African swine fever virus (ASFV) has huge particles and numerous encoded proteins. At present, few of the existing studies on ASFV proteins have investigated the function of p17. Specific antibodies against p17 to promote the development of prevention techniques against African swine fever (ASF) are urgently needed. Herein, we successfully expressed ASFV p17 in CHO cells using a suspension culture system and generated a monoclonal antibody (mAb) against p17. The mAb recognized a novel linear epitope (8LLSHNLSTREGIK20) and exhibited specific reactivity, which was conducive to the identification of ectopically expressed p17, the recombinant porcine reproductive and respiratory syndrome virus expressing p17, and the ASFV-SY18. The epitope was conservative among genotype I and genotype II ASFV strains. Overall, the mAb against p17 revealed efficient detection and promising application prospects, making it a useful tool for future vaccine research on ASF. Determination of the conserved linear epitope of p17 would contribute to the in-depth exploration of the biological function of ASFV antigen protein.

PTBP1 suppresses porcine epidemic diarrhea virus replication via inducing protein degradation and IFN production.

Porcine epidemic diarrhea virus (PEDV) causes severe morbidity and mortality among newborn piglets. It significantly threatens the porcine industry in China and around the globe. To accelerate the developmental pace of drugs or vaccines against PEDV, a deeper understanding of the interaction between viral proteins and host factors is crucial. The RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1), is crucial for controlling RNA metabolism and biological processes. The present work focused on exploring the effect of PTBP1 on PEDV replication. PTBP1 was upregulated during PEDV infection. The PEDV nucleocapsid (N) protein was degraded through the autophagic and proteasomal degradation pathways. Moreover, PTBP1 recruits MARCH8 (an E3 ubiquitin ligase) and NDP52 (a cargo receptor) for N protein catalysis and degradation through selective autophagy. Furthermore, PTBP1 induces the host innate antiviral response via upregulating the expression of MyD88, which then regulates TNF receptor-associated factor 3/ TNF receptor-associated factor 6 expression and induces the phosphorylation of TBK1 and IFN regulatory factor 3. These processes activate the type Ⅰ IFN signaling pathway to antagonize PEDV replication. Collectively, this work illustrates a new mechanism related to PTBP1-induced viral restriction, where PTBP1 degrades the viral N protein and induces type Ⅰ IFN production to suppress PEDV replication.

Screening and identification of the dominant antigens of the African swine fever virus.

African swine fever is a highly lethal contagious disease of pigs for which there is no vaccine. Its causative agent African swine fever virus (ASFV) is a highly complex enveloped DNA virus encoding more than 150 open reading frames. The antigenicity of ASFV is still unclear at present. In this study, 35 proteins of ASFV were expressed by Escherichia coli, and ELISA was developed for the detection of antibodies against these proteins. p30, p54, and p22 were presented as the major antigens of ASFV, positively reacting with all five clinical ASFV-positive pig sera, and 10 pig sera experimentally infected by ASFV. Five proteins (pB475L, pC129R, pE199L, pE184L, and pK145R) reacted well with ASFV-positive sera. The p30 induced a rapid and strong antibody immune response during ASFV infection. These results will promote the development of subunit vaccines and serum diagnostic methods against ASFV.

N protein of PEDV plays chess game with host proteins by selective autophagy.

Macroautophagy/autophagy is a cellular degradation and recycling process that maintains the homeostasis of organisms. The protein degradation role of autophagy has been widely used to control viral infection at multiple levels. In the ongoing evolutionary arms race, viruses have developed various ways to hijack and subvert autophagy in favor of its replication. It is still unclear exactly how autophagy affects or inhibits viruses. In this study, we have found a novel host restriction factor, HNRNPA1, that could inhibit PEDV replication by degrading viral nucleocapsid (N) protein. The restriction factor activates the HNRNPA1-MARCHF8/MARCH8-CALCOCO2/NDP52-autophagosome pathway with the help of transcription factor EGR1 targeting the HNRNPA1 promoter. HNRNPA1 could also promote the expression of IFN to facilitate the host antiviral defense response for antagonizing PEDV infection through RIGI protein interaction. During viral replication, we found that PEDV can, in contrast, degrade the host antiviral proteins HNRNPA1 and others (FUBP3, HNRNPK, PTBP1, and TARDBP) through its N protein through the autophagy pathway. These results reveal the dual function of selective autophagy in PEDV N and host proteins, which could promote the ubiquitination of viral particles and host antiviral proteins and degradation both of the proteins to regulate the relationship between virus infection and host innate immunity.Abbreviations: 3-MA: 3-methyladenine; ATG: autophagy related; Baf A1: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; GPI: glycosyl-phosphatidylinositol; hpi: hours post infection; MARCHF8/MARCH8: membrane-associated ring-CH-type finger 8; MOI: multiplicity of infection; N protein: nucleocapsid protein; PEDV: porcine epidemic diarrhea virus; siRNA: small interfering RNA; TCID50: 50% tissue culture infectious doses.

