Journal: PLoS ONE

Article Title: Searching for Cellular Partners of Hantaviral Nonstructural Protein NSs: Y2H Screening of Mouse cDNA Library and Analysis of Cellular Interactome

doi: 10.1371/journal.pone.0034307

Figure Lengend Snippet: Intersection of closest shared TULV and PUUV NSs -linked nodes. Primary nodes are in red, secondary nodes in pink. Secondary nodes that do not directly connect to two different primary nodes are in white. Metanodes are represented by pink diamonds.

Article Snippet: Samples were incubated 1 h at 37°C with rabbit polyclonal antibodies against TULV NSs-GST fusion protein and mouse monoclonal anti-ACBD3 antibody (Abnova, Taipei, Taiwan).

Techniques:

Journal: PLoS ONE

Article Title: Searching for Cellular Partners of Hantaviral Nonstructural Protein NSs: Y2H Screening of Mouse cDNA Library and Analysis of Cellular Interactome

doi: 10.1371/journal.pone.0034307

Figure Lengend Snippet: Primary cells were infected with TULV strain Lodz for 9 days and fixed on coverslips. Cells were stained for the viral NSs protein, which was seen as bright spots around perinuclear area (A), and ACBD3 protein (B). FRET assay: D pre , donor intensity before bleaching, D post , donor intensity after bleaching, A pre , acceptor intensity before bleaching, A post , acceptor intensity after bleaching, FRET eff , calculated efficiency of FRET.

Article Snippet: Samples were incubated 1 h at 37°C with rabbit polyclonal antibodies against TULV NSs-GST fusion protein and mouse monoclonal anti-ACBD3 antibody (Abnova, Taipei, Taiwan).

Techniques: Infection, Staining

Journal: Journal of Virology

Article Title: Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

doi: 10.1128/JVI.02490-15

Figure Lengend Snippet: SFTS virus NSs induces the formation of endosome-like structures. Ultrastructure analyses of SFTS virus NSs-expressing cells (A) and SFTS virus-infected cells (B) show cytoplasmic structures reminiscent of early endosomes (arrows) in ultrathin sections. Immunogold staining of distinct ultrathin sections shows cytoplasmic structures positive for SFTS virus NSs and Rab5 (C) or SFTS virus NSs and LC3B (D). A solid red arrows indicate SFTS virus NSs in panels C and D, while a red triangle (in panel C) and a green triangle (in panel D) indicate the detection of Rab5 and LC3B, respectively. Ultrathin sections of mock-infected cells were also labeled, as indicated above (not shown), to ensure the specificity of antibody. Representatives images are shown.

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit anti-SFTS virus NSs (1:500; GenScript), mouse anti-SFTS virus NP (1:500), rabbit anti-CD63 (1:100; Abcam), mouse anti-β Tubulin (1:1,000; Abcam), rabbit anti-LC3 (1:1,000; Abcam), and rabbit anti-Rab5 (1:1,000; Abcam).

Techniques: Expressing, Infection, Staining, Labeling

Journal: Journal of Virology

Article Title: Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

doi: 10.1128/JVI.02490-15

Figure Lengend Snippet: Isolated extracellular vesicles are positive for SFTS virus NSs and the exosomal marker CD63. Supernatant was collected from HeLa cells stably expressing mCherry or SFTS virus NSs-mCherry (A) and from HeLa cells mock-infected or infected with SFTS virus (B), and isolation of extracellular microvesicles was performed as depicted in Fig. 3A. The final pellet was resuspended in molecular-grade water. The sample was adsorbed onto Ni grids and negatively stained with 2% aqueous uranyl acetate. Additional grids were incubated with primary antibodies against SFTS virus NSs (rabbit) and CD63 (mouse) and then secondary goat anti-mouse IgG couple to 6-nm colloidal gold and goat anti-rabbit IgG coupled to 15-nm colloidal gold antibodies and then negatively stained with 2% aqueous uranyl acetate. SFTS virus NSs is indicated by red arrows, and CD63 is indicated by green arrows. (C) The number of CD63-positive and CD63/SFTS virus NSs-positive extracellular vesicles was quantified based on immunogold labeling observed from 10 fields of view. A total of 22 vesicles were observed in each experiment, of which an average of 14 were positive for both SFTS virus NSs and CD63. All electron microscopy experiments were repeated three times. Results are expressed as means plus the standard errors of the mean (SEM). Asterisks specify statistically significant difference (P < 0.05) between the indicated groups.

