Journal: PLOS Pathogens

Article Title: N460S in PB2 and I163T in nucleoprotein synergistically enhance the viral replication and pathogenicity of influenza B virus

doi: 10.1371/journal.ppat.1013463

Figure Lengend Snippet: (A-D) HEK 293T cells were transfected with expression plasmids pCAGGS encoding GZ0215-01 derived PB2-Myc (with 460N or 460S) (A and B), or NP (with 163I or 163T) (C and D). As a control, empty pCAGGS vector was used. 24 hours post-transfection, cells were harvested, and relative mRNA levels of PB2 (A) and NP (C) were measured by qRT-PCR. hGAPDH was used as an internal reference. Protein expression levels of PB2-Myc (left panel of B) and NP (left panel of D) were determined by Western blot using rabbit anti-Myc tag polyclonal antibody and rabbit anti-NP polyclonal antibody, respectively. GAPDH was used as a loading control. The band intensities were quantified using ImageJ software ( http://rsb.info.nih.gov/ij/ ) (right panels of B and D). (E-F) HEK 293T cells were transfected with bidirectional expression plasmids pM encoding GZ0215-01 derived PB2-Myc (with 460N or 460S) and NP (with 163I or 163T), along with PB1 and PA. Co-immunoprecipitation was performed using ProteinA/G magnetic beads conjugated to NP antibody. Precipitated eluates and non-precipitated whole cell lysates were subjected to SDS-PAGE, and PB2 and NP were detected by Western blot (E). GAPDH was used as a loading control. The relative levels of input PB2 and NP proteins (normalized by GAPDH, left panel of F) and the relative levels of PB2 in IP samples (normalized by NP protein, right panel of F) were quantified using ImageJ software. (G) Polymerase activity. HEK 293T cells were co-transfected with PB2, PB1, PA, and NP plasmids from the indicated recombinant IBV mutants, along with a firefly luciferase reporter template flanked by the NP untranslated regions of IBV and a Renilla luciferase expression plasmid as an internal control. Dual-luciferase activity was measured 24 hours post-transfection. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: Protein expression was detected using rabbit anti-NP polyclonal antibody (Cat. No. 40438-T62, Sino Biological) or rabbit anti-Myc tag polyclonal antibody (Cat. No. 16286–1-AP, Proteintech), followed by HRP-conjugated secondary antibodies (Cell Signaling Technology).

Techniques: Transfection, Expressing, Derivative Assay, Control, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Software, Immunoprecipitation, Magnetic Beads, SDS Page, Activity Assay, Recombinant, Luciferase

Journal: iScience

Article Title: Synthetic vaccine affords full protection to mice against lethal challenge of influenza B virus of both genetic lineages

doi: 10.1016/j.isci.2021.103328

Figure Lengend Snippet: Intranasal immunization with a prime/boost regimen provides the best protection against lethal IBV challenge compared to subcutaneous immunization (A–I) (A) Schematic diagram of the immunization, viral challenge, and necropsy timeline. DBA2 mice were subcutaneously (B, D, F, and H) or intranasally (C, E, G, and I) vaccinated once or twice before an intranasal challenge with B/Victoria/02/87. Survival (B, C; n = 10), weight (D and E; n = 10), Day 21 anti-HA2 IgG endpoint titer (F and G; n = 3–5), and Day 49 anti-HA2 IgG endpoint titer (H and I; n = 8–10) are shown. Data shown is mean ± SEM; ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 (Mann-Whitney test).

Article Snippet: Briefly, 96-well plates were coated with 100ul/well of 0.5ug/ml of recombinant influenza B/Florida/4/06 HA2 protein (Sino Biological Inc.), overnight at 4°C.

Techniques: MANN-WHITNEY

Journal: iScience

Article Title: Synthetic vaccine affords full protection to mice against lethal challenge of influenza B virus of both genetic lineages

doi: 10.1016/j.isci.2021.103328

Figure Lengend Snippet: rAd-HA2F elicits robust HA2-specific antibody responses with high ADCC activity against multiple IBV strains Serum from DBA2 mice collected 21 days after prime vaccination (Day 21) and 21 days after boost vaccination (Day 49) was used to determine anti-HA2 endpoint titer. IgG1 (A), IgG2a (B), and IgG2b (C) titers are shown. Bronchoalveolar lavage fluid (BALF) was collected on Day 49 to determine anti-HA2 IgG (D) and IgA (E) endpoint titers. Day 49 serum was used to determine the ADCC activity against B/Victoria/2/87 (F), B/Florida/04/06 (G; Yamagata lineage), B/Brisbane/60/08 (H; Victoria lineage), and B/Yamagata/16/88 (I). Data shown is mean ± SEM; n = 6 per group in each experiment; ∗∗p < 0.01 (Mann-Whitney test).

