Publications by authors named "Zhongtian Qi"

53 Publications

High-Throughput Screening of FDA-Approved Drug Library Reveals Ixazomib Is a Broad-Spectrum Antiviral Agent against Arboviruses.

Viruses 2022 06 24;14(7). Epub 2022 Jun 24.

Department of Microbiology, Navy Medical University, Shanghai 200433, China.

The emergence of significant arboviruses and their spillover transmission to humans represent a major threat to global public health. No approved drugs are available for the treatment of significant arboviruses in circulation today. The repurposing of clinically approved drugs is one of the most rapid and promising strategies in the identification of effective treatments for diseases caused by arboviruses. Here, we screened small-molecule compounds with anti-tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus activity from 2580 FDA-approved drugs. In total, 60 compounds showed antiviral efficacy against all four of the arboviruses in Huh7 cells. Among these compounds, ixazomib and ixazomib citrate (inhibitors of 20S proteasome β5) exerted antiviral effects at a low-micromolar concentration. The time-of-drug-addition assay suggested that ixazomib and ixazomib citrate disturbed multiple processes in viruses' life cycles. Furthermore, ixazomib and ixazomib citrate potently inhibited chikungunya virus replication and relieved virus-induced footpad swelling in a mouse model. These results offer critical information which supports the role of ixazomib as a broad-spectrum agent against arboviruses.
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http://dx.doi.org/10.3390/v14071381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9322861PMC
June 2022

Mosquito-Borne Flaviviruses and Current Therapeutic Advances.

Viruses 2022 06 5;14(6). Epub 2022 Jun 5.

Department of Microbiology, Faculty of Naval Medicine, Naval Medical University, Shanghai 200433, China.

Mosquito-borne flavivirus infections affect approximately 400 million people worldwide each year and are global threats to public health. The common diseases caused by such flaviviruses include West Nile, yellow fever, dengue, Zika infection and Japanese encephalitis, which may result in severe symptoms and disorders of multiple organs or even fatal outcomes. Till now, no specific antiviral agents are commercially available for the treatment of the diseases. Numerous strategies have been adopted to develop novel and promising inhibitors against mosquito-borne flaviviruses, including drugs targeting the critical viral components or essential host factors during infection. Research advances in antiflaviviral therapy might optimize and widen the treatment options for flavivirus infection. This review summarizes the current developmental progresses and involved molecular mechanisms of antiviral agents against mosquito-borne flaviviruses.
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http://dx.doi.org/10.3390/v14061226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9229039PMC
June 2022

Antiviral efficacy of selective estrogen receptor modulators against SARS-CoV-2 infection in vitro and in vivo reveals bazedoxifene acetate as an entry inhibitor.

J Med Virol 2022 Jun 22. Epub 2022 Jun 22.

Department of Microbiology, Faculty of Naval Medicine, Naval Medical University, Shanghai, China.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the seventh member of the coronavirus family that can infect humans. Recently, more contagious and pathogenic variants of SARS-CoV-2 have been continuously emerging. Clinical candidates with high efficacy and ready availability are still in urgent need. To identify potent anti-SARS-CoV-2 repurposing drugs, we evaluated the antiviral efficacy of 18 selective estrogen receptor modulators (SERMs) against SARS-CoV-2 infection. Six SERMs exhibited excellent anti-SARS-CoV-2 effects in Vero E6 cells and three human cell lines. Clomifene citrate, tamoxifen, toremifene citrate, and bazedoxifene acetate reduced the weight loss of hamsters challenged with SARS-CoV-2, and reduced hamster pulmonary viral load and interleukin-6 expression when assayed at 4 days postinfection. In particular, bazedoxifene acetate was identified to act on the penetration stage of the postattachment step via altering cholesterol distribution and endosome acidification. And, bazedoxifene acetate inhibited pseudoviruses infection of original SARS-CoV-2, Delta variant, Omicron variant, and SARS-CoV. These results offer critical information supporting bazedoxifene acetate as a promising agent against coronaviruses.
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http://dx.doi.org/10.1002/jmv.27951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9350378PMC
June 2022

Renaming the SARS-CoV-2 Omicron sublineages as SARS-CoV-3 is contrary to nomenclature standards based on evolutionary and serological evidence.

Clin Transl Med 2022 Jun;12(6):e924

Key Laboratory of Medical Molecular Virology (MOE/NHC), Institute of Infectious Disease and Biosecurity, School of Basic Medical Sciences, Fudan University, Shanghai, China.

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http://dx.doi.org/10.1002/ctm2.924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9191866PMC
June 2022

Identification of clinical candidates against West Nile virus by activity screening in vitro and effect evaluation in vivo.

J Med Virol 2022 May 29. Epub 2022 May 29.

Department of Microbiology, Faculty of Naval Medicine, Navy Medical University, Shanghai, People's Republic of China.

