Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-caused coronavirus disease 2019 (COVID-19) is a global crisis with no satisfactory therapies. Vitamin D3 (VD3) is considered a potential candidate for COVID-19 treatment; however, little information is available regarding the exact effects of VD3 on SARS-CoV-2 infection and the underlying mechanism. Herein, we confirmed that VD3 reduced SARS-CoV-2 nucleocapsid (N) protein-caused hyperinflammation in human bronchial epithelial (HBE) cells. Meanwhile, VD3 inhibited the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation in N protein-overexpressed HBE (HBE-N) cells. Notably, the inhibitors of caspase-1, NLRP3, and NLRP3 or caspase-1 small interference RNA (siRNA) enhanced VD3-induced NLRP3 inflammasome inactivation, with subsequent suppression of interleukin-6 (IL6) and IL1 & beta; release in HBE-N cells, which were abolished by the NLRP3 agonist. Moreover, VD3 increased NLRP3 ubiquitination (Ub-NLRP3) expression and the binding of the VDR with NLRP3, with decreased BRCA1/BRCA2-containing complex subunit 3 (BRCC3) expression and NLRP3-BRCC3 association. VD3-induced Ub-NLRP3 expression, NLRP3 inflammasome inactivation, and hyperinflammation inhibition were improved by the BRCC3 inhibitor or BRCC3 siRNA, which were attenuated by the vitamin D receptor (VDR) antagonist or VDR siRNA in HBE-N cells. Finally, the results of the in vivo study in AAV-Lung-enhanced green fluorescent protein-N-infected lungs were consistent with the findings of the in vitro experiment. In conclusion, VD3 attenuated N protein-caused hyperinflammation by inactivating the NLRP3 inflammasome partially through the VDR-BRCC3 signaling pathway.

oxidative/ER stress-mediated apoptosis and pyroptosis 3 Xin Le, Junhao Mu, Weiyan Peng, Jun Tang, Qin Xiang, Shaorong Tian, Yixiao 4 Feng, Sanxiu He, Zhu Qiu, Guosheng Ren, Ailong Huang, Yong Lin, Qian Tao, 5 Tingxiu Xiang 6 7 Key Laboratory of Molecular Oncology and Epigenetics, The First Affiliated 8 Hospital of Chongqing Medical University, Chongqing, China; 9 MOE Key Laboratory of Molecular Biology for Infectious Diseases, 10 Department of Infectious Disease, Chongqing Medical University, China; 11 Lovelace Respiratory Research Institute, Albuquerque, New Mexico, USA 12 Cancer Epigenetics Laboratory, Department of Clinical Oncology, State Key 13 Laboratory of Translational Oncology, Sir YK Pao Center for Cancer and Li Ka 14 Shing Institute of Health Sciences, The Chinese University of Hong Kong, 15 Hong Kong. 16 17

目的:建立干扰素-γ(interferon-γ,IFN-γ)基因敲除小鼠慢性乙型肝炎病-毒(hepatitis B virus,HBV)复制模型.方法:IFN-γ基因敲除(IFN-γ-/-)小鼠繁育并抽提组织DNA进行聚合酶链反应(PCR)及凝胶电泳鉴定基因型.IFN-γ-/-小鼠纯合子9只与野生型C57BL/6小鼠9只同时高压水注射pAAV/HBVl.2质粒,按既定时间点采血检测乙型肝炎表面抗原(hepatitis B virus surface antigen,HBsAg)、乙型肝炎e抗原(hepatitis B virus e antigen,HBeAg)和HBV DNA.血清HBsAg和HBeAg表达水平由电化学发光法进行定量检测.经抽提血清总DNA后,血清HBV DNA由定量PCR进行检测.结果:本实验室繁殖的IFN-γ-/-小鼠均为纯合子基因型.IFN-γ-/小鼠和野生型C57BL/6小鼠血清中HBsAg、HBeAg和HBV DNA持续存在,转染后第40天仍阳性.但是,IFN-γ-/-小鼠血清HBsAg表达水平高于C57BL/6野生小鼠(40天时,P=0.042); IFN-γ-/-小鼠血清HBV DNA持续高水平复制,明显高于C57BL/6野生小鼠(第25天时,P=0.012; 第40天时,P=0.039).两组小鼠血清HBeAg表达水平无差异.结论:IFN-γ-/-小鼠慢性HBV复制模型成功建立,并揭示了IFN-γ在慢性HBV感染中可抑制HBV复制.

Objective To optimize the amplification and sequencing of non structural protein 3 (NS3) gene in patients infected with hepatitis C virus (HCV) genotype 1b. Methods The genotyping after extracting HCV RNA from the serum was detected with type specific primers assay. After designing three pairs of outside primers and three pairs of inner primers, HCV NS3 (HCV genotype 1b) were amplified by a nested PCR assay (option I) with above primers and sequenced. In order to optimize effects of NS3 amplification and sequencing and to reduce the complicated operation, we improved the new nested PCR assay (option II) and redesigned the primers, including a pair of outside primers and a pair of inner primers. We amplified directly HCV NS3 (HCV genotype 1b) with improved nested PCR assay and sequenced NS3 with three forward primers and a reverse primer. Results Compared to the original nested PCR assay (option I), the improved nested PCR assay (option II) is simpler in operation and of higher efficiency. Conclusion The improved nested PCR assay (option II) is more effective and practical for amplification and sequencing of HCV NS3.

OBJECTIVE:This report aims to investigate the Toll-like receptor (TLR) signaling pathways and induced antiviral activity in hepatocytes.METHODS:We isolated primary hepatocytes from wild-type C57BL/6 mice and examined the expression of TLR by realtime RT-PCR. Hepatocytes were stimulated with TLR 1-9 agonists and the supernatants were harvested. The secretion of cytokines were tested by ELISA. The antiviral effectors in supernatants were assayed via virus protection assay (in EMCV system) and the control of HBV replication were assessed via Southern blotting (in HBV system).RESULTS:We demonstrated that hepatocytes expressed TLR1-9. In accordance with these TLR expression profiles, hepatocytes responded to all TLR ligands by producing inflammatory cytokines (TNF-α or IL-6), to TLR -1,-3,-7 and -9 ligands by producing type I IFN (IFN-α or IFN-β). Only TLR 3 and TLR 7 agonists could stimulate the production of high amounts of antiviral mediators by hepatocytes in virus protection assay. By contrast, supernatants from TLR1, -3 and -4 directly stimulated hepatocytes and TLR 3, -7 and -9 transfected hepatocytes were able to potently suppress HBV replication.CONCLUSION:Primary hepatocytes display a unique TLR signaling pathway and can control HBV replication after stimulation by TLR agonists in mice. It may be helpful for the development of TLR-based therapeutic approaches against hepatotropic virus.