Journal: Archives of Virology

Article Title: Single B cells reveal the antibody responses of rhesus macaques immunized with an inactivated enterovirus D68 vaccine

doi: 10.1007/s00705-020-04676-6

Figure Lengend Snippet: Sorting of single EV-D68-specific memory B cells by FACS. PBMCs were stained with an antibody cocktail including anti-CD20 (PE), anti-CD27 (FITC), and anti-EV-D68 (APC) antibodies. Single EV-D68-specific memory B cells (CD20 + /CD27 + /EV-D68 + ) from monkeys were sorted into each well of 96-well plates containing 20 μl of cell lysis buffer. (a) Sample from monkey no. 15239. (b) Sample from monkey no. 15083

Article Snippet: Except for the anti-EV-D68-APC antibody, all antibodies used in flow cytometry experiments were purchased from BD Biosciences, and the anti-EV-D68-APC conjugate was prepared according to the protocols provided by the manufacturer (Innova Bioscience Ltd., USA).

Techniques: Staining, Lysis

Journal: Antiviral Research

Article Title: Comparative analysis of the molecular mechanism of resistance to vapendavir across a panel of picornavirus species

doi: 10.1016/j.antiviral.2021.105177

Figure Lengend Snippet: Vapendavir-resistant isolates carry mutations in VP1 and are cross-resistant to other capsid binders . Antiviral activity of capsid binders was assessed in a CPE-reduction assay. Data are mean values of 3 independent experiments ±SD. NA – not active .

Article Snippet: The EV-D68 strain CU70 was obtained from RIVM (Bilthoven, The Netherlands) and cultured in HeLa cells. hRV2 and hRV14 were kindly provided by Dr. K. Andries (Janssen Pharmaceutica, Belgium) and cultured in HeLa cells.

Techniques: Activity Assay, Virus

Journal: Biomolecules

Article Title: A Novel Peptide from VP1 of EV-D68 Exhibits Broad-Spectrum Antiviral Activity Against Human Enteroviruses

doi: 10.3390/biom14101331

Figure Lengend Snippet: Peptide P11 and P25 exhibit antiviral potency to human enteroviruses. ( a ) Antiviral effects of peptide P1-P30 on EV-A71/FY0805, Echo 30/WZ16, and EV-D68/BCH895A. RD cells were infected with 100 TCID 50 /50 µL human enteroviruses co-incubated with 50 µL peptide P1-P30 at a concentration of 125 µg/mL (about 56 µM). 24 h post-infection, RD cells were stained using crystal violet and measured at 550 nm. The CPE was normalized to the only virus control (0%) and 0.5% DMSO mock (100%), and then converted to a white and blue heatmap. ( b ) The antiviral effects of the P25 homologous segments of VP1 from EV-A71 (P25.A71), Echo 30 (P25.E30), Poliovirus 3 (P25.PV3), Rhinovirus A81 (P25.A81), and Rhinovirus B70 (P25.B70). ( c ) Location of P11 and P25 at VP1 of EV-D68 (PDB: 6CRR). P11 and P25 are shown in yellow and magenta, respectively. ( d ) Peptide parameters of P11 and P25 calculated using the ProtParam tool. The positively charged amino acids were marked in red. P25 has a longer estimated half-life than P11. Sequences of P1-P30 are provided in .

Article Snippet: Peptides (18–20-mers) overlapping by 10 residues and spanning the full length of the VP1 of EV-D68, were designed using the web-based software PeptGen, provided by the Los Alamos National Laboratory ( http://www.hiv.lanl.gov/content/sequence/PEPTGEN/peptgen.html (accessed on 9 January 2023)), and synthesized (Purity > 90%; SciLight, Beijing, China).

Techniques: Infection, Incubation, Concentration Assay, Staining, Virus, Control

Journal: Biomolecules

Article Title: A Novel Peptide from VP1 of EV-D68 Exhibits Broad-Spectrum Antiviral Activity Against Human Enteroviruses

doi: 10.3390/biom14101331

Figure Lengend Snippet: IC 50 of peptides.

Article Snippet: Peptides (18–20-mers) overlapping by 10 residues and spanning the full length of the VP1 of EV-D68, were designed using the web-based software PeptGen, provided by the Los Alamos National Laboratory ( http://www.hiv.lanl.gov/content/sequence/PEPTGEN/peptgen.html (accessed on 9 January 2023)), and synthesized (Purity > 90%; SciLight, Beijing, China).

