Yafeng Qiu

Shanghai Veterinary Research Institute, Shanghai, Shanghai Shi, China

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Publications (14)29.7 Total impact

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    ABSTRACT: Enhanced IAV replication in p53-knockdown cells was associated with attenuated ISG expression.•Pretreatment of p53-knockdown cells with IFN-α failed to inhibit IAV replication.•p53 plays an essential role in enhancing the type I IFN-mediated immune response against IAV infection.
    Biochemical and Biophysical Research Communications 10/2014; · 2.28 Impact Factor
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    ABSTRACT: Japanese encephalitis virus (JEV) has a significant impact on public health. An estimated three billion people in 'at-risk' regions remain unvaccinated and the number of unvaccinated individuals in certain Asian countries is increasing. Consequently, there is an urgent need for the development of novel therapeutic agents against Japanese encephalitis. Nitazoxanide (NTZ) is a thiazolide anti-infective licensed for the treatment of parasitic gastroenteritis. Recently, NTZ has been demonstrated to have antiviral properties. In this study, the anti-JEV activity of NTZ was evaluated in cultured cells and in a mouse model. JEV-infected cells were treated with NTZ at different concentrations. The replication of JEV in the mock- and NTZ-treated cells was examined by virus titration. NTZ was administered at different time points of JEV infection to determine the stage at which NTZ affected JEV replication. Mice were infected with a lethal dose of JEV and intragastrically administered with NTZ from 1 day post-infection. The protective effect of NTZ on the JEV-infected mice was evaluated. NTZ significantly inhibited the replication of JEV in cultured cells in a dose dependent manner with 50% effective concentration value of 0.12 +/- 0.04 mug/ml, a non-toxic concentration in cultured cells (50% cytotoxic concentration = 18.59 +/- 0.31 mug/ml). The chemotherapeutic index calculated was 154.92. The viral yields of the NTZ-treated cells were significantly reduced at 12, 24, 36 and 48 h post-infection compared with the mock-treated cells. NTZ was found to exert its anti-JEV effect at the early-mid stage of viral infection. The anti-JEV effect of NTZ was also demonstrated in vivo, where 90% of mice that were treated by daily intragastric administration of 100 mg/kg/day of NTZ were protected from a lethal challenge dose of JEV. Both in vitro and in vivo data indicated that NTZ has anti-JEV activity, suggesting the potential application of NTZ in the treatment of Japanese encephalitis.
    Virology Journal 01/2014; 11(1):10. · 2.09 Impact Factor
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    ABSTRACT: Influenza A virus (IAV) induces apoptosis of infected cells. In response to IAV infection, p53, a tumor suppressor involved in regulating apoptosis and host antiviral defense, accumulates and becomes activated. This study was undertaken to examine the mechanism of p53 accumulation in IAV-infected cells. Here we show that p53 accumulation in IAV-infected cells results from protein stabilization, which was associated with compromised Mdm2-mediated ubiquitination of p53. In IAV-infected cells, p53 was stabilized and its half-life was remarkably extended. The ladders of polyubiquitinated p53 were not detectable in the presence of the proteasome inhibitor MG132 and were less sensitive to proteasome-mediated degradation. IAV infection did not affect the abundance of Mdm2, a major ubiquitin E3 ligase responsible for regulating p53 ubiquitination and degradation, but weakened the interaction between p53 and Mdm2. Viral nucleoprotein (NP) was able to increase the transcriptional activity and stability of p53. Furthermore, NP was found to associate with p53 and to impair the p53-Mdm2 interaction and Mdm2-mediated p53 ubiquitination, demonstrating its role in inhibiting Mdm2-mediated p53 ubiquitination and degradation.
    Journal of Biological Chemistry 04/2012; 287(22):18366-75. · 4.65 Impact Factor
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    ABSTRACT: Icariin is the major pharmacologically active compound of Herba epimedii which has been used as a tonic, aphrodisiac and an antirheumatic in traditional Chinese medicine. This study analysed the effect of icariin on the expression of Toll-like receptor 9 (TLR9) which plays an important role in regulation of the innate immune response. Stimulation of Ana-1 murine macrophages with icariin induced a significant dose-dependent expression of TLR9, and its mRNA expression which increased from 3 h post-treatment was approximately five-fold that of DMSO-treated cells. Several molecules, such as myeloid differentiation factor 88, tumor necrosis factor-α and interleukin 6, which are involved in the TLR9 downstream signaling pathway, were also significantly up-regulated in response to icariin stimulation. Our findings demonstrated that icariin is able to induce the expression of TLR9.