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit anti-SFTS virus NSs (1:500; GenScript), mouse anti-SFTS virus NP (1:500), rabbit anti-CD63 (1:100; Abcam), mouse anti-β Tubulin (1:1,000; Abcam), rabbit anti-LC3 (1:1,000; Abcam), and rabbit anti-Rab5 (1:1,000; Abcam).

Techniques: Isolation, Marker, Stable Transfection, Expressing, Infection, Staining, Incubation, Labeling, Electron Microscopy

Journal: Journal of Virology

Article Title: Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

doi: 10.1128/JVI.02490-15

Figure Lengend Snippet: Cytoplasmic vesicles containing SFTS virus NSs-mCherry are secreted into the extracellular space and are endocytosed by neighboring cells. Live cell imaging was carried out in HeLa cell line stably expressing SFTS virus NSs-mCherry. Cells were visualized for 16 h using a Prairie Technologies/Nikon multimodal live cell imaging system. The arrow highlights the movement of the vesicle from cell to cell.

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit anti-SFTS virus NSs (1:500; GenScript), mouse anti-SFTS virus NP (1:500), rabbit anti-CD63 (1:100; Abcam), mouse anti-β Tubulin (1:1,000; Abcam), rabbit anti-LC3 (1:1,000; Abcam), and rabbit anti-Rab5 (1:1,000; Abcam).

Techniques: Live Cell Imaging, Stable Transfection, Expressing

Journal: Journal of Virology

Article Title: Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

doi: 10.1128/JVI.02490-15

Figure Lengend Snippet: Isolation and characterization of SFTS virus NSs-positive secreted extracellular vesicles. (A) Schematic representation of the protocol for the isolation of secreted extracellular vesicles by ultracentrifugation. (B) Supernatants from cell lines expressing the mCherry and SFTS virus NSs-mCherry proteins were collected, and isolation of extracellular microvesicles was performed as indicated in panel A. The final pellet was resuspended in lysis buffer, sonicated, resolved by SDS-PAGE electrophoresis, transferred to a PVDF membrane, and blotted for SFTS virus NSs, LC3B, and common markers for microvesicles such as Rab5, β-tubulin, and CD63 (core protein, 26 kDa; glycosylated protein, 30 to 60 kDa). The cell monolayer was used to generate the whole-cell lysate (WCL) and assayed for the detection of the proteins indicated above. Densitometry analysis of CD63, LC3-I, and Rab 5 present in extracellular vesicles isolated from mCherry or SFTS virus NSs-mCherry expressing cells was also conducted. The band signal intensity of each protein was normalized to the signal intensity of β-tubulin and expressed as arbitrary units (A.U.). Signal intensities were obtained by using ImageJ software.

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit anti-SFTS virus NSs (1:500; GenScript), mouse anti-SFTS virus NP (1:500), rabbit anti-CD63 (1:100; Abcam), mouse anti-β Tubulin (1:1,000; Abcam), rabbit anti-LC3 (1:1,000; Abcam), and rabbit anti-Rab5 (1:1,000; Abcam).

Techniques: Isolation, Expressing, Lysis, Sonication, SDS Page, Electrophoresis, Software

Journal: Journal of Virology

Article Title: Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

doi: 10.1128/JVI.02490-15

Figure Lengend Snippet: Characterization of extracellular microvesicles secreted during SFTS virus infection. HeLa cells were mock infected or infected with SFTS virus for 72 h. (A) Supernatant was collected, and isolation of extracellular microvesicles was performed as indicated. (B) The final pellet was resuspended in lysis buffer, sonicated, resolved by SDS-PAGE electrophoresis, transferred to a PVDF membrane, and blotted for SFTS virus NSs, SFTS virus NP, and LC3B, in addition to common markers for microvesicles such as Rab5, CD63, and β-tubulin. The cell monolayer was used to generate the WCL and assayed for the detection of proteins indicated above. Densitometry analysis of CD63, LC3-I, and Rab 5 present in extracellular vesicles isolated from mock infected or SFTS virus-infected cells was carried out. The band signal intensity of each protein was normalized to the signal intensity of β-tubulin and expressed as arbitrary units (A.U.). Signal intensities were obtained by using ImageJ software.

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit anti-SFTS virus NSs (1:500; GenScript), mouse anti-SFTS virus NP (1:500), rabbit anti-CD63 (1:100; Abcam), mouse anti-β Tubulin (1:1,000; Abcam), rabbit anti-LC3 (1:1,000; Abcam), and rabbit anti-Rab5 (1:1,000; Abcam).

Techniques: Infection, Isolation, Lysis, Sonication, SDS Page, Electrophoresis, Software

Journal: Journal of Virology

Article Title: Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

doi: 10.1128/JVI.02490-15

Figure Lengend Snippet: SFTS virus NSs-positive extracellular vesicles harbor infectious SFTS virus virions. (A) Extracellular vesicles were isolated as indicated in Fig. 4A, and samples were collected at each step of the purification process for evaluation by plaque assay. The sample resulting from the immune-selection step using anti-CD63 beads is depicted in the figure as the CD63 selected, whereas those resulting from the subsequent immune-(negative) selection step are shown as the CD63/SFTSV selected. As a final step, the purified vesicles were incubated with SFTS virus mouse hyperimmune ascitic fluid at a 1:1 ratio, and the antibody-virus complex was then removed with magnetic beads. A plaque assay of the resulting purified vesicles was performed, and the results are depicted in the figure as vesicles + anti-SFTS virus. The results of a plaque assay of the virus stock (infected cells from which the extracellular vesicles were derived) are shown as the SFTS virus stock. As a control, a virus stock was also subjected to the immune-negative selection step, followed by incubation with the SFTS virus antibody as indicated above, and a plaque assay was carried out (labeled “SFTS virus+anti-SFTS virus”). A dashed line indicates the limit of detection. The results are expressed as means + the SEM. Asterisks specify statistically significant differences (P < 0.05) between the indicated groups. (B) An electron micrograph of isolated extracellular vesicles reveals the presence of virus-like particles contained within the vesicles (red arrow). The presence of free virions from broken vesicles was also observed (green arrow).

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit anti-SFTS virus NSs (1:500; GenScript), mouse anti-SFTS virus NP (1:500), rabbit anti-CD63 (1:100; Abcam), mouse anti-β Tubulin (1:1,000; Abcam), rabbit anti-LC3 (1:1,000; Abcam), and rabbit anti-Rab5 (1:1,000; Abcam).

Techniques: Isolation, Purification, Plaque Assay, Selection, Incubation, Magnetic Beads, Infection, Derivative Assay, Labeling

Journal: Journal of Virology

Article Title: Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

doi: 10.1128/JVI.02490-15

Figure Lengend Snippet: SFTS virus NSs-positive extracellular vesicles can mediate receptor-independent transmission of SFTS virus. HeLa cells were pretreated with anti-CD63 antibody (A), IgG control (B), anti-SFTS virus antibody (C), or PBS (D) prior to infection with the purified extracellular vesicles, SFTS virus, or the SFTS virus preparation subjected to the immune-negative selection and the antibody incubation step described in Fig. 6. Supernatants were harvested at 0, 24, 48, and 72 hpi, and a plaque assay was performed. The dashed line indicates the limit of detection (4 PFU/ml). All experiments were repeated three times with consistent results. The results presented are expressed as means + the SEM. Asterisks indicate statistically significant differences (P < 0.05) between the indicated groups.

