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: 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

Journal: Lancet Regional Health - Americas

Article Title: Enterovirus D68 in United States wastewater: a longitudinal surveillance study integrating climatic, demographic, and clinical data

doi: 10.1016/j.lana.2026.101446

Figure Lengend Snippet: EV-D68 RNA in wastewater at national and state levels, 2023–2025. Smoothed, PMMoV-normalized EV-D68 RNA concentration for (a) United States, (b) California, and (c) Pennsylvania. The shaded band denotes the EV-D68 activity duration; the vertical line marks the center of the EV-D68 season. For the national series (a), duration was defined as the period when concentrations exceeded a threshold equal to a baseline plus three standard deviations, where the baseline and standard deviation were estimated from the quiet tail (lowest tertile) of the data distribution. For state series (b–c), duration was defined as the continuous run containing the center of season during which values were detectable, with a minimum of at least two consecutive weeks with EV-D68 RNA concentration above 0. Dates with day-of-month annotate the start and end of the duration and the season center in each state. ∗EV-D68 RNA concentration refers to the smoothed, PMMoV-normalized concentration, calculated using a five-day centered trimmed moving average. For each five-day window, the highest and lowest daily values were excluded, and the remaining three were averaged.

Article Snippet: Most EV-D68 infections cause mild or no symptoms and therefore rarely prompt clinical testing, and EV-D68 specific diagnostics are not routinely performed.

Techniques: Concentration Assay, Activity Assay, Standard Deviation

Journal: Lancet Regional Health - Americas

Article Title: Enterovirus D68 in United States wastewater: a longitudinal surveillance study integrating climatic, demographic, and clinical data

doi: 10.1016/j.lana.2026.101446

Figure Lengend Snippet: State-level timing and duration of EV-D68 activity in U.S. wastewater, with WWTP-level geographic gradients. (a) Map shows, for each state, the calendar date of the EV-D68 seasonal center estimated from the state-aggregated weekly series (PMMoV-normalized, 5-day–trimmed), after first aggregating across WWTPs within state and week. Colors run from earlier (Jun ’24) to later (Nov ’24). (b) Map shows the length of the detectable activity window (displayed in months) around the seasonal center of the state, computed on the same state-aggregated weekly series by identifying consecutive weeks with detectable signal (runs of at least two weeks above the non-detect threshold) and converting weeks to months. Gray states do not have wastewater data available. Alaska and Hawaii are shown out of scale for layout. Panels c–f are weighted bivariate regressions displaying gradients between longitude or latitude and EV-D68 center of season (C and d) or activity duration (e and f). Each point is a WWTP within the United States (n = 146) Clinton, Iowa had W = 0 (no detections after normalisation and trimming) and therefore was excluded. Point size denotes the plant weight WW, defined as the sum across weeks of PMMoV-normalised, 5-day–trimmed EV-D68 concentrations, with non-detects set to 0. Larger circles indicate greater cumulative signal and therefore greater influence on the weighted least-squares fits. Lines show weighted least-squares fits with 95% CIs.

Article Snippet: Most EV-D68 infections cause mild or no symptoms and therefore rarely prompt clinical testing, and EV-D68 specific diagnostics are not routinely performed.

Techniques: Activity Assay

Journal: Lancet Regional Health - Americas

Article Title: Enterovirus D68 in United States wastewater: a longitudinal surveillance study integrating climatic, demographic, and clinical data

doi: 10.1016/j.lana.2026.101446

Figure Lengend Snippet: EV-D68 center of season as a function of mean environmental conditions across WWTPs (n = 146). For each plant, the x-axis is the mean of the indicated variable (daily values aggregated to weekly means over the EV-D68 activity window); the y-axis is the calendar date of the season center. Lines are univariate weighted least-squares fits with 95% CIs; weights are proportional to each plant's cumulative EV-D68 signal, with non-detects set to 0. Point sizes are proportional to the weights and the weighted R 2 is shown on each panel.

Article Snippet: Most EV-D68 infections cause mild or no symptoms and therefore rarely prompt clinical testing, and EV-D68 specific diagnostics are not routinely performed.

Techniques: Activity Assay

Journal: Lancet Regional Health - Americas

Article Title: Enterovirus D68 in United States wastewater: a longitudinal surveillance study integrating climatic, demographic, and clinical data

doi: 10.1016/j.lana.2026.101446

Figure Lengend Snippet: Duration of EV-D68 circulation by wastewater treatment plant (WWTP)-level characteristics. Each panel shows the distribution of EV-D68 circulation duration (in weeks) across categories of a single WWTP–level determinant. Points represent individual WWTPs and boxplots show the median, interquartile range (IQR), and whiskers at 1.5 × IQR. Airport presence is dichotomized as absent and present. Hospital and nursing home coverage are split using the dataset medians: 2 hospitals and 8 nursing homes within each WWTP catchment area, respectively. Urbanicity is defined by the proportion of the WWTP catchment area classified as urban (≤50% vs >50%). The proportions of children aged ≤5 years, adults aged ≥65 years, crowded households, birth rate (per capita), childcare density (per km 2 ), and population density (per km 2 ) are grouped into within-study tertiles (“low”, “middle”, “high”). Sample sizes (n) shown in each panel indicate the number of WWTPs included in each comparison group. Kruskal–Wallis test was used to assess overall differences, and pairwise comparisons used Wilcoxon rank-sum tests with Bonferroni adjustment. Horizontal connector bars indicate statistically significant differences; asterisks denote adjusted p-values (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Article Snippet: Most EV-D68 infections cause mild or no symptoms and therefore rarely prompt clinical testing, and EV-D68 specific diagnostics are not routinely performed.