The West Nile virus (WNV) is a member of the flavivirus and is known to cause encephalitis. There is currently no specific treatment for WNV infection. Repurposing of clinically approved drugs appeared promising for rapidly identifying effective, safe, and readily available candidates for antiviral drugs. Here, we screened the small-molecule compounds with anti-WNV activity from 978 Food Drug Administration-approved drugs. Four compounds, including cilnidipine, mycophenolate mofetil, nitazoxanide, and teriflunomide, were found to efficiently abrogate WNV infection in Vero cells and human neuroblastoma SH-SY5Y cells. The four compounds also exert broad-spectrum antiviral activity against the Zika virus, Japanese encephalitis virus, yellow fever virus, tick-borne encephalitis virus, and chikungunya virus. Furthermore, nitazoxanide (a synthetic benzamide) and teriflunomide (an inhibitor of dihydroorotate dehydrogenase, DHODH) protected 20% and 40% of mice from lethal WNV challenge, respectively. Both drugs, which are orally bioavailable and have been approved clinically for many years, may be promising therapeutics for WNV infection. Moreover, the other two DHODH inhibitors, ML390 and vidofludimus, also displayed potent activity against WNV infection in vitro and in vivo.
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http://dx.doi.org/10.1002/jmv.27891DOI Listing
May 2022

Hepatitis C Virus Infection Cycle-Specific MicroRNA Profiling Reveals Stage-Specific miR-4423-3p Targets RIG-I to Facilitate Infection.

Front Cell Infect Microbiol 2022 24;12:851917. Epub 2022 Mar 24.

Department of Microbiology, Faculty of Naval Medicine, Naval Medical University, Shanghai, China.

Hepatitis C virus (HCV) infection is one of the main causes of chronic liver diseases, the disorders of which involve multiple pathological processes and elements including host factors such as non-coding small RNAs. Although several genes have been reported to be correlated with HCV infection, the potential regulatory network has not been deciphered clearly. By small RNA sequencing, we clarified the expression profile of microRNAs (miRNAs) in HCV-infected Huh7 and Huh7.5.1 cells and identified 6 dysregulated miRNAs with the same expression trend and 32 dysregulated miRNAs with different expression trends during different stages of HCV life cycle. By looking into each infection stage, we found that 6 miRNAs were entry stage specific, 4 miRNAs were replication stage specific, and 1 miRNA was related to the transmission stage. Moreover, due to the fact that Huh7.5.1 cells have a retinoic acid-inducible gene 1 (RIG-I) mutation which causes reduced production of interferons (IFNs), we here focused on the miRNAs of different trends to decipher the RIG-I/IFN specific miRNAs. Among them, miR-4423-3p showed a significant promotive effect on HCV infection by suppressing RIG-I/IFN pathway through direct binding to RIG-I mRNA. Together, the results displayed novel insights into the miRNA regulatory networks in HCV infection and progression, thus providing a prosperous perspective into the establishment of novel therapeutic and diagnostic targets of the disease.
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http://dx.doi.org/10.3389/fcimb.2022.851917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8987439PMC
March 2022

Rifapentine is an entry and replication inhibitor against yellow fever virus both in vitro and in vivo.

Emerg Microbes Infect 2022 Dec;11(1):873-884

Department of Microbiology, Faculty of Naval Medicine, Naval Medical University, Shanghai, People's Republic of China.

Yellow fever virus (YFV) infection is a major public concern that threatens a large population in South America and Africa. No specific anti-YFV drugs are available till now. Here, we report that rifapentine is a potent YFV inhibitor in various cell lines by high-throughput drugs screening, acting at both cell entry and replication steps. Kinetic test and binding assay suggest that rifapentine interferes the viral attachment to the target cells. The application of YFV replicon and surface plasmon resonance assay indicates that rifapentine suppresses viral replication by binding to the RNA-dependent RNA polymerase (RdRp) domain of viral nonstructural protein NS5. Further molecular docking suggests that it might interact with the active centre of RdRp. Rifapentine significantly improves the survival rate, alleviates clinical signs, and reduces virus load and injury in targeted organs both in YFV-infected type I interferon receptor knockout A129 and wild-type C57 mice. The antiviral effect in vivo is robust during both prophylactic intervention and therapeutic treatment, and the activity is superior to sofosbuvir, a previously reported YFV inhibitor in mice. Our data show that rifapentine may serve as an effective anti-YFV agent, providing promising prospects in the development of YFV pharmacotherapy.
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http://dx.doi.org/10.1080/22221751.2022.2049983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8942558PMC
December 2022

Discovery of potential anti-SARS-CoV-2 drugs based on large-scale screening in vitro and effect evaluation in vivo.

Sci China Life Sci 2022 06 24;65(6):1181-1197. Epub 2021 Dec 24.

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai, 200433, China.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global crisis. Clinical candidates with high efficacy, ready availability, and that do not develop resistance are in urgent need. Despite that screening to repurpose clinically approved drugs has provided a variety of hits shown to be effective against SARS-CoV-2 infection in cell culture, there are few confirmed antiviral candidates in vivo. In this study, 94 compounds showing high antiviral activity against SARS-CoV-2 in Vero E6 cells were identified from 2,580 FDA-approved small-molecule drugs. Among them, 24 compounds with low cytotoxicity were selected, and of these, 17 compounds also effectively suppressed SARS-CoV-2 infection in HeLa cells transduced with human ACE2. Six compounds disturb multiple processes of the SARS-CoV-2 life cycle. Their prophylactic efficacies were determined in vivo using Syrian hamsters challenged with SARS-CoV-2 infection. Seven compounds reduced weight loss and promoted weight regain of hamsters infected not only with the original strain but also the D614G variant. Except for cisatracurium, six compounds reduced hamster pulmonary viral load, and IL-6 and TNF-α mRNA when assayed at 4 d postinfection. In particular, sertraline, salinomycin, and gilteritinib showed similar protective effects as remdesivir in vivo and did not induce antiviral drug resistance after 10 serial passages of SARS-CoV-2 in vitro, suggesting promising application for COVID-19 treatment.
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http://dx.doi.org/10.1007/s11427-021-2031-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8713546PMC
June 2022

A human cell-based SARS-CoV-2 vaccine elicits potent neutralizing antibody responses and protects mice from SARS-CoV-2 challenge.

