Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-381 protects myocardial cell function in children and mice with viral myocarditis via targeting cyclooxygenase-2 expression

doi: 10.3892/etm.2018.6082

Figure Lengend Snippet: Contents of (A) creatine kinase (CK-MB) and (B) lactate dehydrogenase (LDH) in serum from mice. Mice in control group (n=30) were not infected by any virus and intraperitoneally injected with saline (0.2 ml), while those in viral myocarditis (VM) group (n=30) were intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml). Contents of CK-MB and LDH were determined by automatic biochemical analysis. **P<0.01 compared with control.

Article Snippet: VM group (n=30) was intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml).

Techniques: Infection, Injection

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-381 protects myocardial cell function in children and mice with viral myocarditis via targeting cyclooxygenase-2 expression

doi: 10.3892/etm.2018.6082

Figure Lengend Snippet: Expression of COX-2 (A) mRNA and (B) protein in serum from mice. Mice in control group (n=30) were not infected by any virus and intraperitoneally injected with saline (0.2 ml), while those in viral myocarditis (VM) group (n=30) were intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml). RT-qPCR was used to measure the expression of mRNA in serum, while ELISA was used to determine protein contents in serum. *P<0.05 compared with control group.

Article Snippet: VM group (n=30) was intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml).

Techniques: Expressing, Infection, Injection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-381 protects myocardial cell function in children and mice with viral myocarditis via targeting cyclooxygenase-2 expression

doi: 10.3892/etm.2018.6082

Figure Lengend Snippet: Expression of COX-2 (A) mRNA and (B) protein in myocardial tissues from mice. Mice in control group (n=30) were not infected by any virus and intraperitoneally injected with saline (0.2 ml), while those in viral myocarditis (VM) group (n=30) were intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml). RT-qPCR was used to measure the expression of mRNA, while Western blotting was used to determine protein expression. **P<0.01 compared with control group.

Article Snippet: VM group (n=30) was intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml).

Techniques: Expressing, Infection, Injection, Quantitative RT-PCR, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-381 protects myocardial cell function in children and mice with viral myocarditis via targeting cyclooxygenase-2 expression

doi: 10.3892/etm.2018.6082

Figure Lengend Snippet: Expression of miR-381 (A) serum and (B) myocardial tissues from mice. Mice in control group (n=30) were not infected by any virus and intraperitoneally injected with saline (0.2 ml), while those in viral myocarditis (VM) group (n=30) were intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml). RT-qPCR was used to measure the expression of miR-381 in both samples. **P<0.01 compared with control group.

Article Snippet: VM group (n=30) was intraperitoneally injected with 100 TCID50 CVB3 (0.2 ml; Biopike, Beijing, China), and one hour later, the mice were injected with saline (0.2 ml).

Techniques: Expressing, Infection, Injection, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Mesenteric CD103 + DCs Initiate Switched Coxsackievirus B3 VP1-Specific IgA Response to Intranasal Chitosan-DNA Vaccine Through Secreting BAFF/IL-6 and Promoting Th17/Tfh Differentiation

doi: 10.3389/fimmu.2018.02986

Figure Lengend Snippet: Intranasal immunization with chitosan-formulated DNA enhances specific IgA induction in the gut and B cell IgA CSR in the MLN. (A) Mice were immunized intranasally with chitosan, DNA (pcDNA3-VP1, 50 μg) or chitosan-DNA biweekly for 4 times. VP1-specific IgA in fecal extracts was measured by ELISA at indicated time points. (B) Representative flow cytometry plots (left) and frequency (right) of B220 + IgA + plasmablasts from spleens, MLNs, PPs, and LPs of wk8-chitosan-DNA-immunized mice. Data represent mean ± SEM for six individual mice. Data in A and B is representative of three independent experiments. *** P < 0.001; ** p < 0.01; * P < 0.05; ns, not significant. (C) Representative examples of ELISPOT results from wells coated with VP1 peptides for enumeration of VP1-specific VP1-specific IgA antibody-forming cells (AFCs, plasmablasts) in spleen, MLN, PP, and LP of wk8-immunized mice. Experiments were independently repeated three times ( n = 6). Numbers of AFCs with mean values ±SEM are shown. ** p < 0.01; ns, not significant. (D) IgA + plasma cells, defined as B220 − CD138 + IgA + , within the MLN and LP were ensured by flow cytometry, Representative plots were previously gated on B220 − cells and frequencies of IgA + plasma cells in MLN and LP with mean values ± SEM were shown ( n = 6). ** p < 0.01; * P < 0.05. (E) Representative semi-quantitative (left) or quantitative (right) RT-PCR on sorted B cells from wk8-chitosan-DNA-immunized MLNs for expression of indicated transcripts (Aicda, α-GT, Iμ-Cα, and α-CT). Relative expression of Aicda and α-GT mRNA are displayed as the fold change of respective transcript expression over the expression by Gapdh. Data present mean ± SEM of three independent experiments ( n = 6). ** P < 0.01; * P < 0.05; ns, not significant. (F) MLN B cells were subdivided into B1a (B220 lo CD5 lo ), B1b (B220 lo CD5 neg ), and B2-B (B220 hi CD5 neg ) cells according to B220 and CD5 expression. Bar graph represents the absolute number of B2-B cells. Histograms represent expression of mIgA by MLN B1 and B2-B cells after chitosan-DNA immunization. Data present mean ± SEM of three independent experiments ( n = 6). ** P < 0.01; * P < 0.05.