Techniques:

Journal: Biomolecules

Article Title: A Novel Peptide from VP1 of EV-D68 Exhibits Broad-Spectrum Antiviral Activity Against Human Enteroviruses

doi: 10.3390/biom14101331

Figure Lengend Snippet: Antiviral activities of P25 mutants and its truncated peptides. ( a ) Sequences and parameters of the mutant P25s, parameters were calculated using the ProtParam tool. The positively charged amino acids were marked in red; “ + ” and “-” indicated the antiviral activity positive and negative, respectively; “ ++ ” indicated a broad spectrum of antiviral activity. The peptide P25.A81, was derived from Rhinovirus A81 and only inhibited EV-A71/FY0805 infection. The replacement of the N-terminal with G and F of P25.A81GF extended its antiviral profile. ( b ) RD cells were treated with 1 mg/mL of P25, P25.8, P25.9, P25.M, and P25.R, with serial 2-fold dilution for 24 h, and CC 50 values were assayed using CCK8 reagents. P25.R was the peptide with a reverse sequence of P25 and had a stronger cytotoxic effect. So, the IC 50 was not further tested as listed in . ( c – f ) RD cells were infected with enteroviruses co-incubated with serial 2-fold diluted P25 mutants and its truncated peptides, stained using crystal violet and measured at 550 nm at 24 h post-infection. The CPE was normalized to the only virus control (0%) and a 0.5% DMSO mock (100%), and then converted to a white and blue heatmap. ( c ) Anti-EV-A71/FY0805 infection. ( d ) Anti-Echo 30/WZ16 infection. ( e ) Anti-Poliovirus 3/nOPV3 infection. ( f ) Anti-EV-D68/BCH895A infection.

Article Snippet: Peptides (18–20-mers) overlapping by 10 residues and spanning the full length of the VP1 of EV-D68, were designed using the web-based software PeptGen, provided by the Los Alamos National Laboratory ( http://www.hiv.lanl.gov/content/sequence/PEPTGEN/peptgen.html (accessed on 9 January 2023)), and synthesized (Purity > 90%; SciLight, Beijing, China).

Techniques: Mutagenesis, Activity Assay, Derivative Assay, Infection, Sequencing, Incubation, Staining, Virus, Control

Journal: Biomolecules

Article Title: A Novel Peptide from VP1 of EV-D68 Exhibits Broad-Spectrum Antiviral Activity Against Human Enteroviruses

doi: 10.3390/biom14101331

Figure Lengend Snippet: Co-treatment of P25, P25.8, P25.9, and P25.M reduced CPE caused by human enteroviruses. P25, P25.8, P25.9, and P25.M co-treatment with enteroviruses from species A, B, C, and D at 35 °C for 1 h demonstrated anti-CPE effects. P25.8, P25.9, and P25.M have a broad spectrum of antiviral activity. P25.5 served as the negative control. Anti-CPE effects were nonlinear curve-fitted on four represented enteroviruses at 100 TCID 50 /50 µL. ( a ) EV-A71/SZK2021, which is not a HS-related strain. ( b ) Echo 30/WZ16. ( c ) Poliovirus 3/nOPV3. ( d ) EV-D68/BCH895A. Experiments were performed in triplicate. IC 50 values are shown at . ( e ) 5 × 10 4 RD cells were seeded 12 h before infection, infected with 10 TCID 50 /50 µL EV-D68 in the presence of P25.5, P25.8, P25.9, and P25.M at serial concentration for 1 h, and washed with DMEM twice, then replaced with DMEM for 24 h incubation. Viral VP1 protein was immune-stained and the stained focus was calculated using ImageJ (Version 1.54g).

Article Snippet: Peptides (18–20-mers) overlapping by 10 residues and spanning the full length of the VP1 of EV-D68, were designed using the web-based software PeptGen, provided by the Los Alamos National Laboratory ( http://www.hiv.lanl.gov/content/sequence/PEPTGEN/peptgen.html (accessed on 9 January 2023)), and synthesized (Purity > 90%; SciLight, Beijing, China).