    Phytotherapy Research 04/2011; 25(11):1732-5. · 2.40 Impact Factor
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    ABSTRACT: Japanese encephalitis virus (JEV), as a re-emerging virus that causes 10,000-15,000 human deaths from encephalitis in the world each year, has had a significant impact on public health. Pigs are the natural reservoirs of JEV and play an important role in the amplification, dispersal and epidemiology of JEV. The nonstructural protein 3 (NS3) of JEV possesses enzymatic activities of serine protease, helicase and nucleoside 5'-triphosphatase, and plays important roles in viral replication and pathogenesis. We characterized the NS3 protein of a neurovirulent strain of JEV (SH-JEV01) isolated from a field-infected pig. The NS3 gene of the JEV SH-JEV01 strain is 1857 bp in length and encodes protein of approximately 72 kDa with 99% amino acid sequence identity to that of the representative immunotype strain JaGAr 01. The NS3 protein was detectable 12 h post-infection in a mouse neuroblastoma cell line, Neuro-2a, and was distributed in the cytoplasm of cells infected with the SH-JEV01 strain of JEV. In the brain of mice infected with the SH-JEV01 strain of JEV, NS3 was detected in the cytoplasm of neuronal cells, including pyramidal neurons of the cerebrum, granule cells, small cells and Purkinje cells of the cerebellum. The NS3 protein of a neurovirulent strain of JEV isolated from a pig was characterized. It is an approximately 72 kDa protein and distributed in the cytoplasm of infected cells. The Purkinje cell of the cerebellum is one of the target cells of JEV infection. Our data should provide some basic information for the study of the role of NS3 in the pathogenesis of JEV and the immune response.
    Virology Journal 01/2011; 8:209. · 2.09 Impact Factor
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    ABSTRACT: NS1 protein of influenza A virus is involved in regulating the apoptosis of infected cells. We found that exogenously expressed NS1 was able to associate with the tumor suppressor p53 that plays an essential role in regulating apoptosis of influenza A virus-infected cells. Exogenous expression of NS1 resulted in inhibition of p53-mediated transcriptional activity and apoptosis. The p53 inhibitory domain of NS1 was located between amino acids 144 and 188. This domain is necessary for NS1 to inhibit p53 activity, but it requires additional region(s) to cooperatively exert this inhibitory function.
    Biochemical and Biophysical Research Communications 03/2010; 395(1):141-5. · 2.28 Impact Factor
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    ABSTRACT: Marek's disease virus (MDV) is an oncogenic herpesvirus, which causes malignant lymphoma in chickens. The Meq protein of MDV, which is expressed abundantly in MDV-infected cells and in Marek's disease (MD) tumor cells, functions as a transcriptional activator and has been proposed to play an important role in oncogenic transformation. Preliminary studies demonstrated that Meq is able to bind p53 in vitro, as demonstrated using a protein-binding assay. This observation prompted us to examine whether the interaction between Meq and p53 occurs in cells, and to investigate the biological significance of this interaction. We confirmed first that Meq interacted directly with p53 using a yeast two-hybrid assay and an immunoprecipitation assay, and we investigated the biological significance of this interaction subsequently. Exogenous expression of Meq resulted in the inhibition of p53-mediated transcriptional activity and apoptosis, as analyzed using a p53 luciferase reporter assay and a TUNEL assay. The inhibitory effect of Meq on transcriptional activity mediated by p53 was dependent on the physical interaction between these two proteins, because a Meq deletion mutant that lacked the p53-binding region lost the ability to inhibit p53-mediated transcriptional activity and apoptosis. The Meq variants L-Meq and S-Meq, but not VS-Meq and ∆Meq, which were expressed in MD tumor cells and MDV-infected cells, exerted an inhibitory effect on p53 transcriptional activity. In addition, ∆Meq was found to act as a negative regulator of Meq. The Meq oncoprotein interacts directly with p53 and inhibits p53-mediated transcriptional activity and apoptosis. These findings provide valuable insight into the molecular basis for the function of Meq in MDV oncogenesis.