Article Snippet: CVB3 VP1 237−249 Peptide (FKPKHVKAWIPRP) was synthesized by GL Biochem Corp (Shanghai, China) with purity over 95%.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Enzyme-linked Immunospot, Clinical Proteomics, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Frontiers in Immunology

Article Title: Mesenteric CD103 + DCs Initiate Switched Coxsackievirus B3 VP1-Specific IgA Response to Intranasal Chitosan-DNA Vaccine Through Secreting BAFF/IL-6 and Promoting Th17/Tfh Differentiation

doi: 10.3389/fimmu.2018.02986

Figure Lengend Snippet: Freshly isolated MLN CD103 + DCs induce specific IgA production. (A) CD103 + DCs were sorted from 24 h chitosan-DNA-immunized mice. Naïve MLN IgM + B cells (5 × 10 5 ) were co-cultured with naïve CD4 + Th0 cells (5 × 10 5 ) with FACS-sorted MLN CD103 + DCs (2.5 × 10 4 ) for 7 days in the presence of DNA or chitosan-DNA (5 ug/ml). VP1-specific IgA secreted in the culture was quantified by ELISA. (B) Representative flow cytometry plots of IgA + B cells after co-culture and cumulative data quantifying of the number of IgA + B cells were shown. Data in (A,B) are mean ± SEM representative of three independent experiments. *** p < 0.001; ** P < 0.01; ns, not significant. (C) Quantitative PCR analysis for expression of Aicda and α-GT in co-cultured B cells. mRNA expression is relative to Gapdh. Data are representative of three independent experiments. ** P < 0.01; * P < 0.05. (D,E) IL-6, BAFF, IL-17A, IL-12p40, IL-12p70, and IFNγ production in the CD103 + DC-Th0-B co-culture (D) or CD103 + DCs after stimulation (E) . MACS-purified naive CD4 + Th0 cells (5 × 10 5 ) were co-cultured with MLN CD103 + DCs (2.5 × 10 4 ) and naïve MLN IgM + B cells (5 × 10 5 ) for 48 h in the presence of DNA or chitosan-DNA (5 μg/ml). Sorted CD103 + DCs (1.0 × 10 5 ) were incubated with chitosan, DNA, chitosan-DNA (5 μg/ml) or anti-IgM mAb (5 μg/ml) plus anti-CD40 mAb (5 μg/ml). Supernatants were harvested to measure cytokine levels by ELISA at 48 h. Data are representative of three independent experiments. *** p < 0.001; ** P < 0.01; * p < 0.05; ns, not significant.

Article Snippet: CVB3 VP1 237−249 Peptide (FKPKHVKAWIPRP) was synthesized by GL Biochem Corp (Shanghai, China) with purity over 95%.