Techniques: Activity Assay, Negative Control, Infection, Concentration Assay, Incubation, Staining

Journal: Biomolecules

Article Title: A Novel Peptide from VP1 of EV-D68 Exhibits Broad-Spectrum Antiviral Activity Against Human Enteroviruses

doi: 10.3390/biom14101331

Figure Lengend Snippet: P25s binding and thermostabilization of the virion. ( a ) EV-D68/BCH895A captured using a P25s coating ELISA and detected using anti-EV-D68 polyclonal Abs with a secondary antibody conjugated with HRP, binding affinities were compared by the fold increase normalized to the blank baseline (PBS). The procedure was described as in the Materials and Methods section. ( b – e ) The viral thermostabilization in the presence of P25, P25.5, P25.8, P25.9, and P25.M. About 4 µg of virus were mixed with 1.875 µg of P25, P25.5, P25.8, P25.9, and P25.M in 20 µL at 37 °C for 15 min and the temperature was subsequently increased to 90 °C, recording 10 points of the fluorescence signal at 1˚C intervals. The normalized genome release fluorescence dynamics and the first derivatives of EV-D68 ( b , c ) and Echo 30 ( d , e ) are shown. ( f ) The breakpoint temperature for genome release was calculated using the derivative of the fluorescence signal as the peak value. P25.8, P25.9, and P25.M increased the breakpoint temperature for the release of viral genome compared with P25.5 by approximately 5 °C for EV-D68/BCH895A and by 2–5 °C for Echo 30/WZ16. ( g ) P25.9 and P25.M retained the infectivity of Echo 30/WZ16. 10 6 TCID 50 /mL of Echo 30/WZ16 was co-incubated with an equal volume of the peptide at a concentration of 62.5 µg/mL at 37 °C for 15 min and 45 °C for 2 min, respectively, followed by rapid cooling on ice. The virus titer was determined using TCID 50 as described in the Materials and Methods section. The experiment was repeated in triplicate. Statistical analysis was performed using paired two-tailed t -test. ** indicates p < 0.01.

Article Snippet: Peptides (18–20-mers) overlapping by 10 residues and spanning the full length of the VP1 of EV-D68, were designed using the web-based software PeptGen, provided by the Los Alamos National Laboratory ( http://www.hiv.lanl.gov/content/sequence/PEPTGEN/peptgen.html (accessed on 9 January 2023)), and synthesized (Purity > 90%; SciLight, Beijing, China).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Virus, Fluorescence, Infection, Incubation, Concentration Assay, Two Tailed Test

Journal: Biomolecules

Article Title: A Novel Peptide from VP1 of EV-D68 Exhibits Broad-Spectrum Antiviral Activity Against Human Enteroviruses

doi: 10.3390/biom14101331

Figure Lengend Snippet: P25.M reduced the production of infectious virions. RD cells were infected with 100 TCID 50 of enteroviruses for 1 h and washed using DMEM twice, then replaced with P25.M and P25.5 at a concentration of 62.5 µg/mL for a 24 h treatment, respectively. P25.5 served as the control. Data were presented in three independent experiments. Statistical analysis was performed using a paired two-tailed t -test. * indicates p < 0.05; ** indicates p < 0.01; ( a ) EV-A71/SZK2021. ( b ) Echo 30/WZ16. ( c ) Poliovirus 3/nOPV3. ( d ) EV-D68/BCH895A. ( e ) 5 × 10 4 RD cells were seeded 12 h before infection, infected with 10 TCID 50 /50 µL of EV-D68 for 1 h and washed with DMEM twice, then replaced with P25.5, P25.8, P25.9, and P25.M at serial concentrations for a 24 h treatment. Viral VP1 was immune-stained and the stained area was calculated using ImageJ (Version 1.54g). P25.9 and P25.M at a concentration of 125 µg/mL, significantly inhibited the synthesis of the viral protein. ( f ) Western blotting for viral proteins. The infected cells in the presence of 62.5 µg/mL peptides were harvested, and the density of the viral protein band was calculated using ImageJ and normalized to β-actin as 100%. (The original image can be found in ). The grey, green, pink and black color denotes the treatment with p25.5, p25.8, p25.9 and P25.M, respectively.

Article Snippet: Peptides (18–20-mers) overlapping by 10 residues and spanning the full length of the VP1 of EV-D68, were designed using the web-based software PeptGen, provided by the Los Alamos National Laboratory ( http://www.hiv.lanl.gov/content/sequence/PEPTGEN/peptgen.html (accessed on 9 January 2023)), and synthesized (Purity > 90%; SciLight, Beijing, China).

Techniques: Infection, Concentration Assay, Control, Two Tailed Test, Staining, Western Blot