Journal: Frontiers in Chemistry

Article Title: Identification of 6ω-cyclohexyl-2-(phenylamino carbonylmethylthio)pyrimidin-4(3 H )-ones targeting the ZIKV NS5 RNA dependent RNA polymerase

doi: 10.3389/fchem.2022.1010547

Figure Lengend Snippet: The interaction between 4w and ZIKV NS5 protein. (A) WB detection of ZIKV NS5 protein expression under different temperature gradients after 4w treatment; (B) NS5 protein grayscale analysis and ZIKV NS5 protein expression after 4w treatment observed changes in aggregation temperature; data is the mean (±SD) of three experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane and incubated with ZIKV E (1:2,000) and NS5 antibody (1:2,000) overnight at 4°C (Sino Biological, China) then add the corresponding secondary antibody and incubate at room temperature for 2 h. A specific signal was presented with a chemiluminescent substrate.

Techniques: Expressing

Journal: Frontiers in Chemistry

Article Title: Identification of 6ω-cyclohexyl-2-(phenylamino carbonylmethylthio)pyrimidin-4(3 H )-ones targeting the ZIKV NS5 RNA dependent RNA polymerase

doi: 10.3389/fchem.2022.1010547

Figure Lengend Snippet: Compound 4w inhibited the expression of ZIKV E and NS5 protein. (A) Western blot detected the inhibitory effect of 4w on ZIKV E and NS5 protein under the concentration gradient of 4w ; (B) Grayscale analysis and statistics of the inhibition of 4w on ZIKV E and NS5 protein under the concentration gradient of WB detection; Data is the mean (±SD) of three experiments, with DMSO as a positive contro * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (C) Detected the inhibitory effect of 4w on ZIKV E protein by immunofluorescence.

Article Snippet: Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane and incubated with ZIKV E (1:2,000) and NS5 antibody (1:2,000) overnight at 4°C (Sino Biological, China) then add the corresponding secondary antibody and incubate at room temperature for 2 h. A specific signal was presented with a chemiluminescent substrate.

Techniques: Expressing, Western Blot, Concentration Assay, Inhibition, Immunofluorescence

Journal: Scientific Reports

Article Title: Asian and African lineage Zika viruses show differential replication and innate immune responses in human dendritic cells and macrophages

doi: 10.1038/s41598-019-52307-1

Figure Lengend Snippet: Comparison of virus replication in human DCs and macrophages infected with Asian and African lineage ZIKV strains. The Asian lineage GWUH strain or the African lineage #976 ZIKV strain was used to separately infect human DCs or macrophages from four different donors. ( A , B ) Cells were infected by the viruses at MOI of 75 TCID 50 /cell and collected at different times after infection as indicated in the figure. ( A ) ZIKV RNA expression was quantitated by qRT-PCR using NS5 gene-specific probes. Data is presented as mean +/− SEM from four individual donors and a representative experiment out of two is shown. ( B ) ZIKV-infected cells from the same 4 donors were collected at different times after infection, cells were pooled and proteins were separated on SDS-PAGE, immunoblotted and stained with anti-NS5 and anti-β-Actin antibodies. ZIKV-infected Vero E6 cell extract was used as a positive control in immunoblotting. ( C ) For the measurement of infectious ZIKV production into cell supernatants, cells were infected with low doses (MOI values 0.2, 1 or 5 TCID 50 /cell) of GWUH or #976 strains followed by collection of cell culture supernatants at times indicated in the figure. Viral titers were measured from the supernatant samples using the end-point dilution assay in Vero E6 cells (TCID 50 /ml) and the virus titers were calculated using the Spearman-Karber method. Results represent the means +/− SEM from DC or macrophage cultures from four different blood donors.

Article Snippet: The rabbit or guinea pig polyclonal antibodies against ZIKV NS3 or NS5 antigens were prepared by immunizing the animals with purified antigens (50 μg/immunization) for 4 times at 2 week intervals together with Freund’s incomplete (FCA) adjuvant (Difco laboratories).