Techniques: Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Co-Culture Assay, Real-time Polymerase Chain Reaction, Expressing, Purification, Incubation

Journal: Frontiers in Immunology

Article Title: Mesenteric CD103 + DCs Initiate Switched Coxsackievirus B3 VP1-Specific IgA Response to Intranasal Chitosan-DNA Vaccine Through Secreting BAFF/IL-6 and Promoting Th17/Tfh Differentiation

doi: 10.3389/fimmu.2018.02986

Figure Lengend Snippet: CD103 + DCs induce specific IgA production in an IL-6 and BAFF-dependent manner. (A–C) BAFF receptors expression by immunized MLN B cells. Flow sorted CD19 + B cells from immunized mice were incubated with chitosan, DNA and chitosan-DNA (5 ug/ml). The mRNA expression of BCMA (A) , BAFF-R (B) and TACI (C) were determined by real-time PCR at indicated time point. Data presented as the mean ± SEM of three independent experiments ( n = 6). *** p < 0.001; ** P < 0.01; * P < 0.05; ns, not significant. (D) FACS-sorted MLN CD103 + DCs (2.5 × 10 4 ) were primed with DNA or chitosan-DNA (10 ug/ml) and cultured with MLN CD19 + IgM + B cells (5 × 10 5 ) and CD4 + Th0 cells (5 × 10 5 ) for 7 days. Either isotype control Ab (10 μg/ml), IL-6-neutralizing Ab (10 μg/ml), or TACI-neutralizing Ab (10 μg/ml) was added to some cultures as indicated. VP1-specific IgA secreted in the culture was quantified by ELISA. Data are mean±SEM from three independent experiments. *** P < 0.001; ** P < 0.01; * P < 0.05; ns, not significant.

Article Snippet: CVB3 VP1 237−249 Peptide (FKPKHVKAWIPRP) was synthesized by GL Biochem Corp (Shanghai, China) with purity over 95%.

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Cell Culture, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: The mammalian host protein DAP5 facilitates the initial round of translation of Coxsackievirus B3 RNA

doi: 10.1074/jbc.RA119.009000

Figure Lengend Snippet: DAP5 is required for CVB3 IRES mediated translation. A, schematic of isoforms of eIF4G in mammalian cells. Different domains in individual proteins, the length of the proteins, and the 2A protease cleavage site are indicated. Poly-A binding protein (PABP) interacting domain and Nuclear Localisation Signal (NLS) are indicated in the N-Terminus of the schematic. B, effect of partial silencing of DAP5 on the CVB3 life cycle. Top panel, schematic of CVB3 replicon RNA. F Luc indicated the Firefly Luciferase gene in the CVB3 replicon. CVB3 replicon RNA was transfected in cells transfected previously with the indicated siRNAs, and cells were processed for luciferase activity 8 h after replicon RNA transfection. Center panel, luciferase activity. Bottom panel, Western blot indicating the DAP5 and β-actin protein levels. The band intensities were quantified, and silencing efficiency is represented normalized to β-actin. C, effect of knockdown of DAP5 or eIF4GI or both on the early stage of the CVB3 life cycle. Top panel, luciferase activity indicating translation of the CVB3 IRES. Bottom panel, Western blot showing the DAP5 and β-Actin protein levels upon treatment of siDAP5, sieIF4GI, or both. D, schematic of WT and 2A protease mutant CVB3 replicon RNAs. The G122E mutation in the 2A protease region of the CVB3 replicon RNA is indicated. E, effect of partial silencing of DAP5 on WT and mutant CVB3 replicon RNA translation in early stages. Left panel, luciferase activity indicating translation of the CVB3 IRES. Right panel, Western blotting showing the DAP5 and β-actin protein levels upon siDAP5 treatment. F, effect of partial silencing of DAP5 and eIF4GI on CVB3 IRES activity 1.5 h after CVB3 infection. The CVB3-RLuc virus was used for this experiment, and the top panel represents percentage luciferase activity. All graphs represent the average of three or more independent experiments. Error bars represent standard deviation. *, p ≤ 0.05; **, p ≤ 0.01. Band intensity normalized to β-actin is indicated below the western blots. Similar sets of blots were used to indicate the molecular weight position in the western blots.

Article Snippet: To prepare biotin-labeled CVB3 5′ UTR RNA, biotin-11–UTP was included in in vitro transcription reactions carried out using the Ribomax Kit (Promega).

Techniques: Binding Assay, Luciferase, Transfection, Activity Assay, Western Blot, Mutagenesis, Infection, Standard Deviation, Molecular Weight

Journal: The Journal of Biological Chemistry

Article Title: The mammalian host protein DAP5 facilitates the initial round of translation of Coxsackievirus B3 RNA

doi: 10.1074/jbc.RA119.009000

Figure Lengend Snippet: Role of DAP5 in modulating functions of different IRES elements. A, role of DAP5 and eIF4GI in CVB3 IRES–mediated translation. Top panel, schematic of bicistronic RNAs. B, role of DAP5 and eIF4GI in HCV IRES–mediated translation. C, role of DAP5 and eIF4GI in EMCV IRES–mediated translation. F luc activity represents IRES-mediated translation, and R luc activity represents cap-dependent RNA translation throughout. *, p < 0.05; **, p < 0.01. D and E, Western blots indicating silencing of DAP5 (D) and eIF4GI (E). Band intensity normalized to β-actin is indicated below the western blots. Similar set of blots were used to indicate the molecular weight position in the western blots. F Luc, Firefly luciferase; R Luc, Renilla Luciferase.