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit anti-SFTS virus NSs (1:500; GenScript), mouse anti-SFTS virus NP (1:500), rabbit anti-CD63 (1:100; Abcam), mouse anti-β Tubulin (1:1,000; Abcam), rabbit anti-LC3 (1:1,000; Abcam), and rabbit anti-Rab5 (1:1,000; Abcam).

Techniques: Transmission Assay, Infection, Purification, Selection, Incubation, Plaque Assay

Journal: Journal of Virology

Article Title: Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

doi: 10.1128/JVI.02490-15

Figure Lengend Snippet: Virions harbored within SFTS virus NSs-positive extracellular vesicles are capable of establishing productive infection. HeLa cells were mock infected (top of panels A and B), infected with SFTS virus (A), or infected with supernatants collected from cells infected with the purified, CD63 immune-selected extracellular vesicles described in Fig. 7D (B). Cells were fixed at 0, 24, 48, and 72 hpi. Immunofluorescence was performed using primary antibodies against SFTS virus NP or NSs and Alexa Fluor 488 as the secondary antibody. Nuclei were visualized with Hoechst 33342. Representative images for the mock-infected groups are shown. (C) Percentages of cells positive for SFTS virus NP or NSs calculated by cell counts in 10 fields of view. A total of 150 cells were counted, and the percentage was calculated by dividing the total cells positive for SFTS virus NP or NSs by 150.

Article Snippet: The following primary antibodies were used for immunoblotting: rabbit anti-SFTS virus NSs (1:500; GenScript), mouse anti-SFTS virus NP (1:500), rabbit anti-CD63 (1:100; Abcam), mouse anti-β Tubulin (1:1,000; Abcam), rabbit anti-LC3 (1:1,000; Abcam), and rabbit anti-Rab5 (1:1,000; Abcam).

Techniques: Infection, Purification, Immunofluorescence

Journal: Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology

Article Title: Gold nanoparticle-mediated photothermal therapy: applications and opportunities for multimodal cancer treatment

doi: 10.1002/wnan.1449

Figure Lengend Snippet: Schematics demonstrating the physiological and biological effects of photothermal therapy. (a) The heat generated by AuNPs in response to NIR light enhances the permeability of tumor vasculature and cell membranes to increase the accumulation of secondary therapies, such as chemotherapeutic drugs. (b) PTT can lead to either cellular necrosis or apoptosis, which cause different cellular and immune responses. The mechanism of cell death depends on the applied irradiation parameters.

Article Snippet: 37 Among the various types of AuNPs used for PTT, NSs coated with PEG (also known as AuroShells, which are being commercialized by Nanospectra Biosciences, Inc.) are the furthest along in development and are currently the only AuNP for PTT being evaluated in clinical trials.

Techniques: Generated, Permeability, Irradiation

Journal: NPJ Vaccines

Article Title: Humoral immunity is sufficient to protect mice against Rift Valley fever encephalitis following percutaneous exposure

doi: 10.1038/s41541-025-01200-2

Figure Lengend Snippet: Mice ( n = 3 to 4 per experimental group) were vaccinated with 2 x 10 5 TCID 50 of ΔNSs, ΔNSsΔNSm RVFV or mock-vaccinated (PBS) and then challenged at 28 days post-vaccination with 2 TCID 50 WT RVFV, both via foot pad injection ( A ). Animals were monitored for survival, and survivors were euthanized on day 56 post-vaccination ( B ). Terminal viral RNA loads in liver, spleen and brain were measured ( C ). The dotted line represents the limit of detection (LOD) of the assay, and values below LOD are plotted at LOD (4.1 × 10 6 copies/g). The endpoint titers of total RVFV-specific or RVFV NSs-specific antibodies by ELISA or neutralization titer were measured pre-challenge ( D , E ) and at time of euthanasia ( F , G ). The LOD is depicted as a dotted line at 100 for ELISA and 40 for focus reduction neutralization test (FRNT 80 ). Negative values were plotted at 50 (ELISA) and 20 (FRNT 80 ), respectively. Survivors are displayed as open symbols, and graphs depict the geometric mean and standard deviation (SD) for each group. The timeline ( A ) was generated with BioRender.com.