Techniques: Comparison, Virus, Infection, RNA Expression, Quantitative RT-PCR, SDS Page, Staining, Positive Control, Western Blot, Cell Culture, End-point Dilution Assay, Endpoint Dilution Assay

Journal: Scientific Reports

Article Title: Asian and African lineage Zika viruses show differential replication and innate immune responses in human dendritic cells and macrophages

doi: 10.1038/s41598-019-52307-1

Figure Lengend Snippet: Expression of signaling molecules and antiviral proteins in ZIKV-infected human DCs and macrophages. Human primary ( A ) DCs and ( B ) macrophages were infected with GWUH or #976 ZIKV strains at a MOI of 75 TCID 50 /cell and the cells from four individual blood donors were collected at different time points after infection, pooled and cellular protein samples were prepared for immunoblotting. Uninfected mock samples were used as negative control and influenza A virus-infected cells (MOI 1, 24 h infection) as positive control samples. The expression levels of phospho-IRF3 (P-IRF3), IRF3, phospho-STAT2 (P-STAT2), STAT2 and antiviral MxA proteins were analyzed by immunoblotting using specific antibodies. The expression of ZIKV NS5 protein was analyzed for visualizing the virus replication with ZIKV-infected DCs and macrophages. ZIKV infected (GWUH) Vero E6 cell lysate functioned as a positive control, and GAPDH and β-Actin were stained as loading controls. The data shown is a representative experiment out of two independent experiments (altogether n = 8).

Article Snippet: The rabbit or guinea pig polyclonal antibodies against ZIKV NS3 or NS5 antigens were prepared by immunizing the animals with purified antigens (50 μg/immunization) for 4 times at 2 week intervals together with Freund’s incomplete (FCA) adjuvant (Difco laboratories).

Techniques: Expressing, Infection, Western Blot, Negative Control, Virus, Positive Control, Staining

Journal: Scientific Reports

Article Title: Asian and African lineage Zika viruses show differential replication and innate immune responses in human dendritic cells and macrophages

doi: 10.1038/s41598-019-52307-1

Figure Lengend Snippet: Expression of ZIKV RNA and host innate immune genes in human DCs and macrophages infected with two recent Asian lineage ZIKV strains. Differentiated DCs and macrophages from four independent blood donors were separately infected with GWUH or HPF strains at MOI of 2 and analyzed for the expression of viral RNA (panel A) or host cell antiviral genes (panel B) at different times after infection during a 4-day infection experiment. ( A ) For viral RNA expression cells were collected at different times after infection, cells from different donors were pooled and total cellular RNA was extracted and qRT-PCR analysis for ZIKV RNA (using NS5 gene as a target) was carried out. ( B ) The DCs from different donors were collected, pooled and total cellular RNA was isolated followed by quantitation of IFN-λ1, IFN-β, CXCL10 and MxA mRNA expression by qRT-PCR. As a positive control cells were infected with influenza A/Beijing/353/89 virus at MOI of 1 for 24 h. The results are shown as fold induction over the mock sample and the data is representative of two individual experiments (altogether n = 8).

Article Snippet: The rabbit or guinea pig polyclonal antibodies against ZIKV NS3 or NS5 antigens were prepared by immunizing the animals with purified antigens (50 μg/immunization) for 4 times at 2 week intervals together with Freund’s incomplete (FCA) adjuvant (Difco laboratories).

Techniques: Expressing, Infection, RNA Expression, Quantitative RT-PCR, Isolation, Quantitation Assay, Positive Control, Virus

Journal: Vector Borne and Zoonotic Diseases

Article Title: Mosquito Protein Kinase G Phosphorylates Flavivirus NS5 and Alters Flight Behavior in Aedes aegypti and Anopheles gambiae

doi: 10.1089/vbz.2012.1110

Figure Lengend Snippet: Dengue virus (DENV) nonstructural 5 protein (NS5) is phosphorylated during an infection in c6/36 cells. A. albopictus c6/36 cells were noninfected or infected with DENV [multiplicity of infection (MOI)=10]. The cells were harvested and lysed at 48 h postinfection, and NS5 was immunoprecipitated with α-WNV NS5. (A) The immunoprecipitated samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the NS5 band from DENV-infected cells (boxed) was excised from the gel for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis. (B) The arrow highlights the amino acid 31–42 phosphopeptide on the spectrum. This phosphopeptide's mass, 1475.77 Da, is approximately 160 Da higher than its expected mass, indicating the presence of two phosphorylations (Ser31 and Thr39). (C) The arrow on the spectrum indicates the amino acid 440–457 phosphopeptide. The peptide's mass of 2294.19 Da includes a 79.9-Da phosphate group, corresponding to one phosphorylation (Thr449).

Article Snippet: NS5 was immunoprecipitated from the infected cell lysate using a custom-made rabbit polyclonal antibody against WNV NS5 (Covance, Princeton, NJ).

Techniques: Virus, Infection, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Mass Spectrometry, Phospho-proteomics