Emerg Microbes Infect 2021 Dec;10(1):1555-1573

Shanghai Public Health Clinical Center & Institutes of Biomedical Sciences, Fudan University, Shanghai, People's Republic of China.

To curb the pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), multiple platforms have been employed toward a safe and highly effective vaccine. Here, we develop a novel cell-based vaccine candidate, namely K562-S, by utilizing human cell K562 as a cellular carrier to display Spike (S) protein of SARS-CoV-2 on the membrane. Analogous to the traditional inactivated vaccine, K562-S cells can be propagated to a large scale by culturing and completely lose their viability after exposure to X-ray irradiation or formalin. We in turn demonstrated high immunogenicity of formalin-inactivated K562-S vaccine in both mouse and non-human primates and its protective efficacy in mice. In mice, immunization with inactivated K562-S vaccines can elicit potent neutralizing antibody (nAb) responses persisting longer than 5 months. We consequently showed in a hACE2 mouse model of SARS-CoV-2 infection that a two-shot vaccination with adjuvanted K562-S rendered greater than 3 log reduction in viral lung load and concomitant ameliorated lung pathology. Of importance, the administration of the same regimen in non-human primates was able to induce a neutralizing antibody titer averaging three-fold higher relative to human convalescent serum. These results together support the promise of K562-based, S-protein-expressing vaccines as a novel vaccination approach against SARS-CoV-2. Importantly, with a powerful capacity to carry external genes for cell-based vectors, this platform could rapidly generate two- and multiple-valent vaccines by incorporating SARS-CoV-2 mutants, SARS-CoV, or MERS-CoV.
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http://dx.doi.org/10.1080/22221751.2021.1957400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8366622PMC
December 2021

Chikungunya virus and autoimmunity: Consensus immune epitope analysis between chikungunya virus and arthritis.

Autoimmun Rev 2021 Apr 18;20(4):102789. Epub 2021 Feb 18.

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai, China. Electronic address:

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http://dx.doi.org/10.1016/j.autrev.2021.102789DOI Listing
April 2021

Transcriptome and miRNome Analysis Provide New Insight Into Host Lipid Accumulation, Innate Immunity, and Viral Persistence in Hepatitis C Virus Infection .

Front Microbiol 2020 30;11:535673. Epub 2020 Sep 30.

Shanghai Key Laboratory of Pancreatic Diseases, Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

Hepatitis C virus (HCV)-host cell interaction during infection disturbs cellular homeostasis and culminates in pathological consequences. The processes could be first embodied in gene expression of HCV-infected cells. Here, we investigated transcriptome and miRNA expression (miRNome) alterations in HCV-infected Huh7 cells at 12, 36, and 60 h after infection to systematically explore host responses. The number of deregulated genes in the HCV-infected cells increased with infection duration. The altered biological processes at 36 h were mainly associated with stress and inflammatory response, whereas the most enriched processes at 60 h were predominantly linked to lipid metabolism. Notably, the key genes that participated in lipogenesis were downregulated, and conversely, the genes implicated in fatty acid beta-oxidation were upregulated. Reduced expression of the key genes involved in lipoprotein assembly and secretion pointed to a decreased requirement for and export of lipids, leading to lipid accumulation in HCV-infected hepatocytes. Fluctuation in the expression of host factors, innate immunity genes and transcription factors provided insight into host-directed mechanisms to control viral replication. Furthermore, miRNome presented a comprehensive expression profile of miRNAs in HCV-infected Huh7 cells. The integrated analysis of transcriptome and miRNome suggested that deregulated miR-483, miR-1303, miR-1260a, miR-27a, and miR-21 directly regulated lipid metabolical genes at 60 h. The decreased miR-122 at 60 h was indirectly involved in lipid metabolism and is expected to attenuate rampant replication of HCV and potentially contribute to viral persistence. Our results will help to gain a comprehensive understanding of the molecular mechanisms implicated in HCV-induced pathogenesis.
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http://dx.doi.org/10.3389/fmicb.2020.535673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555709PMC
September 2020

SNORD126 Promotes Hepatitis C Virus Infection by Upregulating Claudin-1 via Activation of PI3K-AKT Signaling Pathway.

Front Microbiol 2020 15;11:565590. Epub 2020 Sep 15.

Department of Microbiology, Second Military Medical University, Shanghai, China.

