Journal: Antiviral research

Article Title: Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

doi: 10.1016/j.antiviral.2017.10.008

Figure Lengend Snippet: a. Schematic representation of the large (L), medium (M), and small (S) genome segments (antigenomic sense) of Crimean-Congo hemorrhagic fever virus (CCHFV). Arrows represent the viral protein coding sequences for RNA-dependent RNA polymerase (RdRp), glycoprotein precursor (GPC), and nucleoprotein (NP). CCHFV strain IbAr10200 S segment was modified by inserting the ZsGreen1 (ZsG) protein coding sequence fused to the P2A sequence (amino acid sequence shown) upstream of the NP coding region (rS). b. Western blot analysis of viral protein expression in Huh7 cells infected with CCHFV/ZsG at MOI 1. Cell lysates were harvested 72 h post infection (hpi) to determine cellular expression levels of NP and ZsG, using tubulin as a loading control. c. Cellular localization of virally expressed proteins. Huh7 cells were infected with either CCHFV or CCHFV/ZsG at MOI 1, and imaged 48 hpi. CCHFV NP was detected using immunofluorescence, and ZsG fluorescence was imaged using standard fluorescent microscopy. d. Viral growth curves in Huh7, SW13, and HAP1 cells infected with either CCHFV or CCHFV/ZsG at MOI 0.1. Titers (TCID50) were determined at 24, 48, and 72 hpi, with ZsG fluorescence determined concurrently at the same time points (RLU, relative light units). Representative fluorescent microscopy images of CCHFV/ZsG-infected cell monolayers at 72 hpi are presented.

Article Snippet: Primary antibodies used were: α-CCHFV NP (IBT Bioservices, #04-0011), α-ZsGreen1 (Clonetech, #632598), and anti-α-tubulin (Sigma-Aldrich, #T5168).

Techniques: Virus, Modification, Sequencing, Western Blot, Expressing, Infection, Control, Immunofluorescence, Fluorescence, Microscopy

Journal: Antiviral research

Article Title: Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

doi: 10.1016/j.antiviral.2017.10.008

Figure Lengend Snippet: a. Dose-response curves in Huh7 or SW13 cells treated with a 2-fold serial dilution of ribavirin before infection with CCHFV/ZsG at MOI 0.1. At 72 h post treatment, the reduction in ZsG fluorescence (green) was determined, and all values were normalized to those in mock-treated (DMSO only) cells. Cell viability was determined concurrently using the same serial-fold dilution of compound, with ATP content (blue) normalized to content in mock-treated cells. Each point represents the mean of quadruplicate wells, with error bars indicating standard deviation. Graphs are representative of 3 independent experiments. b. Representative concentration-response curves in Huh7 cells infected with CCHFV/ZsG and treated with T-705, 2′-deoxy-2′-fluorocytidine (2′-dFC), mycophenolic acid, or mycophenolate mofetil. Each point represents the mean of quadruplicate wells, with error bars indicating standard deviation. Graphs are representative of 3 independent experiments. c. Confirmatory counter-screen titer reduction assays were performed using wild-type CCHFV. Huh7 cells were treated with 4 dilutions of each compound centered around their calculated EC50 value 1 h prior to infection with CCHFV at MOI 0.1. At 48 hpi, virus-containing supernatants were harvested, and viral titers were determined by TCID50. Each point represents the mean of triplicate wells, with error bars indicating standard deviation. Dotted line represents the limit of detection for this assay (1.89 × 101 TCID50/mL). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used were: α-CCHFV NP (IBT Bioservices, #04-0011), α-ZsGreen1 (Clonetech, #632598), and anti-α-tubulin (Sigma-Aldrich, #T5168).

Techniques: Serial Dilution, Infection, Fluorescence, Standard Deviation, Concentration Assay, Virus

Journal: Antiviral research

Article Title: Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

doi: 10.1016/j.antiviral.2017.10.008

Figure Lengend Snippet: Compounds evaluated in the antiviral screening assay, showing the 50% effective concentrations (EC 50 ) and 50% cytotoxicity concentrations (CC 50 ) of each compound in Huh7 cells. All values are in μM; selectivity indices (SI) shown in parentheses. RdRp, RNA-dependent RNA polymerase; IMPDH, inosine monophosphate dehydrogenase.

Article Snippet: Primary antibodies used were: α-CCHFV NP (IBT Bioservices, #04-0011), α-ZsGreen1 (Clonetech, #632598), and anti-α-tubulin (Sigma-Aldrich, #T5168).

Techniques: Screening Assay

Journal: Antiviral research

Article Title: Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

doi: 10.1016/j.antiviral.2017.10.008

Figure Lengend Snippet: Dose-response curve in SW13 cells treated with a 2-fold serial dilution of 2′-dFC before infection with CCHFV at MOI 0.1. At 48 hpi, cells were fixed and stained with anti-CCHFV polyclonal antibodies, CellMask Red, and Nuc-Blue to visualize viral proteins, cell cytoplasm, and cell nuclei, respectively. a. The percentage of infected cells at each concentration of compound was calculated by determining the proportion of cells expressing CCHFV proteins compared to total cell number (based on CellMask Red). b. Cell viability assessed by determining the number of Nuc-Blue-stained nuclei. c. Representative images at indicated 2′-dFC concentrations show the reduction in CCHFV protein production (green) in the absence of any cytotoxicity (Nuc-Blue-stained nuclei). CellMask Red staining was omitted from these images for clarity. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies used were: α-CCHFV NP (IBT Bioservices, #04-0011), α-ZsGreen1 (Clonetech, #632598), and anti-α-tubulin (Sigma-Aldrich, #T5168).

Techniques: Serial Dilution, Infection, Staining, Concentration Assay, Expressing

Journal: Antiviral research

Article Title: Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

doi: 10.1016/j.antiviral.2017.10.008

Figure Lengend Snippet: Huh7 cells were treated with a 6 × 6 compound combination matrix prior to infection with CCHFV/ZsG at MOI 0.1. At 72 h post treatment, the reduction in ZsG fluorescence was determined, and all values normalized to mock-treated (DMSO only) cells. The compound combinations evaluated were: a. T-705 with ribavirin; b. T-705 with 2′-dFC; and c. 2′-dFC with ribavirin. Dose-response surface curves were created for each combination (Bliss model), from which Bliss synergy and antagonism surface isograms were generated. Matrix tables indicate the maximum synergy value for each combination (% synergy effect observed over expected), with standard deviation indicated. Significance of synergy effect over expected results is indicated following a one-sample t-test (*p < 0.05; **p < 0.01, ***p < 0.001).

Article Snippet: Primary antibodies used were: α-CCHFV NP (IBT Bioservices, #04-0011), α-ZsGreen1 (Clonetech, #632598), and anti-α-tubulin (Sigma-Aldrich, #T5168).

Techniques: Infection, Fluorescence, Generated, Standard Deviation

Journal: Antiviral research

Article Title: Identification of 2′-deoxy-2′-fluorocytidine as a potent inhibitor of Crimean-Congo hemorrhagic fever virus replication using a recombinant fluorescent reporter virus

doi: 10.1016/j.antiviral.2017.10.008

Figure Lengend Snippet: a. Huh7 cells were treated with the indicated compounds either individually or in combination at the following concentrations: T-705, 2 μM; 2′-dFC, 200 nM; ribavirin, 10 μM. Cell were infected with wild-type CCHFV at MOI 0.1, and viral titers were determined at 48 hpi. Each point represents the mean of triplicate wells, with error bars indicating standard deviation. Dotted line represents the limit of detection for this assay (1.89 × 101 TCID50/mL). b. Cell viability (ATP content) was determined concurrently and normalized to ATP content in mock-treated (DMSO only) Huh7 cells.

Article Snippet: Primary antibodies used were: α-CCHFV NP (IBT Bioservices, #04-0011), α-ZsGreen1 (Clonetech, #632598), and anti-α-tubulin (Sigma-Aldrich, #T5168).

Techniques: Infection, Standard Deviation

Journal: PLoS Neglected Tropical Diseases

Article Title: Development of a multiplex microsphere immunoassay for the detection of antibodies against highly pathogenic viruses in human and animal serum samples

doi: 10.1371/journal.pntd.0008699

Figure Lengend Snippet: Previously characterised serum samples were used as a validation panel to confirm the ability of the MMIA to detect anti-NP antibodies generated in response to infection with these viruses. These serum samples were assayed by MMIA under the same conditions that were used to assay the serum panels from the German blood bank and Guinea, thus providing a range of MFI values for each viral NP coupled microsphere region for known positive serum samples. For EBOV and MARV validation, serum taken from each NHP prior to experimental infection (pre) is compared to serum taken at various time points post-infection. For the RVFV NHP serum and for CCHFV and DOBV human serum, samples taken at a single time point after infection are compared to presumed negative human serum samples.

Article Snippet: Hyperimmune sheep serum containing anti-CCHFV NP antibodies was generated by AltaBiosciences Ltd. (United Kingdom) using 1 mg purified CCHFV NP (highly similar to strain Baghdad 12) in a 1-ml volume [ , ].

Techniques: Biomarker Discovery, Generated, Infection