HCV interference with ISG expression upon IFNα treatment in chimeric mice.

<p>We analyzed the differences in expression levels of intrahepatic human ISGs among uninfected (NI), IFNα-treated chimeric mice and IFN-treated chimeric mice infected with either HCVgt1a or HCVgt2b. The uninfected IFNα-treated animals were set as the baseline for comparison. Each column in the heat map (<b>A</b>) represents the results in a mouse in each group. <i>P</i>-values in <b>B</b> were calculated using unpaired t-tests. In <b>B</b>, significantly (<i>P <</i> .05) upregulated and downregulated expression of ISGs compared to the uninfected IFNα-treated animal controls is indicated, respectively, by red and green highlighting in the tables. No highlighting indicates no significant difference in comparison to the uninfected IFNα-treated control group.</p

Effect of the poor-responder genotypes of <i>IL28B/IFNL4</i> SNPs on HCV responses to IFNα in mice.

<p>We infected chimeric mice populated with human hepatocytes from two donors, Hu4109 (<b>A, B</b>) and FLO (<b>C, D</b>), with HCVgt1a (<b>A, C</b>) or HCVgt2b (<b>B, D</b>). We administered exogenous human IFNα-2b for 14 days starting at 5 weeks post-infection. We administered saline to control chimeric mice on the same schedule. We collected sera weekly to measure viremia. Results for mice treated with saline are represented by black solid lines and data for mice treated with human IFNα are represented by blue dashed lines. The period of IFNα/saline treatment is shaded in gray.</p

Intrahepatic IFN or ISG expression in mice infected with the two HCV strains.

<p>We infected chimeric mice produced with hepatocytes from donor Hu8063 with either HCVgt1a or HCVgt2b. Seven weeks p.i., we euthanized the mice and determined the expression levels of human-specific endogenous IFN genes (<b>A</b>) and ISGs (<b>B</b>) relative to a house-keeping gene HPRT-1 using the 2^-ΔΔCt method. In uninfected donor-matched chimeric mice, the mean value of each gene mRNA level was normalized to 1. Data in <b>A</b> are mean ± the standard error of the mean (SEM); n = 3 to 5. We determined <i>P</i>-values using a one-way ANOVA calculation. In <b>B</b>, each column represents an experimental mouse. Increased and decreased expression of specific genes compared to the uninfected control group is shown in red (Fold >1 to ≥4) and green (Fold <1 to ≤0.25), respectively, whereas black indicates no change (Fold = 1).</p

Responses of the two HCV strains to IFNα in mice populated with a single donor.

<p>We infected chimeric mice produced with a single hepatocyte donor, Hu8063, with 2 HCV strains for 5 weeks. We then subcutanously injected exogenous human IFNα-2b daily at 1,350 IU/gram for 14 days. We treated control chimeric mice with saline. We took sera from the mice weekly in order to measure viremia. Six hours after the last IFNα injection, we terminated the mice and measured intrahepatic HCV load. Data for mice treated with saline are represented by black solid lines and data for mice treated with IFNα are represented by blue dashed lines in <b>A</b> (HCVgt1a viremia titer) and <b>B</b> (HCVgt2b viremia titer). Each line in <b>A</b> and <b>B</b> represents a single mouse. The period of human IFNα/saline treatment is shaded in gray. A comparison of intrahepatic HCV RNA levels for different treatment groups at week 7 p.i. is shown in <b>C</b>. <i>P</i>-values in <b>C</b> were calculated using unpaired t-tests.</p

Aptamer Binding Assay for the E Antigen of Hepatitis B Using Modified Aptamers with G‑Quadruplex Structures

The e antigen of hepatitis B (HBeAg) is positively associated with an increased risk of developing liver cancer and cirrhosis in chronic hepatitis B (CHB) patients. Clinical monitoring of HBeAg provides guidance to the treatment of CHB and the assessment of disease progression. We describe here an affinity binding assay for HBeAg, which takes advantage of G-quadruplex aptamers for enhanced binding and stability. We demonstrate a strategy to improve the binding affinity of aptamers by modifying their sequences upon their G-quadruplex and secondary structures. On the basis of predicting a stable G-quadruplex and a secondary structure, we truncated 19 nucleotides (nt) from the primer regions of an 80-nt aptamer, and the resulting 61-nt aptamer enhanced binding affinity by 19 times (Kd = 1.2 nM). We mutated a second aptamer (40 nt) in one loop region and incorporated pyrrolo-deoxycytidine to replace deoxycytidine in another loop. The modified 40-nt aptamer, with a stable G-quadruplex and two modified loops, exhibited a 100 times higher binding affinity for HBeAg (Kd = 0.4 nM) than the unmodified original aptamer. Using the two newly modified aptamers, one serving as the capture and the other as the reporter, we have developed an improved sandwich binding assay for HBeAg. Analyses of HBeAg in serum samples (concentration ranging from 0.1 to 60 ng/mL) of 10 hepatitis B patients, showing consistent results with clinical tests, demonstrate a successful application of the aptamer modification strategy and the associated aptamer binding assay