Hepatitis C virus (HCV) infection involves a variety of viral and host factors, some of which promote the infection process. A small nucleolar RNA, C/D box 126 (SNORD126), was previously shown to be associated with hepatocellular carcinoma (HCC). However, the role of SNORD126 in HCV infection, which is one of the primary reasons for HCC development, has not been elucidated. In the present study, using small nucleolar RNA profiling, we observed that SNORD126 was significantly downregulated during HCV infection in both Huh7 and Huh7.5.1 cells. In addition, overexpression of SNORD126 enhanced HCV entry into host cells, whereas SNORD126 knockdown showed the opposite effect, suggesting that SNORD126 promotes HCV infection, especially through viral entry. Further functional analysis revealed that SNORD126 could enhance the expression level of claudin-1 (CLDN1), a key HCV entry factor, by increasing the levels of phosphorylated AKT. Additionally, the function of SNORD126 in HCV infection was associated with ribonucleoprotein (RNP) complexes. In summary, our findings demonstrate that oncogenic SNORD126 levels are decreased during HCV infection probably due to the host defense reaction, and SNORD126 may be important to promote viral entry by increasing CLDN1 expression through activation of the PI3K-AKT pathway, the mechanism of which is partly associated with SNORD126-mediated snoRNA RNP (snoRNP) function. Our work here provides initial evidence that endogenous snoRNA takes part in HCV infection and shows potential as a diagnostic or antiviral agent.
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http://dx.doi.org/10.3389/fmicb.2020.565590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522514PMC
September 2020

Ezrin is essential for the entry of Japanese encephalitis virus into the human brain microvascular endothelial cells.

Emerg Microbes Infect 2020 12;9(1):1330-1341

Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Naval Medical University (Second Military Medical University), Shanghai, People's Republic of China.

Japanese encephalitis virus (JEV) remains the predominant cause of viral encephalitis worldwide. It reaches the central nervous system upon crossing the blood-brain barrier through pathogenic mechanisms that are not completely understood. Here, using a high-throughput siRNA screening assay combined with verification experiments, we found that JEV enters the primary human brain microvascular endot ezrin, an essential host factor for JEV entry based on our screening, in caveolae-mediated JEV internalization was investigated. We observed that JEV internalization in HBMEC is largely dependent on ezrin-mediated actin cytoskeleton polymerization. Moreover, Src, a protein predicted by a STRING database search, was found to be required in JEV entry. By a variety of pharmacological inhibition and immunoprecipitation assays, we found that Src, ezrin, and caveolin-1 were sequentially activated and formed a complex during JEV infection. A combination of kinase assay and subcellular analysis demonstrated that ezrin is essential for Src-caveolin-1 interactions. , both Src and ezrin inhibitors protected ICR suckling mice against JEV-induced mortality and diminished mouse brain viral load. Therefore, JEV entry into HBMEC requires the activation of the Src-ezrin-caveolin-1 signalling axis, which provides potential targets for restricting JEV infection.
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http://dx.doi.org/10.1080/22221751.2020.1757388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473060PMC
December 2020

A cross-sectional serum investigation of a clustering hepatitis C virus infection in Southwest China.

J Med Virol 2019 03 9;91(3):508-513. Epub 2018 Oct 9.

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai, China.

Serum samples were collected in a village with a clustering hepatitis C virus (HCV) infection. HCV antibody, HCV RNA loads, liver function indexes, HCV envelope antibody, and neutralizing activity were assessed. Among 851 adult sera, 342 samples were positive for anti-HCV. Of these positive samples, 254 (74.3%) were HCV RNA positive (≥800 copies/mL). None of the 69 children's sera were positive for HCV antibody or RNA. Among the HCV antibody positive sera, alanine aminotransferase, and aspartate aminotransferase levels increased with the higher virus loads, but decreased when virus loads were higher than 1 × 10  copies/mL. HCV envelope antibody and neutralizing antibody levels increased with viral load.
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http://dx.doi.org/10.1002/jmv.25315DOI Listing
March 2019

Zika Virus Induces Autophagy in Human Umbilical Vein Endothelial Cells.

Viruses 2018 05 15;10(5). Epub 2018 May 15.

Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai 200433, China.

Autophagy is a common strategy for cell protection; however, some viruses can in turn adopt cellular autophagy to promote viral replication. Zika virus (ZIKV) is the pathogen that causes Zika viral disease, and it is a mosquito-borne virus. However, its pathogenesis, especially the interaction between ZIKV and target cells during the early stages of infection, is still unclear. In this study, we demonstrate that infecting human umbilical vein endothelial cells (HUVEC) with ZIKV triggers cellular autophagy. We observed both an increase in the conversion of LC3-I to LC3-II and increased accumulation of fluorescent cells with LC3 dots, which are considered to be the two key indicators of autophagy. The ratio of LC3-II/GAPDH in each group was significantly increased at different times after ZIKV infection at different MOIs, indicating that the production of lipidated LC3-II increased. Moreover, both the ratio of LC3-II/GAPDH and the expression of viral NS3 protein increased with increasing time of viral infection. The expression level of p62 decreased gradually from 12 h post-infection. Expression profile of double fluorescent protein labelling LC3 indicated that the autophagy induced by ZIKV infection was a complete process. We further investigated the role of autophagy in ZIKV replication. We demonstrated that either the treatment with inhibitors of autophagosomes formation or short hairpin RNA targeting the Beclin-1 gene, which is critical for the formation of autophagosomes, significantly reduced viral production. Taken together, our results indicate that ZIKV infection induces autophagy of HUVEC, and inhibition of ZIKV-induced autophagy restrains viral replication.
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http://dx.doi.org/10.3390/v10050259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5977252PMC
May 2018

Overexpression of Annexin A2 Receptor Inhibits Neovascularization via the Promotion of Krüppel-Like Transcription Factor 2.

Cell Physiol Biochem 2018 24;46(4):1617-1627. Epub 2018 Apr 24.