The rPRRSV-E2 strain exhibited a low level of potential risk for virulence reversion.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Classical Swine Fever Virus (CSFV) are two important pathogens, which cause serious impact on swine industry worldwide. In our previous research, rPRRSV-E2, the recombinant PRRSV expressing CSFV E2 protein, could provide sufficient protection against the lethal challenge of highly pathogenic PRRSV and CSFV, and could maintained genetically stable in vitro. Here, to evaluate the virulence reversion potential risk, rPRRSV-E2 had been continuously passaged in vivo, the stability of E2 expression and virulence of the passage viruses were analyzed. The results showed that no clinical symptoms or pathological changes could be found in the inoculated groups, and there were no significant differences of viraemia among the test groups. Sequencing and IFA analysis showed that the coding gene of exogenous CSFV E2 protein existed in the passaged viruses without any sequence mutations, deletions or insertions, and could expressed steadily. It could be concluded that the foreign CSFV E2 gene in the genome of rPRRSV-E2 could be maintained genetically stable in vivo, and rPRRSV-E2 strain had relatively low level of potential risk for virulence reversion.

PSMB1 Inhibits the Replication of Porcine Reproductive and Respiratory Syndrome Virus by Recruiting NBR1 To Degrade Nonstructural Protein 12 by Autophagy.

The nonstructural proteins (Nsps) of porcine reproductive and respiratory syndrome virus (PRRSV) play essential roles in virus replication-a multistep process that requires the participation of host factors. It is of great significance for the development of antiviral drugs to characterize the host proteins that interact with PRRSV Nsps and their functions in PRRSV replication. Here, we determined that proteasome subunit ß type 1 (PSMB1) interacted with viral Nsp12 to inhibit PRRSV replication in target and permissive cells. PSMB1 could be downregulated by PRRSV infection through interaction with the transcription factor EBF1. Proteasome and autophagy inhibitor assays showed that PSMB1 was regulated by the autophagic pathway to degrade Nsp12. Cotransfection of PSMB1 and Nsp12 increased the level of intracellular autophagy; both molecules were colocated in lysosomes. We also found that the selective autophagy cargo receptor protein NBR1 and E3 ubiquitin ligase STUB1 interacted with PSMB1 and Nsp12, respectively, in the autophagic degradation of Nsp12. Furthermore, the degradation of Nsp12 by PSMB1 was mainly dependent on the ubiquitination of Nsp12 at lysine site 130. Our results indicate for the first time that PSMB1 is an anti-PRRSV host protein that inhibits the replication of PRRSV by degradation of Nsp12 through the selective autophagy pathway. IMPORTANCE PRRS is a major threat to the global pig industry and urgently requires an effective and sustainable control strategy. PRRSV Nsps have important roles in viral RNA synthesis, proteinase activity, induction of replication-associated membrane rearrangements, replicative endoribonuclease activity, determination of virulence, and regulation of host immune response. Research associated with PRRSV Nsps can provide vital guidance to modify the PRRSV genome through reverse genetics in the development of vaccines and diagnostics. The function of Nsp12, which generally plays essential roles in virus replication, remains unclear. We demonstrated that PSMB1 interacted with and degraded Nsp12 through an autophagic pathway to inhibit PRRSV replication. Our data confirmed a novel antiviral function of PSMB1 and allowed us to elaborate on the roles of Nsp12 in PRRSV pathogenesis. These findings suggest a valid and highly conserved candidate target for the development of novel therapies and more effective vaccines and demonstrate the complex cross talk between selective autophagy and PRRSV infection.

PRPF19 Limits Porcine Epidemic Diarrhea Virus Replication through Targeting and Degrading Viral Capsid Protein.

Porcine epidemic diarrhea (PED) indicates the disease of the acute and highly contagious intestinal infection due to porcine epidemic diarrhea virus (PEDV), with the characteristics of watery diarrhea, vomiting, and dehydration. One of the reasons for diarrhea and death of piglets is PEDV, which leads to 100% mortality in neonatal piglets. Therefore, it is necessary to explore the interaction between virus and host to prevent and control PEDV. This study indicated that the host protein, pre-mRNA processing factor 19 (PRPF19), could be controlled by the signal transducer as well as activator of transcription 1 (STAT1). Thus, PEDV replication could be hindered through selective autophagy. Moreover, PRPF19 was found to recruit the E3 ubiquitin ligase MARCH8 to the N protein for ubiquitination. For the purpose of degradation, the ubiquitin N protein is acknowledged by the cargo receptor NDP52 and transported to autolysosomes, thus inhibiting virus proliferation. To conclude, a unique antiviral mechanism of PRPF19-mediated virus restriction was shown. Moreover, a view of the innate immune response and protein degradation against PEDV replication was provided in this study. IMPORTANCE The highly virulent porcine epidemic diarrhea virus (PEDV) emerged in 2010, and causes high mortality rates in newborn pigs. There are no effective and safe vaccines against the highly virulent PEDV. This virus has caused devastating economic losses in the pork industry worldwide. Studying the relationship between virus and host antiviral factors is important to develop the new antiviral strategies. This study identified the pre-mRNA processing factor 19 (PRPF19) as a novel antiviral protein in PEDV replication and revealed its viral restriction mechanisms for the first time. PRPF19 recruited the E3 ubiquitin ligase MARCH8 to the PEDV N protein for ubiquitination, and the ubiquitin N protein was acknowledged by the cargo receptor NDP52 and transported to autolysosomes for degradation. Our findings provide new insights in host antiviral factors PRPF19 that regulate the selective autophagy protein degradation pathway to inhibit PEDV replication.