Article Snippet: To prepare biotin-labeled CVB3 5′ UTR RNA, biotin-11–UTP was included in in vitro transcription reactions carried out using the Ribomax Kit (Promega).

Techniques: Activity Assay, Western Blot, Molecular Weight, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: The mammalian host protein DAP5 facilitates the initial round of translation of Coxsackievirus B3 RNA

doi: 10.1074/jbc.RA119.009000

Figure Lengend Snippet: Interaction of DAP5 with CVB3 IRES. A, UV cross-linking experiment carried out with the radiolabeled CVB3 5′ UTR and recombinant DAP5 protein in the presence of unlabeled CVB3 5′ UTR RNA (lanes 2 and 3) or HCV IRES RNA (lanes 4 and 5) as competitor RNAs. NC, no competition. B, schematic of stem loop V in the CVB3 IRES. The binding site of eIF4GI is indicated in the box, and this site is mutated from CCG to AAA. C, UV cross-linking experiment carried out using a radiolabeled CVB3 RNA probe with recombinant DAP5 protein in the presence of the WT unlabeled CVB3 5′ UTR and CCG→AAA mutant CVB3 5′ UTR RNA as competitor RNAs. Normalized band intensities are indicated under individual lanes. D, RNA immunoprecipitation experiment carried out 1.5 h after CVB3 replicon RNA transfection. Top panel, CVB3 positive-strand RNA after immunoprecipitation. Bottom panel, protein levels by Western blotting (WB). All graphs indicate the average of normalized band intensities from three independent experiments. Error bars represent standard deviation. *, p ≤ 0.05; **, p ≤ 0.01.

Article Snippet: To prepare biotin-labeled CVB3 5′ UTR RNA, biotin-11–UTP was included in in vitro transcription reactions carried out using the Ribomax Kit (Promega).

Techniques: Recombinant, Binding Assay, Mutagenesis, Immunoprecipitation, Transfection, Western Blot, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: The mammalian host protein DAP5 facilitates the initial round of translation of Coxsackievirus B3 RNA

doi: 10.1074/jbc.RA119.009000

Figure Lengend Snippet: Affinity of DAP5 and C-terminal eIF4GI proteins for binding CVB3 IRES. A and B, sensorgrams representing the interaction of DAP5 (A) or C-terminal eIF4GI (B) with CVB3 5′ UTR RNA at the indicated protein concentrations. The y axis represents the change in RUs during association and dissociation phases. The average Kd is indicated in the inset.

Article Snippet: To prepare biotin-labeled CVB3 5′ UTR RNA, biotin-11–UTP was included in in vitro transcription reactions carried out using the Ribomax Kit (Promega).

Techniques: Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The mammalian host protein DAP5 facilitates the initial round of translation of Coxsackievirus B3 RNA

doi: 10.1074/jbc.RA119.009000

Figure Lengend Snippet: Role of DAP5 in the initial round of translation. Shown is the proposed model for the initial round of translation in type I IRES. Upon infection, DAP5 interacts with CVB3 IRES and leads to the basal level of translation. This produces viral protein 2A protease, which can now cleave eIF4GI. The C-terminal eIF4GI now interacts with CVB3 IRES and leads to robust activation of translation, which is essential for rapid progression of the viral life cycle.

Article Snippet: To prepare biotin-labeled CVB3 5′ UTR RNA, biotin-11–UTP was included in in vitro transcription reactions carried out using the Ribomax Kit (Promega).

Techniques: Infection, Activation Assay

Journal: Viruses

Article Title: Development of Group B Coxsackievirus as an Oncolytic Virus: Opportunities and Challenges

doi: 10.3390/v13061082

Figure Lengend Snippet: Number of registered clinical trials on oncolytic viral studies from clinicaltrials.gov in top 10 countries ( A ) from 2001 to 2021 ( B ). Note that the data of 2021 only cover clinical studies registered until 31 May 2021. Search keywords ‘oncolytic’ and ‘tumor’ found 182 records in total with five excluded for not involving oncolytic virus.