Article Snippet: For RVFV NSs-specific ELISA, plates were coated with SARS-CoV-2 N protein as the negative control or recombinant RVFV NSs (custom, Genscript) at 200 ng of protein per well in sterile PBS and incubated at 4 °C overnight.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Neutralization, Standard Deviation, Generated

Journal: NPJ Vaccines

Article Title: Humoral immunity is sufficient to protect mice against Rift Valley fever encephalitis following percutaneous exposure

doi: 10.1038/s41541-025-01200-2

Figure Lengend Snippet: Mice ( n = 3 per experimental group) were vaccinated with either 20 TCID 50 or 2000 TCID 50 of ΔNSsΔNSm RVFV and then challenged at 28 days post-vaccination with 2 TCID 50 WT RVFV, both via foot pad injection ( A ). Animals were monitored for survival, and survivors were euthanized on day 56 post-vaccination ( B ). Terminal viral RNA loads in liver, spleen and brain were measured for the L and S segment for vaccinated animals and samples from Mock infected animals from Fig. were included for comparison ( C , D ). The dotted lines represent the LOD of the assays (L segment: 3.6 x 10 6 copies/g; S segment: 8 x 10 6 copies/g). Data points marked with x indicate that no virus could be isolated from brain tissues while the data point with the check indicates that virus could be isolated from brain tissue ( C ). The endpoint titers of total RVFV-specific or RVFV NSs-specific antibodies by ELISA or neutralization titers were measured pre-challenge ( E , F ) and at the time of euthanasia ( G , H ). The LOD is depicted as a dotted line at 100 for ELISA and 40 for FRNT 80 . Negative values were plotted at 50 for ELISA. Survivors are displayed as open symbols, and graphs depict the geometric mean and SD for each group. The timeline ( A ) was generated using BioRender.com.

Article Snippet: For RVFV NSs-specific ELISA, plates were coated with SARS-CoV-2 N protein as the negative control or recombinant RVFV NSs (custom, Genscript) at 200 ng of protein per well in sterile PBS and incubated at 4 °C overnight.

Techniques: Injection, Infection, Comparison, Virus, Isolation, Enzyme-linked Immunosorbent Assay, Neutralization, Generated

Journal: NPJ Vaccines

Article Title: Humoral immunity is sufficient to protect mice against Rift Valley fever encephalitis following percutaneous exposure

doi: 10.1038/s41541-025-01200-2

Figure Lengend Snippet: Splenocytes were collected from vaccinated mice (day 28) or vaccinated and WT RVFV challenged mice (day 56) and tested in ex vivo peptide stimulation assays ( n = 4 per group). The number of RVFV-specific T cells in total splenocytes was assessed by IFN-γ ELISPOT assay, expressed as the number of spot-forming units (SFUs) per 1x10 5 splenocytes for RVFV N, Gn (Gn1 + Gn2), and Gc (Gc1 + Gc2) ( A ). The gating strategy for flow cytometry is displayed ( B ). The percentage of RVFV-specific CD4+ and CD8+ T cells was determined by flow cytometry by staining for CD44 and IFN-γ ( C , D ). Graphs depict mean and SD per group. Statistical comparison was performed using non-parametric Mann-Whitney test with * p < 0.05.

Article Snippet: For RVFV NSs-specific ELISA, plates were coated with SARS-CoV-2 N protein as the negative control or recombinant RVFV NSs (custom, Genscript) at 200 ng of protein per well in sterile PBS and incubated at 4 °C overnight.