    Virology Journal 01/2010; 7:348. · 2.09 Impact Factor
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    ABSTRACT: M2 protein of influenza A virus is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in influenza virus replication. It is also a target molecule of anti-virus drugs. We extracted the viral genome RNAs from MDCK cells infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M2 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into a prokaryotic expression vector pET-28a(+) (designated pET-28a(+)-M2) and a eukaryotic expression vector p3xFLAG-CMV-7.1 (designated p3xFLAG-CMV-7.1-M2), respectively. The resulted constructs were confirmed by restriction enzyme digestion and DNA sequencing analysis. We then transformed the plasmid pET-28a(+)-M2 into Escherichia coli BL21 (DE3) strain and expressed it by adding 1 mmol/L of IPTG (isopropyl-beta-D-thiogalactopyranoside). The recombinant M2 protein was purified from the induced bacterial cells using Ni(2+) affinity chromatography. Wistar rats were immunized with the purified M2 protein for producing polyclonal antibodies specific for it. Western blotting analysis and immunofluorescence analysis showed that the produced antibodies were capable of reacting with M2 protein expressed in p3xFLAG-CMV-7.1-M2-transfected cells as well as that synthesized in SIV-infected cells. We also transfected plasmid p3xFLAG-CMV-7.1-M2 into Vero cells and analyzed its subcellular localization by immunofluorescence. The M2 protein expressed in the Vero cells was 20 kDa in size and dominantly localized in the cytoplasm, showing a similar distribution to that in SIV-infected cells. Western blotting analysis of SIV-infected cells suggested that M2 was a late phase protein, which was detectable 12 h post-infection, later than NS1, NP and M1 proteins. It would be a potential molecular indicator of late phases replication of virus. Our results would be useful for studying the biological function of M2 protein in SIV replication.
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 01/2010; 26(1):16-21.
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    ABSTRACT: The rabies virus (RV) glycoprotein (G protein) induces neutralizing antibodies, which are important in protection against rabies. In the present study, three gene fragments that encode polypeptides (corresponding to amino acid residues 19-60, 181-219, and 300-458) comprising the linear neutralization sites of the G protein were spliced together in tandem by PCR-based site-directed mutagenesis and heterologously expressed in Escherichia coli (DE3). The recombinant protein (designated rRVg) was purified under denaturing conditions and solubilized by stepwise dialysis against an alkaline buffer (0.05 M Na(2)CO(3) pH 9.6). Western blot analysis of the antigenicity of rRVg showed that it was recognized by rabies-immune serum. Competitive neutralization assay revealed that rRVg significantly reduced the RV-neutralizing activity of the rabies-immune serum. These results show potential of rRVg as a diagnostic antigen for detecting RV-neutralizing antibodies in immunized hosts.
    Protein Expression and Purification 09/2009; 70(2):179-83. · 1.43 Impact Factor
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    ABSTRACT: Tumor suppressor p53, the major cellular defense against tumor development, has recently been implicated in host antiviral defense. Previous studies have shown that p53 was induced at the apoptotic stage of influenza virus-infected cells. However, we found that p53 was induced not only at the apoptotic stage but also at the beginning phase of infection, showing a biphasic pattern with a first transient elevation apparent at the beginning phase of infection and a second elevation observable at the middle-late phase of infection. This up-regulation of p53 was independent of increased p53 transcription, but dependent on virus adsorption and replication. The increased p53 was active and able to transactivate its downstream target genes, such as interferon regulatory factor 9 (IRF9) and Bax. To our knowledge, this is the first report to describe a biphasic pattern of p53 accumulation in influenza virus-infected cells.