Department of Ophthalmology, Changhai Hospital, Second Military Medical University, Shanghai, China.

Background/aims: Annexin A2 receptor (AX2R) can mediate annexin A2 signalling and induce apoptosis in a variety of cells, but its role in neovascularization (NV) remains unclear. Krüppel-like transcription factor 2 (KLF2) is known to be expressed in a range of cell types and to participate in a number of processes during development and disease, such as endothelial homeostasis, vasoregulation and vascular growth/remodelling. The aim of our study was to investigate the role of AX2R in NV and the plausible molecular mechanism.

Methods: We constructed a eukaryotic overexpression plasmid for AX2R (Lenti-AX2R) by using polymerase chain reaction (PCR). The full-length human AX2R gene was transfected into human retinal endothelial cells (HRECs) and human umbilical vein endothelial cells (HUVECs) using lentivirus vectors to overexpress AX2R. All experiments were divided into three groups: control, negative control (Lenti-EGFP), and Lenti-AX2R.Cell proliferation, cell migration, tube formation, mouse aortic ring assays and mouse matrigel plug assay were applied to analyse the effect of AX2R in NV. Furthermore, we conducted flow cytometry to evaluate whether AX2R could influence the cell cycle. A series of cell cycle-related proteins including cyclin A1, cyclin B1, cyclin D1, cyclin E1, CDK1, and p-CDC2 were detected by WB. The mRNA and protein levels of KLF2, vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) were further quantified by RT-PCR and WB to reveal the possible mechanism.

Results: Overexpression of AX2R significantly inhibited cell proliferation, migration and tube formation in both types of endothelial cells (ECs), HRECs and HUVECs. It also suppressed vessel sprouting in the mouse aortic ring assay and NV in mouse matrigel plug assay. Furthermore, infection with Lenti-AX2R lentivirus arrested the cell cycle in S/G2 and influenced the expression of a series of cell cycle-related proteins. We also found that the overexpression of AX2R increased the expression of KLF2, mediating VEGF and VEGFR2.

Conclusions: Overexpression of AX2R contributes to the inhibition of NV via suppressing KLF2 ubiquitin-dependent protein degradation, which might therefore be a therapeutic option for NV. It could be considered more broadly as an anti-angiogenic agent in the treatment of neovascular-related diseases in the future.
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http://dx.doi.org/10.1159/000489209DOI Listing
July 2018

H-NS represses transcription of the flagellin gene lafA of lateral flagella in Vibrio parahaemolyticus.

Can J Microbiol 2018 Jan 1;64(1):69-74. Epub 2017 Nov 1.

c State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.

Swarming motility is ultimately mediated by the proton-powered lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf genes is tightly regulated by a number of environmental conditions and regulatory factors. The nucleoid-associated DNA-binding protein H-NS is a small and abundant protein that is widely distributed in bacteria, and H-NS-like protein-dependent expression of laf genes has been identified in Vibrio cholerae and V. parahaemolyticus. The data presented here show that H-NS acts as a repressor of the swarming motility in V. parahaemolyticus. A single σ-dependent promoter was detected for lafA encoding the flagellin of the lateral flagella, and its activity was directly repressed by H-NS. Thus, H-NS represses swarming motility by directly acting on lafA. Briefly, this work revealed a novel function for H-NS as a repressor of the expression of lafA and swarming motility in V. parahaemolyticus.
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http://dx.doi.org/10.1139/cjm-2017-0315DOI Listing
January 2018

E3 Ubiquitin Ligase Nedd4 Promotes Japanese Encephalitis Virus Replication by Suppressing Autophagy in Human Neuroblastoma Cells.

Sci Rep 2017 03 28;7:45375. Epub 2017 Mar 28.

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai 200433, China.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that causes the most prevalent viral encephalitis in Asia. Since JEV is a neurotropic virus, it is important to identify key molecules that mediate JEV infection in neuronal cells and to investigate their underlying mechanisms. In this study, the critical role of Nedd4, an E3 ubiquitin ligase that is highly expressed in the central nervous system, was examined in JEV propagation. In SK-N-SH neuroblastoma cells, Nedd4 was up-regulated in response to JEV infection. Moreover, down-regulation of Nedd4 resulted in a significant decrease in JEV replication without alterations in virus attachment and internalization or in JEV pseudotyped virus infection, suggesting that Nedd4 participates in the replication but not in the entry stage of JEV infection. Further functional analysis showed that Nedd4 attenuated JEV-induced autophagy, which negatively regulates virus replication during infection. These results suggest that Nedd4 facilitates the replication of JEV by suppressing virus-induced autophagy. Taken together, our results indicate that Nedd4 plays a crucial role in JEV infection of neuronal cells, which provides a potential target for the development of novel treatment to combat JEV infection.
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http://dx.doi.org/10.1038/srep45375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5368976PMC
March 2017

Exosomal MicroRNAs Derived From Umbilical Mesenchymal Stem Cells Inhibit Hepatitis C Virus Infection.

Stem Cells Transl Med 2016 09 5;5(9):1190-203. Epub 2016 Aug 5.

Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai, People's Republic of China

Unlabelled: : Hepatitis C virus (HCV) is a significant global public health problem, causing more than 350,000 deaths every year. Although the development of direct-acting antivirals has improved the sustained virological response rate in HCV patients, novel anti-HCV agents with higher efficacy as well as better tolerance and cheaper production costs are still urgently needed. Cell-based therapy, especially its unique and strong paracrine ability to transfer information to other cells via extracellular vesicles such as exosomes, has become one of the most popular therapeutic methods in recent years. In our study, exosomes secreted from umbilical mesenchymal stem cells (uMSCs), which are widely used in regenerative medicine, inhibited HCV infection in vitro, especially viral replication, with low cell toxicity. Our analysis revealed that microRNAs (miRNAs) from uMSC-derived exosomes (uMSC-Exo) had their unique expression profiles, and these functional miRNAs, mainly represented by let-7f, miR-145, miR-199a, and miR-221 released from uMSC-Exo, largely contributed to the suppression of HCV RNA replication. These four miRNAs possessed binding sites in HCV RNA as demonstrated by the target prediction algorithm. In addition, uMSC-Exo therapy showed synergistic effect when combined with U.S. Food and Drug Administration-approved interferon-α or telaprevir, enhancing their anti-HCV ability and thus improving the clinical significance of these regenerative substances for future application as optimal adjuvants of anti-HCV therapy.

Significance: This work reported, for the first time, the identification of stem cell-derived exosomes of antiviral activity. Umbilical mesenchymal stem cell-secreted exosomes inhibited hepatitis C virus infection through transporting a mixture of microRNAs complementing the viral genomes to the host cells. This finding provides insights and prospects for physiologically secreted substances for antiviral therapy.
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http://dx.doi.org/10.5966/sctm.2015-0348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996444PMC
September 2016

Overexpression of Annexin II Receptor-Induced Autophagy Protects Against Apoptosis in Uveal Melanoma Cells.

Cancer Biother Radiopharm 2016 May;31(4):145-51

1 Department of Ophthalmology, Changhai Hospital, Second Military Medical University , Shanghai, China .

Uveal melanoma is the most common primary malignant intraocular tumor in adults and still lacks effective systemic therapies. Annexin A2 receptor (AXIIR), a receptor for Annexin II, was demonstrated to play an important role in multiple cells, but its role in uveal melanoma cells remains exclusive. Herein, the authors reported that overexpression of AXIIR was able to reduce cell viability and activate apoptosis apparently in the Mum2C uveal melanoma cell line. Meanwhile, overexpression of AXIIR could induce autophagy and increase autophagy flux. After autophagy was inhibited by chloroquine, enhanced apoptosis and cytotoxicity could be detected. In summary, these data highlighted the crucial role of AXIIR in reducing Mum2C cell viability through inducing apoptosis, while autophagy played a protective role in this process. Interference of this gene may be a promising method for uveal melanoma therapy and combination with specific inhibitor of autophagy may serve as a supplementary.
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http://dx.doi.org/10.1089/cbr.2016.1991DOI Listing
May 2016

Caveolin-1-mediated Japanese encephalitis virus entry requires a two-step regulation of actin reorganization.

Future Microbiol 2016 10 17;11:1227-1248. Epub 2016 Mar 17.

Department of Microbiology, Second Military Medical University, Shanghai Key Laboratory of Medical Biodefense, Shanghai 200433, China.

Aim: To investigate the detailed mechanism of Japanese encephalitis virus (JEV) cell entry.

Materials & Methods: Utilize a siRNA library targeting cellular membrane trafficking genes to identify key molecules that mediate JEV entry into human neuronal cells.

Results: JEV enters human neuronal cells by caveolin-1-mediated endocytosis, which depends on a two-step regulation of actin cytoskeleton remodeling triggered by RhoA and Rac1: RhoA activation promoted the phosphorylation of caveolin-1, and then Rac1 activation facilitated caveolin-associated viral internalization. Specifically, virus attachment activates the EGFR-PI3K signaling pathway, thereby leading to RhoA activation.

Conclusion: This work provides a detailed picture of the entry route and intricate cellular events following the entry of JEV into human neuronal cells, and promotes a better understanding of JEV entry.
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http://dx.doi.org/10.2217/fmb-2016-0002DOI Listing
October 2016

Long non-coding RNA GAS5 inhibited hepatitis C virus replication by binding viral NS3 protein.

Virology 2016 May 21;492:155-65. Epub 2016 Mar 21.

Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, 800th Xiangyin Road, Shanghai 200433, PR China. Electronic address:

HCV infection has a complex and dynamic process which involves a large number of viral and host factors. Long non-coding RNA GAS5 inhibits liver fibrosis and liver tumor migration and invasion. However, the contribution of GAS5 on HCV infection remains unknown. In this study, GAS5 was gradually upregulated during HCV infection in Huh7 cells. In addition, GAS5 attenuated virus replication with its 5' end sequences, as confirmed by different GAS5 truncations. Moreover, this 5' end sequences showed RNA-protein interaction with HCV NS3 protein that could act as a decoy to inhibit its functions, which contributed to the suppression of HCV replication. Finally, the innate immune responses remained low in HCV infected Huh7 cells, ruling out the possibility of GAS5 to modulate innate immunity. Thus, HCV stimulated endogenous GAS5 can suppress HCV infection by acting as HCV NS3 protein decoy, providing a potential role of GAS5 as a diagnostic or therapeutic target.
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http://dx.doi.org/10.1016/j.virol.2016.02.020DOI Listing
May 2016

Serum adsorption, cellular internalization and consequent impact of cuprous oxide nanoparticles on uveal melanoma cells: implications for cancer therapy.

Nanomedicine (Lond) 2015 15;10(24):3547-62. Epub 2015 Oct 15.

Department of Ophthalmology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.

Aim: To investigate the biological fate of cuprous oxide nanoparticles (Cu2O-NPs) and to evaluate their potential in uveal melanoma therapy.

Materials & Methods: The protein corona, cellular uptake mechanism and localization of Cu2O-NPs were investigated. Furthermore, the effect of Cu2O-NPs on uveal melanoma cell proliferation, migration and invasion, and possible mechanisms were studied in detail.

Results: Cu2O-NPs are able to adsorb serum proteins in cell culture medium, which are then internalized by uveal melanoma cells mainly through lipid raft-mediated endocytosis. Furthermore, Cu2O-NPs selectively inhibit cancer cell growth and impair the ability of uveal melanoma cell migration, invasion and the cytoskeleton assembly. The mechanism may be that Cu2O-NPs located in and damage mitochondria, autophagolysosomes and lysosomes, leading to elevated reactive oxygen species level and over-stimulated apoptosis and autophagy.

Conclusion: The data provide detailed information of Cu2O-NPs for further application and indicate that Cu2O-NPs could be a potential agent for uveal melanoma therapy.
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http://dx.doi.org/10.2217/nnm.15.178DOI Listing
September 2016

Antiviral activity of cuprous oxide nanoparticles against Hepatitis C Virus in vitro.

J Virol Methods 2015 Sep 25;222:150-7. Epub 2015 Jun 25.

Department of Infectious Disease, Changzheng Hospital, Second Military Medical University, Shanghai 200433, China. Electronic address:

Small molecular inhibitors in combination with or without interferon have improved sustained antiviral responses against Hepatitis C Virus (HCV) infection. Nonetheless, resistance to these inhibitors is expected to emerge rapidly due to the high mutation rate of the virus. Thus, new antiviral drugs, in combination with currently available therapies, are urgently needed to treat HCV infection. In the present study, we evaluated the antiviral efficacy of cuprous oxide nanoparticles (CO-NPs) against HCV in the HCVcc/Huh7.5.1 cell culture system. CO-NPs were able to significantly inhibit the infectivity of HCVcc at a non-cytotoxic concentration. In addition, CO-NPs inhibited the entry of HCV pseudoparticle (HCVpp), including genotypes 1a, 1b, and 2a, while no effect on HCV replication was observed. Further time-of-addition experiment indicated that CO-NPs blocked HCV infection both at the attachment and entry stages. In conclusion, we report that CO-NPs can act as an anti-HCV agent by targeting the binding of infectious HCV particles to hepatic cells and the virus entry into the cells. These findings suggest that CO-NPs may have novel roles in the treatment of patients with chronic hepatitis C.
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http://dx.doi.org/10.1016/j.jviromet.2015.06.010DOI Listing
September 2015

SiRNA directed against annexin II receptor inhibits angiogenesis via suppressing MMP2 and MMP9 expression.

Cell Physiol Biochem 2015 30;35(3):875-84. Epub 2015 Jan 30.

Department of Ophthalmology, Changhai Hospital, Second Military University, Shanghai, China.

Background/aims: Annexin II receptor (AXIIR) is able to mediate Annexin II signal and induce apoptosis, but its role in angiogenesis remains unclear. This study tries to investigate the role of AXIIR in angiogenesis and the plausible molecular mechanism.

Methods/results: RNA interference technology was used to silence AXIIR, and the subsequent effects in vitro and in vivo were evaluated thereafter. Our data indicated that human umbilical vein endothelial cells (HUVECs) expressed AXIIR and knockdown of AXIIR significantly inhibited HUVECs proliferation, adhesion, migration, and tube formation in vitro and suppressed angiogenesis in vivo. Furthermore, AXIIR siRNA induced cell arrest in the S/G2 phase while had no effect on cell apoptosis. We found that these subsequent effects might be via suppressing the expression of matrix metalloproteinase 2and matrix metalloproteinase 9.

Conclusion: AXIIR participates in angiogenesis, and may be a potential therapeutic target for angiogenesis related diseases.
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http://dx.doi.org/10.1159/000369745DOI Listing
November 2015

Serologic assay for avian-origin influenza A (H7N9) virus in adults of Shanghai, Guangzhou and Yunnan, China.

J Clin Virol 2014 Jul 13;60(3):305-8. Epub 2014 Apr 13.

Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai, China. Electronic address:

Background And Objective: To investigate serologic status for novel avian influenza A (H7N9) virus in different areas in China, we examined serum samples collected in winter of 2011 from adult population of Shanghai (eastern China), Guangzhou (southern China) and Yunnan (southwest China) for the antibody responses to this virus.

Study Design: A total of 900 stored serum samples of adult outpatients (300 samples for each area) were subjected to anti-hemagglutinin (HA) antibodies assay using enzyme-linked immunosorbent assay (ELISA) and neutralizing antibodies assay using H7N9 pseudotyped particles (H7N9pp) and authentic H7N9 virus based neutralization test.