Techniques: Ex Vivo, Enzyme-linked Immunospot, Flow Cytometry, Staining, Comparison, MANN-WHITNEY

Journal: NPJ Vaccines

Article Title: Humoral immunity is sufficient to protect mice against Rift Valley fever encephalitis following percutaneous exposure

doi: 10.1038/s41541-025-01200-2

Figure Lengend Snippet: Mice ( n = 3 to 9 per experimental group) were challenged with 2 TCID 50 WT RVFV via foot pad injection and later received passive transfer (PT) of normal or immune mouse serum via intraperitoneal (i.p.) injection at indicated timepoints ( A ). Animals were monitored for survival and survivors were euthanized on day 28 post-infection ( B ). Statistical comparison was performed using Mantel-Cox test with * p < 0.05, ** p < 0.01, ns- not significant. Terminal viral RNA loads in liver, spleen and brain were measured for the L segment for all animals ( C ) and for the S segment for the survivors ( D ). The dotted lines represent the LOD of the assays (L segment: 1.1 × 10 6 copies/g; S segment: 1 × 10 8 copies/g). Data points marked with x indicate that no virus could be isolated from brain tissues while the data point with the check indicates that virus could be isolated from brain tissue ( C ). The endpoint titers of total RVFV-specific (all animals) or RVFV NSs-specific (survivors only) antibodies by ELISA or neutralization titers were measured one day following i.p. injection ( E , F ) and at time of euthanasia ( G , H ). The limit of LOD is depicted as a dotted line at 100 for ELISA and 40 for FRNT 80 . Negative values were plotted at 50 (ELISA) and 20 (FRNT 80 ), respectively. Survivors are displayed as open symbols, and graphs depict the geometric mean and SD for each group. The timeline ( A ) was generated using BioRender.com.

Article Snippet: For RVFV NSs-specific ELISA, plates were coated with SARS-CoV-2 N protein as the negative control or recombinant RVFV NSs (custom, Genscript) at 200 ng of protein per well in sterile PBS and incubated at 4 °C overnight.

Techniques: Injection, Infection, Comparison, Virus, Isolation, Enzyme-linked Immunosorbent Assay, Neutralization, Generated

Journal: NPJ Vaccines

Article Title: Humoral immunity is sufficient to protect mice against Rift Valley fever encephalitis following percutaneous exposure

doi: 10.1038/s41541-025-01200-2

Figure Lengend Snippet: Mice ( n = 3 per experimental group) were challenged with 2 TCID 50 WT RVFV via foot pad injection and later received passive transfer of various dilutions (1:20, 1:100, 1:500) of immune mouse serum via i.p. injection at 2dpi ( A ). Animals were monitored for survival, and survivors were euthanized on day 28 post-infection ( B ). Statistical comparison to PT D2 normal serum animals (Fig. ) was performed using Mantel-Cox test with * p < 0.05, ns- not significant. Terminal viral RNA loads in liver, spleen and brain were measured for the L and S segment ( C , D ). The dotted lines represent the LOD of the assays (L segment: 3.9 × 10 5 copies/g; S segment: 4.7 × 10 6 copies/g). Data points marked with x indicate that no virus could be isolated from brain tissues ( C ). The endpoint titers of total RVFV-specific (all animals) or RVFV NSs-specific (survivors only) antibodies by ELISA or neutralization titers were measured one day following i.p. injection ( E , F ) and/or at the time of euthanasia ( G , H ). The limit of detection (LOD) is depicted as a dotted line at 100 for ELISA, 20 for post-PT FRNT 80,50 and 40 for terminal FRNT 80 . Negative values were plotted at 50 for ELISA, 10 for post-PT FRNT 80,50 and 20 for terminal FRNT 80 . Survivors are displayed as open symbols, and graphs depict the geometric mean and SD for each group. The timeline ( A ) was generated using BioRender.com.

Article Snippet: For RVFV NSs-specific ELISA, plates were coated with SARS-CoV-2 N protein as the negative control or recombinant RVFV NSs (custom, Genscript) at 200 ng of protein per well in sterile PBS and incubated at 4 °C overnight.

Techniques: Injection, Infection, Comparison, Virus, Isolation, Enzyme-linked Immunosorbent Assay, Neutralization, Generated