Article Snippet: Briefly, 96-well plates were coated with 100ul/well of 0.5ug/ml of recombinant influenza B/Florida/4/06 HA2 protein (Sino Biological Inc.), overnight at 4°C.

Techniques: Activity Assay, MANN-WHITNEY

Journal: iScience

Article Title: Synthetic vaccine affords full protection to mice against lethal challenge of influenza B virus of both genetic lineages

doi: 10.1016/j.isci.2021.103328

Figure Lengend Snippet: Truncated vaccines elicit higher Th2 antibody isotypes than rAd-HA2F affecting ADCC activity Serum from DBA2 mice collected 21 days after boost vaccination (Day 49) was used to determine anti-HA2 endpoint titer. IgG1 (A), and IgG2a (C) endpoint titers, and IgG1:IgG2a ratios (B) are shown. (D) Day 49 serum was used to determine the ADCC activity against B/Victoria/2/87. Fold induction over ‘no antibody’ controls is shown. Data shown is mean ± SEM; n = 6 per group in each experiment; ∗p < 0.05, ∗∗p < 0.01 (one-way ANOVA with Tukey's post-test in panels (A–C) and two-way ANOVA with Tukey's post-test in panel (D)). In (D), rAd-HA2ΔN30F is significantly different from rAd-HA2F at the first 2 points (dilution (log) = 1.48 and 1.95).

Article Snippet: Briefly, 96-well plates were coated with 100ul/well of 0.5ug/ml of recombinant influenza B/Florida/4/06 HA2 protein (Sino Biological Inc.), overnight at 4°C.

Techniques: Vaccines, Activity Assay

Journal: iScience

Article Title: Synthetic vaccine affords full protection to mice against lethal challenge of influenza B virus of both genetic lineages

doi: 10.1016/j.isci.2021.103328

Figure Lengend Snippet:

Article Snippet: Briefly, 96-well plates were coated with 100ul/well of 0.5ug/ml of recombinant influenza B/Florida/4/06 HA2 protein (Sino Biological Inc.), overnight at 4°C.

Techniques: Virus, Recombinant, Bioassay, Luciferase, Expressing, Multiplex Assay, Plasmid Preparation, Software

Journal: Proceedings of the Royal Society B: Biological Sciences

Article Title: An experimental test of a fundamental food web motif

doi: 10.1098/rspb.2009.2191

Figure Lengend Snippet: Estimates of interaction strength and resulting measures of stability for each experimental treatment. Here, n is the number of replicate microcosms. Numbers presented are means ± s.e. Letters (A, B, C) represent significant differences from ANOVA (d.f. = 2, 13) with Tukey's HSD analysis ( p ≤ 0.05). ACF, autocorrelation function.

Article Snippet: Treatments were: (i) B. calyciflorus (Florida Aqua Farms Inc., Dade City, USA) and S. obliquus (University of Toronto Culture Collection (UTCC), Toronto, Canada, number 157); (ii) B. calyciflorus and C. vulgaris (UTCC number 90); (iii) B. calyciflorus and both S. obliquus and C. vulgaris .

Techniques: Algae

Journal: Proceedings of the Royal Society B: Biological Sciences

Article Title: An experimental test of a fundamental food web motif

doi: 10.1098/rspb.2009.2191

Figure Lengend Snippet: Correlation coefficients between the density of C. vulgaris and S. obliquus in communities with and without B. calyciflorus. Here we show mean ± s.e. Communities with B. calyciflorus are significantly more negatively correlated than communities without B. calyciflorus (two-tailed t-test, d.f. = 8, p < 0.001).

Article Snippet: Treatments were: (i) B. calyciflorus (Florida Aqua Farms Inc., Dade City, USA) and S. obliquus (University of Toronto Culture Collection (UTCC), Toronto, Canada, number 157); (ii) B. calyciflorus and C. vulgaris (UTCC number 90); (iii) B. calyciflorus and both S. obliquus and C. vulgaris .

Techniques: Two Tailed Test

Journal: Proceedings of the Royal Society B: Biological Sciences

Article Title: An experimental test of a fundamental food web motif

doi: 10.1098/rspb.2009.2191

Figure Lengend Snippet: Sample population dynamics of B. calyciflorus for each experimental treatment. Presented are population dynamics (solid line) and model dynamics (dotted line) for each of the three treatments: (a) the strong consumer–resource interaction; (b) the weak consumer–resource interaction and (c) the weak and strong consumer–resource interaction.

Article Snippet: Treatments were: (i) B. calyciflorus (Florida Aqua Farms Inc., Dade City, USA) and S. obliquus (University of Toronto Culture Collection (UTCC), Toronto, Canada, number 157); (ii) B. calyciflorus and C. vulgaris (UTCC number 90); (iii) B. calyciflorus and both S. obliquus and C. vulgaris .

Techniques: