Journal: Journal of Virology

Article Title: Ceramide synthase 4 interferes with replication of influenza virus but is downregulated by infection

doi: 10.1128/jvi.01563-25

Figure Lengend Snippet: Transient overexpression of CerS4 impairs productive infection of influenza viruses. ( A ) HEK293FT cells were transfected with control plasmid (−) or plasmid encoding Myc-tagged CerS4 (+). The cells were mock-infected or infected with IAV (pH1N1) at an MOI of 1 at 18 hours post-transfection. At 24 hpi, cells were harvested, and Western blotting was performed to detect the levels of IAV-HA, PB1, NS1, M1, Myc-tagged CerS4, and GAPDH. Densitometric values are shown, with relative expression of viral protein/GAPDH in vector control and infected samples set to 1.0. The effect of CerS4 overexpression in infected cells was compared. ( B ) A549 cells were transfected with vector control or CerS4-encoding plasmid. At 18 hours after transfections, cells were mock-infected or infected with pH1N1 at an MOI of 3 for 8 hours. Expression levels of viral proteins were assessed as shown in panel ( A ). ( C–E ) A549 cells were transfected with a vector control plasmid (Control) or plasmid encoding CerS4 (CerS4) or plasmid encoding CerS1 (CerS1, only). In ( C ), at 18 hours post-transfection, the cells were infected with IAV pH1N1 at an MOI of 0.01. At 1 ( n = 7/group), 2 ( n = 3/group), or 3 ( n = 6/group) dpi, cellular supernatants were harvested. Control vs CerS1 and Control vs CerS4 titers were statistically analyzed as indicated. In ( D ), A549 cells were infected with IAV pH1N1 at an MOI of 3 for 8 hours, followed by supernatant collection for titration by plaque assay ( n = 3/group). In ( E ), A549 cells were infected with IAV WSN at an MOI of 0.001. Cell supernatants were collected at 2 and 3 dpi for assessing viral titers using plaque assay on MDCK cells ( n = 3/group). Statistical significance was determined by a t -test and is indicated by * P < 0.05, ** P < 0.01, and *** P < 0.001. Data are expressed as means ± SD. ( F ) HEK293FT cells were transfected with control plasmid (−) or plasmid encoding Myc-tagged CerS4 (+) and were infected with influenza B/Lee/40 virus (IBV) or A/Hong Kong/8/68 (H3N2) at an MOI of 1 at 18 hours post-transfection. At 24 hpi, cells were harvested, and Western blotting was performed to detect the levels of IBV NP, IAV NS1, Myc-tagged CerS4, and GAPDH. The data are representative of three independent experiments.

Article Snippet: Both the A/Hong Kong/8/68 (H3N2) virus (ATCC VR-1679) and the B/Lee/40 (IBV) virus (ATCC VR-1535) were purchased through ATCC.

Techniques: Over Expression, Infection, Transfection, Control, Plasmid Preparation, Western Blot, Expressing, Titration, Plaque Assay, Virus

Journal: Nature Communications

Article Title: Characterization of two non-competing antibodies to influenza H3N2 hemagglutinin stem reveals its evolving antigenicity

doi: 10.1038/s41467-025-65595-1

Figure Lengend Snippet: A The binding activities of AG2-G02 and 2F02 against recombinant HA proteins from the specified influenza strains were measured by ELISA. The absorbance values at 450 nm are shown as a heatmap. B , C The neutralization activity of AG2-G02 and 2F02 against recombinant H3N8 and different recombinant H3N2 viruses was measured by a microneutralization assay, with antibodies ( B ) continuously present in the medium throughout the experiment, or ( C ) removed at one-hour post-infection (hpi). MIC: minimal inhibitory concentration. Each bar represents the mean of two independent biological replicates. Each data point represents one replicate. A–C Strain names are abbreviated as follows: H3N2 A/Hong Kong/1/1968 (HK/1968), H3N2 A/Philippines/2/1982 (Philippines/1982), H3N2 A/Beijing/109/1992 (Beijing/1992), H3N2 A/Wuhan/359/1995 (Wuhan/1995), H3N2 A/Moscow/10/1999 (Moscow/1999), H3N2 A/New York/55/2004 (NY/2004), H3N2 A/Wisconsin/67/2005 (Wisconsin/2005), H3N2 A/Uruguay/716/2007 (Uruguay/2007), H3N2 A/Switzerland/9715293/2013 (Switzerland/2013), H3N2 A/Darwin/6/2021 (Darwin/2021), H3N8 A/mallard/Alberta/362/2017 (Alberta/2017), H4N6 A/mallard/Alberta/455/2015 (Alberta/2015), H7N3 A/Canada/rv444/2004 (Canada/2004), H7N9 A/Hong Kong/125/2017 (HK/2017), H7N9 A/Anhui/1/2013 (Anhui/2013), H14N5 A/mallard/Astrakhan/263/1982 (Astra/1982), and H15N2 A/Australian shelduck/Western Australia/1756/1983 (WAus/1983).

Article Snippet: HA proteins of the following strains were purchased from SinoBiological: H3N2 A/HongKong/1/1968, H3N2 A/Beijing/109/1992, H3N2 A/Wuhan/359/1995, H3N2 A/Moscow/10/1999, H3N2 A/Switzerland/9715293/2013, H14N5 A/mallard/Astrakhan/263/1982, and H15N2 A/Australian shelduck/Western Australia/1756/1983.

Techniques: Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Neutralization, Activity Assay, Microneutralization Assay, Infection, Concentration Assay, Western Blot

Journal: Nature Communications

Article Title: Characterization of two non-competing antibodies to influenza H3N2 hemagglutinin stem reveals its evolving antigenicity

doi: 10.1038/s41467-025-65595-1

Figure Lengend Snippet: Female BALB/c mice at six weeks old were injected intraperitoneally with 5 mg/kg of the indicated antibody four hours prior to challenge with 5 × LD 50 of H3N2 Philippines/1982 (X-79). CR9114 was used as a positive control for protection. A Kaplan-Meier survival curves are presented ( n = 7 for control, n = 5 for experimental groups). B The mean percentage of body weight change post-infection is shown ( n = 7 for the control group, n = 5 for each experimental group). The humane endpoint was defined as a 25% weight loss from the initial weight on day 0. Data are presented as mean ± standard deviation. C Lung viral titers on day 3 post-infection are shown ( n = 3 per group). Each bar represents the mean. Each data point represents one mouse.

Article Snippet: HA proteins of the following strains were purchased from SinoBiological: H3N2 A/HongKong/1/1968, H3N2 A/Beijing/109/1992, H3N2 A/Wuhan/359/1995, H3N2 A/Moscow/10/1999, H3N2 A/Switzerland/9715293/2013, H14N5 A/mallard/Astrakhan/263/1982, and H15N2 A/Australian shelduck/Western Australia/1756/1983.

Techniques: Injection, Positive Control, Control, Infection, Standard Deviation

Journal: Nature Communications

Article Title: Characterization of two non-competing antibodies to influenza H3N2 hemagglutinin stem reveals its evolving antigenicity

doi: 10.1038/s41467-025-65595-1

Figure Lengend Snippet: A Occurrence of different amino acid variants at HA2 position 32 of human H3N2 strains from 1968 to the present was analyzed based on 33,000 human H3N2 HA sequences from the GSAID database . The x-axis represents the years, while the y-axis shows the percentage of strains containing a particular amino acid at HA2 position 32 in a given year. Thr, Ile, and Arg are represented by orange, brown, and green lines, respectively. B The structures of AG2-G02 binding to HA stem with I32 HA2 and R32 HA2 are modeled. The binding affinity ( K d ) of AG2-G02 Fab to different HA stem variants is indicated in parentheses. C–E The binding of plasma samples from ( C ) pre-2003 adults ( n = 20), ( D ) post-2011 adults ( n = 20), and ( E ) post-2011 infants ( n = 15) to H3 stem with either T32 HA2 , I32 HA2 or R32 HA2 is measured by ELISA. The y-axis represents the area under the curve (AUC) of four serial 10-fold dilutions of serum (1:100, 1:1,000, 1:10,000, and 1:100,000). P -values were determined using a paired two-tailed Student’s t -test.

Article Snippet: HA proteins of the following strains were purchased from SinoBiological: H3N2 A/HongKong/1/1968, H3N2 A/Beijing/109/1992, H3N2 A/Wuhan/359/1995, H3N2 A/Moscow/10/1999, H3N2 A/Switzerland/9715293/2013, H14N5 A/mallard/Astrakhan/263/1982, and H15N2 A/Australian shelduck/Western Australia/1756/1983.

Techniques: Binding Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Two Tailed Test