Article Snippet: H Luo is collaborating with Virogin Biotech Ltd for the development of oncolytic CVB3 under the MITACS Accelerate program agreement between the university and the company.

Techniques: Clinical Proteomics, Virus

Journal: Viruses

Article Title: Development of Group B Coxsackievirus as an Oncolytic Virus: Opportunities and Challenges

doi: 10.3390/v13061082

Figure Lengend Snippet: Structure and genome of group B coxsackieviruses. ( A ) Genome structure of coxsackievirus. ( B ) CVB3 structures are generated using the program PyMOL based on the report data by Muchkelbauer et al. . ( C ) Life-cycle of CVB. Following coxsackievirus-adenovirus receptor (CAR)-mediated endocytosis, viral genome is translated into a polyprotein, which is then processed by virus-encoded proteinases 2A and 3C into individual structural and non-structural proteins. Subsequently, cellular machinery is hijacked for viral genome replication through the action of the viral RNA-dependent RNA polymerase 3D. Finally, the viral genome is packaged inside asymmetric protein capsid, followed by viral particle release.

Article Snippet: H Luo is collaborating with Virogin Biotech Ltd for the development of oncolytic CVB3 under the MITACS Accelerate program agreement between the university and the company.

Techniques: Generated, Virus

Journal: Viruses

Article Title: Development of Group B Coxsackievirus as an Oncolytic Virus: Opportunities and Challenges

doi: 10.3390/v13061082

Figure Lengend Snippet: Mechanism of action and strategies applied or proposed to enhance the safety and potency of oncolytic CVBs. CVBs preferentially replicate in and kill tumor cells with limited replication and lysis capacity in healthy cells. Several approaches have been or may be used to further improve the safety profile of CVBs, including insertion of organ-specific/tumor-suppressive miRNA target sequences to viral genome, utilization of non-pathogenic strains of CVBs, selection of non-toxic CVB variants through directed evolution, and production of less toxic CVBs by genetic modification. CVBs enter tumor cells through the coxsackievirus-adenovirus receptor (CAR, primary receptors for all CVBs) and/or heparan sulfate (the PD strain of CVB3). After replication, oncolytic CVBs induce tumor cell lysis, leading to the release of viral progeny that can infect adjacent and distant tumor cells, and the leakage of danger-associated molecular patterns (DAMPs), pathogen-associated molecular patterns (PAMPs), tumor-associated antigens, and cytokines to modulate the tumor microenvironment and activate systemic anti-tumor immune response. A combinational treatment of oncolytic CVB with an immunotherapy, such as checkpoint inhibitor and CAR-T cell therapy, or use of “armed” recombinant CVBs that express immunomodulatory transgenes are expected to yield an additive anti-tumor effect.

Article Snippet: H Luo is collaborating with Virogin Biotech Ltd for the development of oncolytic CVB3 under the MITACS Accelerate program agreement between the university and the company.

Techniques: Lysis, Selection, Modification, Recombinant

Journal: Viruses

Article Title: Development of Group B Coxsackievirus as an Oncolytic Virus: Opportunities and Challenges

doi: 10.3390/v13061082

Figure Lengend Snippet: Schematic illustration of the strategy for generating a non-toxic CVB for oncolytic purpose. ( A ) Viral RNA is prepared by RNA extraction from cell cultures. ( B ) Viral cDNA is generated via reverse transcription using poly T primer, followed by PCR amplification with a primer pair containing cloning sites (CS) and T7 promoter sequence (red) in the forward primer. ( C ) Viral cDNA is then rescued into a bacterial plasmid (e.g., pUC18/19). ( D ) Viral genome is modified through insertion of tumor-suppressive and/or organ-specific miRNA target sequences into the 5′UTR and/or 3′UTR of viral genome or by genetic mutagenesis of the viral nucleotides or amino acid codon. ( E ) Finally, the viral RNA is prepared by in vitro transcription with T7 RNA polymerase, and subsequently transfected into CVB susceptible cells to prepare the viral particles for further propagation. CDS, coding sequence.

Article Snippet: H Luo is collaborating with Virogin Biotech Ltd for the development of oncolytic CVB3 under the MITACS Accelerate program agreement between the university and the company.

Techniques: RNA Extraction, Generated, Reverse Transcription, Amplification, Cloning, Sequencing, Plasmid Preparation, Modification, Mutagenesis, In Vitro, Transfection