Techniques: Comparison

Journal: Lancet Regional Health - Americas

Article Title: Enterovirus D68 in United States wastewater: a longitudinal surveillance study integrating climatic, demographic, and clinical data

doi: 10.1016/j.lana.2026.101446

Figure Lengend Snippet: National and within-state correlations between wastewater EV-D68 RNA concentrations and clinical diagnoses. (a–d) National time-series comparing wastewater EV-D68 RNA concentrations (black lines, right axis) with proportions of clinical diagnoses (bars, left axis) for wheezing in children ≤5 years (A), wheezing in adults ≥65 years (b), enterovirus-specific encounters (c), and acute flaccid myelitis (AFM) (d). (e–h) State-level Spearman correlations between wastewater EV-D68 and AFM (e), enterovirus-specific encounters (f), wheezing ≤5 years (g), and wheezing ≥65 years (h). Points represent correlation coefficients, with colors denoting effect size and significance (adjusted p < 0.05, Bonferroni). Correlations at the state level were performed using data filtered to the EV-D68 seasonal duration defined by wastewater and extended ±2 weeks for clinical diagnoses, and ±2 months for AFM.

Article Snippet: Most EV-D68 infections cause mild or no symptoms and therefore rarely prompt clinical testing, and EV-D68 specific diagnostics are not routinely performed.

Techniques:

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Preparation and characterization of IWV and VLP (A) Schematic representation of the construction of plasmids encoding EV-D68 P1 and 3CD. (B) Workflow for the expression and purification of VLP. (C–F) SDS-PAGE analysis of purified IWV and VLP. (C and D) VLP expressed in (C) Expi293F cells and (D) ExpiCHO-S cells purified with sucrose. The Hsp and Hsc identified by mass spectrometry are labeled in red lines. (E) VLP was expressed in ExpiCHO-S cells and further purified using both sucrose and iodixanol (OptiPrep) gradients. (F) IWV was purified by sucrose gradient ultracentrifugation. (G) IWV and VLP particle size distribution was measured by dynamic light scattering. (H) Representative negative-stain TEM images of IWV and VLP. Scale bars, 100 nm. (I) The thermal stability of the non-inactivated virus, IWV, and VLP was assessed using differential scanning fluorimetry (DSF). Data represent the mean of four independent measurements ( n = 4) for each sample.

Article Snippet: The use of EV-D68 was reviewed and approved by the Institutional Review Board of the Research Institute for Microbial Diseases, The University of Osaka (protocol number: BIKEN-00184-004).

Techniques: Expressing, Purification, SDS Page, Mass Spectrometry, Labeling, Staining, Virus

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Epitope specificities of IgG induced by IWV and VLP vaccines (A) Localization of neutralizing antigenic sites I–IV and corresponding epitope peptide sequences. Neutralizing antigenic sites I–IV were mapped onto a single icosahedral asymmetric unit of the EV-D68 MO strain capsid, based on the previously reported cryo-EM structure (PDB: 6CSG ). VP1, VP2, and VP3 are shown in gray, pink, and cyan, respectively. Neutralizing antigenic sites I, II, III, and IV are highlighted in yellow, green, blue, and magenta, respectively. The accompanying table lists the sequences of epitope peptides spanning the antigenic sites; residues constituting the neutralizing antigenic sites are indicated in red. Molecular graphics were generated using UCSF ChimeraX v1.9. (B) Plasma IgG levels specific to epitope peptides following boost immunization with either IWV or VLP. (C) Plasma IgG reactivity to mutant VLPs following boost immunization with IWV or wild-type VLP. Then, 1,000-fold diluted plasma samples were used. Details of the mutations in each mutant are summarized in the table on the right. (B–C) n = 5 per group. Data are presented as mean ± SD. (B) Statistical comparisons were performed using 50-fold diluted plasma samples. (B) “ns” indicates not significant. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001, as determined by Tukey’s test. (C) ∗ p < 0.05 and ∗∗∗∗ p < 0.0001, as determined by Dunnett’s multiple comparison test.

Article Snippet: The use of EV-D68 was reviewed and approved by the Institutional Review Board of the Research Institute for Microbial Diseases, The University of Osaka (protocol number: BIKEN-00184-004).

Techniques: Vaccines, Cryo-EM Sample Prep, Generated, Clinical Proteomics, Mutagenesis, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative immunogenic and structural analysis of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

doi: 10.1016/j.omtn.2026.102957

Figure Lengend Snippet: Cryo-EM structure of EV-D68 MO strain VLP (A) Cryo-EM density maps showing the overall structures of the mature virion (PDB: 6CSG ), empty particle (PDB: 6CRU ), and VLP of the EV-D68 MO strain. A schematic illustration of the viral particle is shown in the left-most. The 5-, 3-, and 2-fold symmetry axes are indicated by a pentagon, triangle, and circle, respectively. (B) Ribbon representations of the mature virion (PDB: 6CSG ), empty particle (PDB: 6CRU ), and VLP structures around the 2-fold axis. The 3-fold and 2-fold axes are denoted by a triangle and circle, respectively. (C) Structural comparison of the icosahedral asymmetric units of the VLP with those of the mature virion (left, PDB: 6CSG ) and the empty particle (middle, PDB: 6CRU ). VP1, VP0, and VP3 of the VLP were superimposed onto the corresponding subunits of the mature virion and the empty particle. Representative structures are shown based on superposition via VP1. All structures are depicted as ribbon models. The table on the right summarizes the number of pruned atom pairs and root-mean-square deviation (RMSD) values for each superposition.

Article Snippet: The use of EV-D68 was reviewed and approved by the Institutional Review Board of the Research Institute for Microbial Diseases, The University of Osaka (protocol number: BIKEN-00184-004).

Techniques: Cryo-EM Sample Prep, Comparison