Antiviral Activity of Various 1-(2′-Deoxy-β-d-lyxofuranosyl), 1-(2′-Fluoro-β-d-xylofuranosyl), 1-(3′-Fluoro-β-d-arabinofuranosyl), and 2′-Fluoro-2′,3′-didehydro-2′,3′-dideoxyribose Pyrimidine Nucleoside Analogues against Duck Hepatitis B Virus (DHBV) and Human Hepatitis B Virus (HBV) Replication

Despite the existence of successful vaccine and antiviral therapies, infection with hepatitis B virus (HBV) continues to be a major global cause of acute and chronic liver disease and high mortality. We synthesized and evaluated several lyxofuranosyl, 2′-fluoroxylofuranosyl, 3′-fluoroarabinofuranosyl, and 2′-fluoro-2′,3′-didehydro-2′,3′-dideoxyribose pyrimidine nucleoside analogues for antiviral activities against hepatitis B virus. Among the compounds examined, 1-(2-deoxy-β-d-lyxofuranosyl)thymine (23), 1-(2-deoxy-β-d-lyxofuranosyl)-5-trifluoromethyluracil (25), 1-(2-deoxy-2-fluoro-β-d-xylofuranosyl)uracil (38), 1-(2-deoxy-2-fluoro-β-d-xylofuranosyl)thymine (39), 2′,3′-dideoxy-2′,3′-didehydro-2′-fluorothymidine (48), and 2′,3′-dideoxy-2′,3′-didehydro-2′-fluoro-5-ethyluridine (49) were found to possess significant anti-HBV activity against DHBV in primary duck hepatocytes with EC50 values of 4.1, 3.3, 40.6, 3.8, 0.2, and 39.0 μM, respectively. Compounds 23, 25, 39, 48, and 49 (EC50 = 41.3, 33.7, 19.2, 2.0−4.1, and 39.0 μM, respectively) exhibited significant activity against wild-type human HBV in 2.2.15 cells. Intriguingly, 25, 39, 48, and 49 retained sensitivity against lamivudine-resistant HBV containing a single mutation (M204I) and 48 emerged as an effective inhibitor of drug-resistant HBV with an EC50 of 4.1 μM. In contrast, 50% inhibition could not be achieved by lamivudine at 44 μM concentration in the drug-resistant strain. The compounds investigated did not show cytotoxicity to host cells up to the highest concentrations tested

Interferon response in Huh7.5 and PDLIM2 K/O cells.

A-H) The expression of 35 ISGs in either Huh7.5 (black outlined bars) or PDLIM knockout cells (red bars) was examined after IFNα2 treatment using a custom TaqMan OpenArray. Six time points are displayed. To examine the initial IFN response, cells were untreated, or treated with IFNα2 for 2 h or 7 h; to examine the subsequent refractory interferon response cells were treated with IFNα2 12 h followed by 12 h rest, then retreated with IFNα2 for 2 h or 7h. A-B) Genes involved in initiation of the innate interferon response, C) inhibitors of the innate immune response. D-F) genes involved in extracellular signaling during the innate interferon response, G-H) effector genes involved in the innate interferon response. HPRT was used to normalize mRNA levels and the average of the duplicate untreated sample was used to determine the fold increase after interferon treatment. Error bars indicate SEM. A 2 way anova analysis was used to determine significance of both IFN addition and the differences between the response seen in Huh7.5 cells and that in PDLIM K/O cells, * denotes p<0.05, ** denotes p<0.01, *** denotes p<0.001, and **** denotes p<0.0001. I) Huh7.5 cells and PDLIM2 K/O cells were infected with VSV for 24 h and then visualized by phase contrast microscopy, or trypsinized, stained with trypan blue and counted. Experiments were performed in duplicate. Scale bars are 40 μm. Error bars indicate standard deviation.</p

Confocal microscopy reveals lower STAT1 and STAT2 levels in HCV infected cells both <i>in vivo</i>, in chimeric mouse livers, and <i>in vitro</i>, in Huh7.5 cells.

Confocal microscopy was performed on A) liver sections or B) and C) Huh7.5 cells either uninfected or infected with HCV. A) SCID/Alb-uPA mice transplanted with human hepatocytes were infected and liver sections were stained using antibodies directed against HCV (red) and antibodies specific for either STAT1 or STAT2 (green). Isotype controls are shown in S1 Fig. The amount of STAT1 or STAT2 was quantified in cells that stained positive or negative for HCV within an infected liver. For STAT1, 58 infected and 79 uninfected cells were analyzed from 10 fields. For STAT2, 42 infected and 74 uninfected cells were analyzed from 4 fields using Metamorph software. To compare cells among several infected fields the average green (STAT) fluorescence of uninfected cells from a single field was arbitrarily set to 1 and the infected cells in that field were scaled appropriately. In B) and C), confocal microscopy was performed on uninfected or HCV infected Huh7.5 cells that were either left untreated or treated with IFNα2 for the indicated times prior to fixation. Cells were stained using antibodies directed against HCV (Red) and antibodies specific for either B) STAT1 or C) STAT2 (green). For quantification, at least 511 cells in multiple fields for each treatment were quantified. Nuclei were stained with DAPI (blue). The scale bars are 10μm. For reference, white borders were drawn around infected cells in the red channel and copied into the green channel; red arrows mark some HCV infected cells, and white arrows mark some uninfected cells in the last panel. Unpaired t-tests were used to determine significance, and *** denotes p<0.001.</p

Huh7.5 cells lacking PDLIM2 are more resistant to viral infection than parental Huh7.5 cells.

A) HCV: Top Panel: Confocal microscopy of Huh7.5 or PDLIM2 knockout cells infected with 0.1GE/cell of HCV. After 7 days the cells were fixed and HCV core was visualized (green). Nuclei were stained with Hoechst (blue). Scale bars are 10 μm. Middle Panel: Cells were first infected with 1GE/cell of HCV and then qRT-PCR for HCV RNA from cells (bar graph) or supernatants (line graph) was performed after 3, 6, and 7 days of infection. Intracellular HCV RNA levels were normalized to HPRT mRNA levels. Lower Panel: Cells were infected with 8 replicates of the indicated MOI in a 96 well plate. After 4 days cells were stained for the presence of HCV NS5a protein and the percentage of wells containing infected cells was determined. B) Huh7.5 cells and PDLIM2 K/O cells were infected with HAV for 4 days. Cells were fixed and HAV capsid was visualized (Top panel, green) or intracellular HAV RNA quantified by qRT-PCR normalized to HPRT mRNA (Lower panel, bar graph) or extracellular HAV quantified by qRT-PCR (Lower panel, line graph). Experiments were done twice. C) Huh7.5 and PDLIM2 K/O cells were infected with ZIKV at the indicated MOI (pfu/cell) for 24h. Intracellular ZIKV RNA was quantified by qRT-PCR and normalized to HPRT mRNA (bar graph). Extracellular ZIKV was quantified by plaque assay (line graph). Experiments were done 3 times. Error bars indicate SEM. D) Huh7.5 cells or their PDLIM2 K/O derivatives were infected with the indicated MOI of DENV for 36h, fixed, and DENV capsid protein was visualized (Upper panels, green). The percent of DENV infected cells is shown in the bar graph (lower panel). A minimum of 15 fields were counted with an average of 44 cells/field. A representative field at an MOI of 0.1 is shown in the upper panels.</p

A Refined Model of the HCV NS5A Protein Bound to Daclatasvir Explains Drug-Resistant Mutations and Activity against Divergent Genotypes

Many direct-acting antiviral agents (DAAs) that selectively block hepatitis C virus (HCV) replication are currently under development. Among these agents is Daclatasvir, a first-in-class inhibitor targeting the NS5A viral protein. Although Daclatasvir is the most potent HCV antiviral molecule yet developed, its binding location and mode of binding remain unknown. The drug exhibits a low barrier to resistance mutations, particularly in genotype 1 viruses, but its efficacy against other genotypes is unclear. Using state-of-the-art modeling techniques combined with the massive computational power of Blue Gene/Q, we identified the atomic interactions of Daclatasvir within NS5A for different HCV genotypes and for several reported resistant mutations. The proposed model is the first to reveal the detailed binding mode of Daclatasvir. It also provides a tool to facilitate design of second generation drugs, which may confer less resistance and/or broader activity against HCV