Results: Anti-H7 antibodies were detected in 164, 186 and 123 samples from three areas above, respectively. Among anti-H7 positive sera, 20, 42 and 13 samples had neutralizing titers of ≥10 when 8×10(2) focus-forming units (FFU) of H7N9 pseudotyped particles (pp) were adopted in neutralizing assay, respectively. When neutralizing antibodies were assayed using classic microneutralization (MN) test, MN titers of ≥10 were found in 7 samples from Guangzhou, but none from Shanghai and Yunnan.

Conclusion: Low levels of protective immunity pre-existed in some general adult population of the three areas, and pre-existing immunity against H7N9 in Guangzhou appears stronger than that in Shanghai and Yunnan.
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http://dx.doi.org/10.1016/j.jcv.2014.04.006DOI Listing
July 2014

Cross-reactive antibody responses to the novel avian influenza A H7N9 virus in Shanghai adults.

J Infect 2014 Jul 22;69(1):96-8. Epub 2014 Feb 22.

Clinical Laboratory, Yangsi Hospital of Pudong District, Shanghai, China.

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http://dx.doi.org/10.1016/j.jinf.2014.02.010DOI Listing
July 2014

Cuprous oxide nanoparticles inhibit angiogenesis via down regulation of VEGFR2 expression.

Nanoscale 2014 Mar 5;6(6):3206-16. Epub 2014 Feb 5.

Department of Ophthalmology, Changhai Hospital, Second Military Medical University, Shanghai, 200433, P.R. China.

Angiogenesis is a process that forms new blood capillaries from existing vessels, which is of great physiological and pathological significance. Although recent studies provide evidence that cuprous oxide nanoparticles (CO-NPs) may have biomedical potential, the mechanisms of CO-NPs in angiogenesis have not been investigated to date. We have studied the anti-angiogenic properties of CO-NPs on primary human umbilical vein endothelial cells (HUVECs). We found that CO-NPs were able to induce cell morphology changes and suppress cell proliferation, migration and tube formation in vitro and in vivo dose dependently. Furthermore, CO-NPs could induce cell apoptosis both at the early and late apoptotic stage and induce cell cycle arrest at S phase in a dose dependent manner. As signalling via the vascular endothelial growth factor receptor-2 (VEGFR2) is critical for angiogenic responses, we further explored the expression of VEGFR2 after the treatment of CO-NPs. They were found to inhibit VEGFR2 expression dose and time dependently both at the protein and mRNA level while had no effect on VEGF and VEGFR1 expression. Together, we report for the first time that CO-NPs can act as an anti-angiogenic agent by suppressing VEGFR2 expression, which may be a potential nanomedicine for angiogenesis therapy.
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http://dx.doi.org/10.1039/c3nr04363kDOI Listing
March 2014

Alanine scanning mutagenesis of hepatitis C virus E2 cysteine residues: Insights into E2 biogenesis and antigenicity.

Virology 2014 Jan 31;448:229-37. Epub 2013 Oct 31.

Department of Microbiology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.

Envelope glycoprotein 2 (E2) of hepatitis C virus contains 18 conserved cysteine (Cys) residues in its ectodomain. By cysteine-alanine mutagenesis and function analysis, six Cys in H77 E2 (C494, C508, C552, C564, C607 and C644) were found to be indispensable for recognition by conformation-dependent mAb H53. Removal of any of these Cys residues did not affect E2 heterodimerization with E1, but notably reduced E1E2 transmembrane transportation. These Cys together with C429 and C503 were required for conformation-dependent mAb H48 recognition. All of the above Cys except C607 were required for H77 and Con1 E2 binding to CD81. None of individual mutation of above Cys affected the ability of E2 to induce neutralizing antibodies in mice. Mouse antibodies mainly recognize E2 linear epitopes and are unrelated to epitopes recognized by human E2 antibodies. The findings provide new insights for understanding the biogenesis of functional HCV envelope proteins and HCV neutralizing immunity.
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http://dx.doi.org/10.1016/j.virol.2013.10.020DOI Listing
January 2014

Rapid and sensitive detection of H7N9 avian influenza virus by use of reverse transcription-loop-mediated isothermal amplification.

J Clin Microbiol 2013 Nov 4;51(11):3760-4. Epub 2013 Sep 4.

Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing, China.

An epidemic of human H7N9 influenza virus infection recently emerged in China whose clinical features include high mortality and which has also resulted in serious economic loss. The novel reassortant avian-origin influenza A (H7N9) virus which was the causative agent of this epidemic raised the possibility of triggering a large-scale influenza pandemic worldwide. It seemed likely that fast molecular detection assays specific for this virus would be in great demand. Here, we report a one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method for rapid detection of the hemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus, the minimum detection limit of which was evaluated using in vitro RNA transcription templates. In total, 135 samples from clinical specimens (from either patients or poultry) were tested using this method in comparison with the real-time PCR recommended by the World Health Organization (WHO). Our results showed that (i) RT-LAMP-based trials can be completed in approximately 12 to 23 min and (ii) the detection limit for the H7 gene is around 10 copies per reaction, similar to that of the real-time PCR, whereas the detection limit for its counterpart the N9 gene is 5 copies per reaction, a 100-fold-higher sensitivity than the WHO-recommended method. Indeed, this excellent performance of our method was also validated by the results for a series of clinical specimens. Therefore, we believe that the simple, fast, and sensitive method of RT-LAMP might be widely applied for detection of H7N9 infections and may play a role in prevention of an influenza pandemic.
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http://dx.doi.org/10.1128/JCM.01907-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3889764PMC
November 2013
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