    Biochemical and Biophysical Research Communications 06/2009; 382(2):331-5. · 2.28 Impact Factor
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    ABSTRACT: Influenza A virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in the virus life cycle. In the present study, we extracted the viral genome RNAs from allantoic fluid of 9-day-old embryonated chicken eggs infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M1 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into an expression vector pET-28a (+) (designated pET-28a-M1) and confirmed by DNA sequencing analysis. We then transformed the plasmid pET-28a-M1 into Escherichia coli BL21 strain for heterologous expression. The expression of M1 was induced by 1mM isopropyl-beta-D-thiogalactopyranoside. SDS-PAGE analysis of the induced bacterial cells revealed that the recombinant M1 protein was expressed in high yield level. Next, we purified the expressed recombinant M1 using Ni2+ affinity chromatography and immunized Wistar rat with the purified M1 protein for producing polyclonal antibodies specific for M1. Western blotting analysis showed that the produced antibodies were capable of reacting with M1 protein expressed in Escherichia coli as well as that synthesized in SIV-infected cells. We further cloned the amplified M1 cDNA into a eukaryotic expression plasmid p3xFLAG-CMV-7.1 to construct the recombinant plasmid p3xFLAG-CMV-M1 for expressing M1 in eukaryotic cells. Western blotting analysis revealed that the M1 protein was expressed in p3xFLAG-CMV-M1-transfected Vero cells and recognized by the produced anti-M1 antibodies. Using the produced anti-M1 antibodies, we analyzed the kinetics of M1 protein in the virus-infected cells during influenza virus infection and estimated the possibility of M1 as an indicator of influenza virus replication. The recombinant M1 protein, anti-M1 antibodies and recombinant expression plasmids would provide useful tools for studies of biological function of M1 protein and the basis of SIV replication.
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 06/2009; 25(5):672-8.
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    ABSTRACT: Although the tumor suppressor protein p53 is important in the control of various cellular activities, the analysis of p53 in the porcine model has been hampered by a lack of a suitable antibody that is specific for porcine p53. Using a recombinant porcine p53, we generated a rabbit polyclonal antibody (designated SH0797) that is directed against porcine p53. The results of the study show that the antibody is capable of detecting recombinant p53 protein expressed in Escherichia coli, as well as FLAG-tagged p53 that is expressed in the transfected cells. This demonstrates that the antibody is specific for the porcine p53 protein. The antibody also showed the ability to immunoprecipitate p53 protein from extracts of porcine cells and to cross-react with human p53 protein. In addition, expression of porcine p53 could be induced readily in porcine cells and detected using this new tool. This antibody is a useful tool for use in studies of the cellular pathways that involve p53 in the porcine model.
    Biochemical and Biophysical Research Communications 11/2008; 377(1):151-5. · 2.28 Impact Factor
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    ABSTRACT: Myeloid differentiation factor 88 (MyD88) is an adaptor protein involved in the interleukin-1 receptor- and Toll-like receptor-induced activation of nuclear factor-kappaB (NF-kappaB). A novel isoform of chicken MyD88, designated chicken MyD88-2, has been cloned and functionally characterized. Its open reading frame is of length 900bp, and it encodes a predicted 299 residue protein, similar in length to its mammalian orthologues, but, respectively, 77 and 69 amino acids shorter than the previously described chicken MyD88-1 and -3. The amino acid sequence of chicken MyD88-2 displays 96.9%, 96.9%, 70.4% and 70.2% identity with, respectively, chicken MyD88-1, -3, human and mouse MyD88. Chicken MyD88-2 expression was detected in a range of tissues tested, but no expression of either chicken MyD88-1 or -3 was observed. The over-expression of chicken MyD88-2 significantly induced the activation of NF-kappaB in vitro, suggesting that chicken MyD88-2 plays an important role in the innate immune responses of chicken.
    Developmental & Comparative Immunology 07/2008; 32(12):1522-30. · 3.24 Impact Factor
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    ABSTRACT: A modified polymerase chain reaction (PCR)-based site-directed mutagenesis method used to splice together different regions of a gene by deleting hundreds of nucleotides of undesired sequences is described. This method was inspired by a PCR-based site-directed mutagenesis method developed by Stratagene (La Jolla, CA, USA); the procedure and primer design were modified to enable the method to generate deletions several hundreds of nucleotides in length with an efficiency of 80-100%, and to delete two DNA fragments simultaneously in a single PCR. This method should be useful for deletion of large DNA fragments from a gene.
    Analytical Biochemistry 03/2008; 373(2):398-400. · 2.58 Impact Factor